Biosynthesis of mycobacterial lipids by polyketide synthases and beyond.

http://informahealthcare.com/bmg ISSN: 1040-9238 (print), 1549-7798 (electronic) Editor: Michael M. Cox Crit Rev Biochem Mol Biol, 2014; 49(3): 179–211 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10409238.2014.896859

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REVIEW ARTICLE

Biosynthesis of mycobacterial lipids by polyketide synthases and beyond Luis E. N. Quadri Department of Biology, Brooklyn College, City University of New York, Brooklyn, NY, USA

Abstract

Keywords

Over a decade ago, the analysis of the complete sequence of the genome of the human pathogen Mycobacterium tuberculosis revealed an unexpectedly high number of open reading frames encoding proteins with homology to polyketide synthases (PKSs). PKSs form a large family of fascinating multifunctional enzymes best known for their involvement in the biosynthesis of hundreds of polyketide natural products with diverse biological activities. The surprising polyketide biosynthesis capacity of M. tuberculosis has been investigated since its initial inference from genome analysis. This investigation has been based on the genes found in M. tuberculosis or their orthologs found in other Mycobacterium species. Today, the majority of the PKS-encoding genes of M. tuberculosis have been linked to specific biosynthetic pathways required for the production of unique lipids or glycolipid conjugates that are critical for virulence and/or components of the extraordinarily complex mycobacterial cell envelope. This review provides a synopsis of the most relevant studies in the field and an overview of our current understanding of the involvement of PKSs and several other polyketide production pathway-associated proteins in critical biosynthetic pathways of M. tuberculosis and other mycobacteria. In addition, the most relevant studies on PKS-containing biosynthetic pathways leading to production of metabolites from mycobacteria other than M. tuberculosis are reviewed.

Cell wall, lipid, mycobacterium, polyketide, siderophore, virulence

Introduction Mycobacterium tuberculosis, the obligate pathogen causative of tuberculosis, is responsible for devastating morbidity and mortality worldwide. The 2012 global tuberculosis report of the World Health Organization indicates that there are close to 9 million new cases of tuberculosis per year, and the disease claims 1.4 million lives annually (WHO, 2012). In 1998, Cole and coworkers reported the complete sequence of the chromosome of the archetypal strain of M. tuberculosis, H37Rv Pasteur (Cole et al., 1998). The analysis of the M. tuberculosis H37Rv genome represents a remarkable milestone that signals the opening of the post-genomic era in M. tuberculosis research and led to a drastic increase and diversification in research undertakings to better understand the biology of this formidable global pathogen. The chromosome of M. tuberculosis H37Rv comprises 4 411 529 bp and includes 4018 protein-encoding genes. Notably, the scrutiny

Address for correspondence: Luis E. N. Quadri, Department of Biology, Brooklyn College, City University of New York, 2900 Bedford Avenue, Brooklyn, NY 11210, USA. E-mail: [email protected]

History Received 27 November 2013 Revised 18 February 2014 Accepted 18 February 2014 Published online 14 March 2014

of this genome revealed a remarkable abundance of genes encoding enzymes with predicted involvement in lipid metabolism, including biosynthesis of several polyketide metabolites. Since the release of the genome information, there have been significant advances in our ability to connect the bioinformatically predicted capacity for polyketide product biosynthesis encoded in M. tuberculosis with the recognized presence of an unusual lipid and glycolipid array in the unique cell envelope of this relentless human pathogen (Figure 1). Today, most of the PKS-encoding genes of M. tuberculosis have been linked to specific biosynthetic pathways required for the production of unique lipids or glycolipid conjugates that are critical for virulence and/or components of the extraordinarily complex outer membrane of the mycobacterial cell envelope (Figure 2). The studies leading to this knowledge have been based on functional interrogation of genes and proteins from M. tuberculosis or their corresponding orthologs in other Mycobacterium species. Mutational analyses coupled with homology-based protein function predictions have provided the critical clues leading to the development of the current working models for biosynthesis of these lipids or glycolipid conjugates. It should be noted, however, that the lack of genetic complementation controls in several mutational analyses (particularly in older literature) has been a major issue in the field. The gene-to-polyketide

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Polyketide synthases at a glance

Figure 1. Proposed layers in the cell envelope of M. tuberculosis. The layer of extractable lipids and glycolipids is thought to be organized as a membrane outer leaflet that partners with a mycolic acid-based membrane inner leaflet to form an asymmetric lipid bilayer that constitutes the characteristic mycobacterial outer membrane. Cell envelope-associated proteins and a possible periplasmic space between the plasma membrane and the peptidoglycan layer are not represented.

production pathway assignments derived from these analyses need to be considered in light of this issue. When complementation information is not explicitly mentioned in the sections of this review outlining these functional assignments, the reader should interpret that such information is not available in the cited literature. In vitro enzymology studies and other biochemical approaches continue to provide experimental evidence supporting and validating these working models. This review presents an overview of our current understanding of the involvement of PKSs and several other polyketide production pathway-associated enzymes in critical biosynthetic pathways of M. tuberculosis and other mycobacteria. Polyketide synthase associated proteins (Paps) and fatty acyl-AMP ligases (FAALs) are among the polyketide production pathway-associated enzymes covered herein. Remarkably, both Paps and FAALs are enzyme families first recognized during studies of polyketide biosynthesis in mycobacteria. Paps form a distinct family of acyltransferases that catalyze the direct transfer of a polyketide product thioesterified on the terminal carrier protein domain of a specific PKS partner to hydroxyl groups in acyl chain acceptor cosubstrates (Onwueme et al., 2004; Trivedi et al., 2005). FAALs represent a new class of adenylation enzymes that adenylate long-chain fatty acids, which are then transferred to cognate PKS partners for acyl chain extension (Trivedi et al., 2004; Arora et al., 2009).

PKSs form a large family of fascinating multifunctional enzymes that are mechanistically and structurally related to fatty acid synthases (Crawford & Townsend, 2010; Fischbach & Walsh, 2006; Hertweck, 2009; Hopwood & Sherman, 1990; Khosla, 2009; Shen, 2003; Staunton & Weissman, 2001; Van Lanen & Shen, 2008; Walsh, 2004). PKSs are involved in the biosynthesis of hundreds of structurally and functionally diverse polyketide and polyketide-derived natural products. Many of these natural products have important medical or agricultural applications (O’Hagen, 1991; Staunton & Weissman, 2001; Weissman & Leadlay, 2005). Others, on the other hand, are disease-associated toxins or virulence factors of pathogenic microbes (George et al., 1999; Huffman et al., 2010; Onwueme et al., 2005a; Quadri, 2000). Most PKSs fall within one of three architectural types, i.e. type I, type II or type III. Type I PKSs are large polypeptides that contain multiple catalytic domains. Based on their biosynthetic strategy, type I PKSs can be further classified into two subtypes; modular and iterative. In the canonical modular type I PKS, distinct functional domains on a single protein are organized in a so-called module, and each domain in a module is utilized only once during polyketide product formation. In contrast, in the canonical iterative type I PKS, each functional domain of those clustered on a single protein is used repetitively during polyketide product assembly. Type II PKSs are multienzyme complexes, with each protein in the complex carrying a single and distinct catalytic domain that is utilized iteratively during formation of the polyketide product. The biosynthetic mechanism utilized by both type I and type II PKSs requires the involvement of an acyl carrier protein (ACP) domain or an ACP to hold and present acyl chains as covalently tethered phosphopantetheinyl-thioester intermediates during polyketide product biosynthesis. Type III PKSs, also known as chalcone synthase-like PKSs, represent the latest addition to the PKS superfamily. Type III PKSs are homodimeric enzymes structurally and mechanistically divergent from type I and type II PKSs. Most notably, the biosynthetic mechanism utilized by type III PKSs does not require the involvement of ACP domains or ACPs to hold and present acyl chain intermediates during polyketide product biosynthesis, as seen in type I and type II PKSs. Type III PKSs catalyze consecutive condensation reactions and act on acyl substrates thioesterified to coenzyme A (CoA) instead. Several excellent reviews dedicated to the enzymology of type I, type II and/or type III PKSs have been previously published (Crawford & Townsend, 2010; Fischbach & Walsh, 2006; Hertweck, 2009; Hopwood, 1997; Hopwood & Sherman, 1990; Khosla, 2009; Shen, 2003; Staunton & Weissman, 2001; Van Lanen & Shen, 2008; Walsh, 2004). The reader is referred to this literature for a more extensive coverage of PKS enzymology than the one provided above.

Polyketide synthases in M. tuberculosis The initial analysis of the genome of M. tuberculosis strain H37Rv revealed an unexpectedly large number of open reading frames encoding proteins with unambiguous homology to PKSs. The genome of M. tuberculosis H37Rv, as


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Biosynthesis of mycobacterial polyketides

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Figure 2. Distribution of polyketide synthase-encoding genes in the chromosome of M. tuberculosis H37Rv Pasteur and their involvement in biosynthesis of specific lipids. The distribution represented is derived from the TubercuList Database (http://tuberculist.epfl.ch/) (Lew et al., 2011). a H37Rv strains do not produce phenolic glycolipids due to a sequence polymorphism that splits the parental pks15/1 gene. bpks16 is currently annotated as a putative polyketide synthase-encoding gene. An NCBI CD-Search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) reveals that Pks16 contains a FAAL conserved domain (cd05931), suggesting that pks16 encodes a class I adenylate forming enzyme, rather than a polyketide synthase. c Strain H37Rv Pasteur does not produce polyacyltrehaloses due to a sequence polymorphism that splits the parental pks3/4 gene. dThe pyrone metabolites are synthesized by the PKS in vitro, but they have not been isolated from mycobacteria. ePredicted pks5 orthologs in other Mycobacterium species are involved in production of lipooligosaccharides, but M. tuberculosis is lipooligosaccharide-deficient and the role of its pks5 gene remains unknown. fIt is unclear whether one or both genes are required for fatty acid production. The megabase pair scale (mbp) is depicted in the inner side of the circular diagram of the chromosome. bp, base pairs.

currently annotated, contains a total of 24 genes encoding PKS homologs (excluding the incorrectly annotated pks16, see below). These PKS-encoding genes are distributed throughout the mycobacterial chromosome (Figure 2), and most of them are located in what appear to be biosynthetic pathway-dedicated gene clusters (Figure 3). Altogether, these PKS-encoding genes amount to 103 644 bp, which represents a surprising 2.6% of the total protein coding bases (4 027 296 bp) of the M. tuberculosis chromosome. The unexpectedly rich repertoire of PKSs in M. tuberculosis includes several modular and iterative type I PKSs with canonical PKS domains predicted from their respective amino acid sequences (Figure 4). Some of these type I PKSs are remarkably large when measured against the average protein length for M. tuberculosis, i.e. 334 amino acids. In particular, Pks12, with 4151 amino acid residues, represents the largest polypeptide encoded in the genome of M. tuberculosis. The ACP domains in Pks12 and in other type I PKSs of M. tuberculosis are predicted to be post-translationally phosphopantetheinylated by the essential phosphopantetheinyl transferase PptT (Chalut et al., 2006; Leblanc et al., 2012). M. tuberculosis PptT (Rv2794c, 227 amino acids) is an Sfp-type phosphopantetheinyl transferase (Quadri et al., 1998b) whose enzymatic activity was first validated and

characterized in the context of its involvement in the biosynthesis of mycobactin/carboxymycobactin siderophores (Quadri et al., 1998a). M. tuberculosis also has several predicted type III PKSs (Figure 4). These are relatively small condensing enzymes (5390 amino acids) that do not require post-translational phosphopantetheinylation and belong to the chalcone synthase superfamily. As noted above, the PKS family is well known for its involvement in the biosynthesis of polyketide natural products with a wide range of biological activities, thus raising the possibility that polyketide biosynthetic pathways could play critical roles in mycobacterial biology and pathogenesis. This intriguing idea has fostered numerous studies to investigate the potential participation of mycobacterial polyketide biosynthetic pathways in the production of complex mycobacterial lipids and to probe the relevance of these pathways to virulence. Since the release of the M. tuberculosis H37Rv genome information (Cole et al., 1998), there has been astonishing progress towards elucidating the biological functions of M. tuberculosis PKSs. The insights in the field have been gained directly from studies with a variety of strains of M. tuberculosis or generated indirectly from work carried out with other species of mycobacteria (e.g. M. bovis and M. marinum) that have orthologs of the PKSs found in

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Figure 3. Chromosomal loci of Mycobacterium tuberculosis H37Rv and other mycobacteria containing biosynthetic pathways involving polyketide synthases. Genes are displayed as horizontal arrows. Polyketide synthase-encoding genes are highlighted with a diagonal stripe pattern. The genome coordinates for the 50 -end and the 30 -end of the chromosomal segments represented are indicated. The size of each genomic chromosomal segment is shown in parentheses. The information represented for M. tuberculosis [panels (a) through (i) and panel (k)] is primarily derived from the TubercuList Database (Lew et al., 2011) and (Tatham et al., 2012). The information represented for M. smegmatis [panel (j)] is primarily derived from the SmegmaList Database (http://mycobrowser.epfl.ch/smegmalist.html; Kapopoulou et al., 2011) and (Ripoll et al., 2007; Vats et al., 2012). The gene pairs pks15 and pks1 (a) and pks3 and pks4 (e) are each fused into a single gene in other strains (see text).


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Figure 4. Domain organization in the polyketide synthases of Mycobacterium tuberculosis H37Rv and other mycobacteria. The synthases are shown grouped in the panels (a) through (l) according to biosynthetic pathways or gene clustering patterns. Each protein is labeled following the nomenclature shown in Figure 3 and noted in the text. The number of amino acids (aa) of each protein is shown in parentheses under the protein name. The numbers displayed above each domain indicate the N-terminal and C-terminal boundaries of the predicted domain in the amino acid sequence of the protein. Protein domains and their respective N-terminal and C-terminal boundaries were predicted using the Domain Analysis tool at the web interfaces of MAPSI Database [an integrated web database for management and analysis for polyketide synthases (Tae et al., 2009)] and the CD-Search tool of NCBI’s Conserved Domain Database (CDD) (Marchler-Bauer et al., 2011). The domain arrangements depicted are in accordance with earlier predictions noted in the text. Domain abbreviations: A, adenylation; ACP, acyl carrier protein; AT, acyltransferase; CHS, chalcone synthase-like; DH dehydratase; ER, enoylreductase; KR, ketoreductase; KS, ketosynthase; TE, thioesterase. Possible docking domains are not marked at the N- or Cterminus of the PKSs. In (j), the presence of an N-terminus ACP domain (*) in Pks is not predicted by sequence analysis using the domain search tools noted above, but its presence has been proposed by others (Vats et al., 2012). In (l), the DH domains presented with dotted lines are predicted to be inactive based on sequence analysis (Stinear et al., 2004). The protein pairs Pks15 and Pks1 (a) and Pks3 and Pks4 (e) are each fused into a single protein in other strains (see text).

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M. tuberculosis. The ensuing sections present a synopsis of the most relevant studies in the field and an overview of our current understanding of the involvement of PKSs and several other polyketide production pathway-associated enzymes in critical biosynthetic pathways of M. tuberculosis and other species of the Mycobacterium genus.


Biosynthesis of dimycocerosate esters Dimycocerosate esters at a glance Mycobacterium tuberculosis and several other mycobacterial pathogens produce two structurally related groups of unique


lipid diesters comprised of b-diol-containing long-chain aliphatic polyketides (e.g. phthiocerols, phenolphthiocerols and related congeners) esterified on their b-diol functionality with long-chain multimethyl-branched fatty acids (e.g. mycocerosic acids; Figure 5a). These lipids are often and herein collectively referred to as dimycocerosate esters and display species-specific structural subtleties that have been extensively reviewed elsewhere (Onwueme et al., 2005a). Dimycocerosate esters are among the most exotic free (glyco)lipids found in the extractable (glycol)lipid layer (and to a much lesser extent in the loosely attached capsule layer) of the unique mycobacterial cell envelope

Figure 5. Representative structures of mycobacterial compounds synthesized with participation of polyketide synthases. (a) PDIMs and PGLs from M. tuberculosis; (b) mycolic acid from M. tuberculosis; (c) sulfolipid-1 from M. tuberculosis; (d) mannosyl-phosphomycoketide from M. tuberculosis; (e) polyacyltrehaloses from M. tuberculosis; (f) lipooligosaccharide from M. canettii; (g) triketide (left) and tetraketide (center) pyrones synthesized in vitro by Pks18 and Pks11 and methyl-branched alkylpyrone (right) synthesized by Pks11 in vitro; (h) glycopeptidolipid from M. smegmatis; (i) mycobactin from M. tuberculosis; (j) mycolactone from M. ulcerans.



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(Daffe´, 2008) (Figure 1). The degree of conservation of their biosynthetic genes in multiple mycobacterial species has been previously highlighted (Onwueme et al., 2005a). Dimycocerosate esters have been proven to be major mycobacterial virulence factors with complex molecular mechanisms of action (Astarie-Dequeker et al., 2009; Camacho et al., 1999; Cambier et al., 2014; Cox et al., 1999; Passemar et al., 2014; Reed et al., 2004; Tsenova et al., 2005) and contributors to the permeability barrier of the cell envelope to antimicrobial drugs and others substances (Alibaud et al., 2011; Camacho et al., 2001; Chavadi et al., 2011a; Yu et al., 2012). The phthiocerol dimycocerosates (and related minor structural variants such as those derived from phthiodiolones) are herein collectively referred to as PDIMs. PDIMs are believed to be produced by all M. tuberculosis strains, with the exception of those strains that have lost PDIM production capacity due to spontaneous mutations that emerged during culturing in vitro (Andreu & Gibert, 2008; Domenech & Reed, 2009; Kirksey et al., 2011; Zheng et al., 2008). The phenolphthiocerol dimycocerosates (and related minor variants such as those derived from phenolphthiodiolones) are glycosylated lipids and often collectively referred to as phenolic glycolipids (PGLs). Unlike PDIMs, PGLs are only produced by a subset of M. tuberculosis strains [e.g. strains of the W-Beijing family (Tsolaki et al., 2005)]. In most M. tuberculosis strains lacking PGL-production capacity, the deficiency has been attributed to natural mutations in a PKS-encoding gene required for biosynthesis of phenolphthiocerols and phenolphthiodiolones (pks15/1 locus, see below) (Constant et al., 2002; He et al., 2009; Reed et al., 2004). Notably, PGL production has been suggested as a trait predisposing M. tuberculosis strains of the W-Beijing family to their characteristic epidemic spread and increased likelihood of developing drug resistance (Reed et al., 2004). The sections below outline the genetic and biochemical studies linking PKSs and other pathway-associated proteins to dimycocerosate ester production. Genetic studies linking the ppsABCDE–mas–pks15/1 chromosomal region to production of dimycocerosate esters ppsABCDE The modular type I PKS-encoding gene cluster ppsABCDE and the iterative type I PKS-encoding gene mas have been implicated in production of dimycocerosate esters (Figure 3a). The first genetic evidence connecting the ppsABCDE cluster and mas to dimycocerosate ester production emerged in the 1990’s from the ground-breaking work of Kolattukudy and coworkers with M. bovis Bacille–Calmette–Guerin (BCG), a PDIM- and PGL-producing species. Kolattukudy and coworkers postulated the involvement of the ppsABCDE cluster (then known as pps12345) in biosynthesis of (phenol)phthiocerols and proposed mas as the mycocerosic acid synthase-encoding gene (Mathur & Kolattukudy, 1992; Rainwater & Kolattukudy, 1985). The investigators demonstrated that replacement of a ppsB-ppsC segment from the ppsABCDE cluster of M. bovis BCG with a hygromycinresistance gene cassette (hyg) renders a mutant incapable of producing PDIMs and PGLs (Azad et al., 1997).

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They also showed that replacement of mas with the hyg marker produces a M. bovis BCG mutant lacking mycocerosic acids as well as PDIMs and PGLs (Azad et al., 1996). More recently, Guilhot, Chalut, Astarie-Dequeker and coworkers constructed and analyzed a ppsE deletion mutants of M. bovis BCG and M. tuberculosis (Astarie-Dequeker et al., 2009; Passemar et al., 2014; Simeone et al., 2007b). These studies revealed that the M. bovis BCG ppsE mutant lacks both PDIMs and PGLs and showed that the M. tuberculosis ppsE mutant are PDIM deficient, a deficiency complemented by the wild-type allele. The observation that M. marinum mutants in which the orthologs of ppsA, ppsB, ppsD, ppsE and mas were individually disrupted by transposon insertions are unable to produce the PDIM and PGL variants characteristic of this species [with multimethyl-branched fatty acids that are dextrorotatory enantiomers of the mycocerosic acids seen in M. tuberculosis (Onwueme et al., 2005a)] provides further support for the proposed role of the pps and mas genes in dimycocerosate ester production (Yu et al., 2012). pks15/1 The initial link between the pks15/1 locus (Figure 3a), which encodes an iterative type I PKS, and production of dimycocerosate esters was established by Constant and coworkers in 2002 (Constant et al., 2002). They noted that the genes pks1 and pks15 of PGL-deficient M. tuberculosis strains of the Euro-American lineage (e.g. strains H37Rv, Erdman, Mt103 and CDC1551) correspond to a single gene, pks15/1, in PGL-producing M. tuberculosis strains (e.g. strain 210) and other PGL-producing Mycobacterium species (i.e. M. canettii and M. bovis strains BCG and AF2122/97). Constant and coworkers proposed that the pks1 and pks15 genes arise from natural sequence polymorphisms that split the parental pks15/1 gene. They found that a 7-bp deletion or a 1-bp deletion in PGL-deficient M. tuberculosis strains relative to the PGL-producing M. tuberculosis strains or M. bovis strains, respectively, is responsible for the splitting of pks15/1. Further evidence of the polymorphic nature of the pks15/1 locus among members of the M. tuberculosis complex has been provided more recently by other investigators (Chaiprasert et al., 2006; Martinez-Gamboa et al., 2008; Pang et al., 2012; Tsolaki et al., 2005). Genetic experiments by Constant and coworkers demonstrated that introduction of the fused pks15/1 gene from M. bovis BCG into M. tuberculosis H37Rv restores PGL production and that, on the other hand, disruption of pks15/1 in M. bovis BCG leads to PGL deficiency, while retaining PDIM production (Constant et al., 2002). Additional genetic evidence establishing the requirement of pks15/1 for PGL production was provided by Reed and coworkers (Reed et al., 2004). They confirmed that the targeted disruption of pks15/1 in M. tuberculosis HN878 (a W-Beijing family strain) renders the expected PGL-deficient mutant, a defect rectified by introduction of the wild-type gene in the mutant. They also showed that the M. tuberculosis strains W4 and W10 of the W-Beijing family have the intact pks15/1 allele and produce PGLs. More recently, other investigators have confirmed the pks15/1 gene-dependent restoration of PGL production in

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M. tuberculosis H37Rv (Sinsimer et al., 2008) and shown that transposon-based disruption of pks15/1 in M. bovis (virulent isolate) leads to PGL deficiency (Hotter et al., 2005). Intriguingly, Pang et al. (2012) have shown that transposon disruption of pks1 in M. tuberculosis H37Rv renders a strain with reduced capacity for biofilm formation in an in vitro pellicle biofilm assay, a defect corrected by complementation with pks1. The involvement of pks1 in lipid biosynthesis remains to be demonstrated. Other pks genes Sirakova et al. (2003b) reported that the disruption of pks12 in M. tuberculosis H37Rv leads to dimycocerosate ester deficiency. The dimycocerosate ester deficiency observed by Sirakova and coworkers contrasts with the data of Matsunaga et al. (2004), which have clearly shown that pks12 is not required for dimycocerosate ester production, but is essential for the biosynthesis of mannosyl-b-1-phosphomycoketides instead (see below). The pks12 gene is not located in the ppsABCDE-mas-pks15/1 chromosomal region (Figure 2). The dimycocerosate ester deficiency of the pks12 mutant constructed by Sirakova and coworkers is likely due to spontaneous mutations that emerge during culturing of the strain in vitro and lead to loss of dimycocerosate ester production capacity, a recently documented common phenomenon that confounds mutant analysis (Andreu & Gibert, 2008; Domenech & Reed, 2009; Kirksey et al., 2011; Zheng et al., 2008). Dimycocerosate ester deficiency has been observed in engineered pks7, pks10 and pks11 mutants of M. tuberculosis H37Rv as well (Rousseau et al., 2003b; Sirakova et al., 2003a; Waddell et al., 2005). These genes are part of the pks10–pks7–pks8–pks17–pks9–pks11 gene cluster (Figure 3i), which is not in proximity to the ppsABCDE– mas–pks15/1 chromosomal region (Figure 2). Unidentified spontaneous mutations in legitimate dimycocerosate ester biosynthetic genes in the ppsABCDE–mas–pks15/1 chromosomal region are known to emerge at relatively high frequency during in vitro culturing, and such mutations are likely to be the underlying cause of the dimycocerosate ester deficiency of the pks7, pks10 and pks11 mutant strains. Additional experiments are needed to evaluate this possibility. Non-pks genes Several non-PKS encoding genes located adjacent or in close proximity to ppsABCDE, mas or pks15/1 have been implicated (conclusively or tentatively due to lack of genetic complementation controls) in production of PDIMs and/or PGLs by various mutational studies. At the forefront of these studies are those based on M. tuberculosis transposon insertion mutants originated from various mutant libraries of strains of the Euro-American lineage that are naturally deficient in PGL biosynthesis (strains Erdman, MT103 or H37Rv). Collectively, these studies reported the analysis of mutants with transposon insertions in fadD26 (FAAL gene) or its promoter region, fadD28 (FAAL gene), tesA (thioesterase gene), mmpL7 (RND-type transporter gene), drrA (ABC-type transporter gene), drrB (ABC-type transporter gene), drrC (ABC-type transporter gene) and lppX (membrane lipoprotein gene) (Figure 3a) (Camacho et al., 2001; Cox et al., 1999;


Sulzenbacher et al., 2006; Murry et al., 2009; Waddell et al., 2005). Overall, these studies indicated that insertions directly upstream of fadD26 reduce or eliminate PDIM production, insertions in fadD26, fadD28, tesA or drrB abrogate PDIM production, and insertions in mmpL7, drrA, drrC or lppX render mutants where PDIM localization to the cell envelope is impaired. It should be noted, however, that genetic complementation controls were only reported for transposon mutants of fadD26, fadD28, drrA, drrC and lppX. The involvement of several other non-PKS encoding genes located in the ppsABCDE-mas-pks15/1 chromosomal region of M. tuberculosis in production of PDIMs and/or PGLs has been investigated using marked or unmarked, gene-specific deletions generated by targeted allelic exchange mutagenesis approaches. The genes probed in these targeted mutational analysis studies include a FAAL gene (fadD29) (Simeone et al., 2010), a predicted oxidoreductase gene (rv2951c) (Onwueme et al., 2005b; Simeone et al., 2007b), two predicted methyltransferase genes (rv2952 and rv2959c) (Perez et al., 2004a), a predicted enoylreductase gene (rv2953) (Simeone et al., 2007a), three predicted glycosyltransferase genes (rv2957, rv2958c and rv2962c) (Perez et al., 2004b) and an acyltransferase gene (papA5) (Onwueme et al., 2004) (Figure 3a). The studies included genetic complementation controls of only the fadD29, rv2951c, rv2953, rv2958c and papA5 mutants. The effect of the fadD29, rv2951c, rv2952, rv2953, rv2957, rv2958c, rv2959c and rv2962c knockouts on dimycocerosate ester biosynthesis was analyzed in M. tuberculosis H37Rv and H37Rv transformed with pks15/1 to afford PGL production capacity. The analysis of these mutants indicated that: (i) fadD29 is required for biosynthesis of PGLs, yet dispensable for PDIM production; (ii) rv2951c is needed for the reduction of (phenol)phthiodiolones to (phenol)phthiotriols, which are the substrates of the methyltransferase encoded by rv2952; (iii) rv2953 is involved in the reduction of the double bond left in (phenol)phthiocerol chains by PpsD; (iv) rv2959c is required for O-methylation of the hydroxyl group at position 2 of the first rhamnosyl residue in PGLs; and (v) rv2962, rv2958c and rv2957 participate in the sequential addition of the three monosaccharide units in M. tuberculosis PGLs. The unmarked deletion of the acyltransferase gene papA5 noted above was constructed in the Erdman strain of M. tuberculosis, and the analysis of the papA5 mutant indicated that the gene is essential for production of PDIMs (Onwueme et al., 2004). Since M. tuberculosis Erdman is naturally deficient in PGL production, the papA5 mutant of the Erdman strain did not afford information as to the role of papA5 in production of this glycolipid. However, the papA5 ortholog from M. marinum has recently been shown to be required for production of both PDIMs and PGLs by a mutational analysis that included a complementation control (Chavadi et al., 2012). Genetic studies by Onwueme et al. (2005b) in nontuberculous mycobacteria support the role of the predicted ketoreductase encoded by rv2951c in the reduction of (phenol)phthiodiolones to (phenol)phthiotriols. Furthermore, the studies of Onwueme et al. (2005b) established that the deficiency of (phenol)phthiocerol-derived dimycocerosates documented in M. kansasii and M. ulcerans strains



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is due to natural mutations abrogating ketoreductase function, and that this deficiency can be corrected by cross-complementation with M. tuberculosis rv2951c. Dimycocerosate ester deficiency has also been documented in a M. bovis mutant with a transposon insertion in mb0101, the ortholog of M. tuberculosis rv0097 (possible oxidoreductase gene, unknown function) (Hotter et al., 2005). However, a connection of this gene (or its neighbors) with dimycocerosate ester production is not clearly predicted, and its involvement in the pathway remains to be verified by complementation analysis. Additional mutational analyses with corresponding complementation controls have been conducted in species other than M. tuberculosis. Inactivation of the ortholog of fadD22 in M. bovis BCG selectively eliminates PGL production (Ferreras et al., 2008). Disruption of the fadD26 ortholog in M. marinum or in M. bovis BCG selectively abolishes PDIM production (Simeone et al., 2010; Yu et al., 2012). Inactivation of the orthologs of fadD28 or tesA in M. marinum leads to loss of both PDIMs and PGLs (Alibaud et al., 2011; Chavadi et al., 2011a; Yu et al., 2012). Biochemical studies supporting predictions derived from analyses of the ppsABCDE–mas–pks15/1 region Mas The first insight into the enzymology of dimycocerosate ester biosynthesis emerged from the work of Rainwater and coworkers in the mid-1980’s (Rainwater & Kolattukudy, 1983, 1985). This pioneering work demonstrated the biosynthesis of mycocerosic acids in cell-free extracts of M. bovis BCG and by mycocerosic acid synthase (Mas) purified from lysates of M. bovis BCG cells. Rainwater and coworkers showed that Mas can extend n-fatty acyl primers derived from n-fatty acyl-CoA thioester substrates using methylmalonylCoA as donor of the methylmalonyl extender unit to produce mycocerosic acids (Rainwater & Kolattukudy, 1985). More recently, Trivedi and coworkers characterized the mycocerosic acid synthase activity of Mas from M. tuberculosis H37Rv generated recombinantly in Escherichia coli (Trivedi et al., 2005). To generate phosphopantetheinylated (holo) Mas, Trivedi and coworkers coexpressed Mas with Sfp, a robust phosphopantetheinyl transferase from Bacillus subtilis capable of acting on a wide range of heterologous carrier protein domain substrates (Quadri et al., 1998b). Overall, the mycocerosic acid synthase activity demonstrated for the Mas proteins from M. bovis BCG and M. tuberculosis H37Rv (99% identity) validates Mas as an iterative type I PKS and is consistent with the domain organization predicted for the protein (Mathur & Kolattukudy, 1992) (Figure 4a). PpsA, PpsB and PpsE The domain arrangement and functional role of each of the proteins of the modular type I PKS system PpsABCDE in the synthesis of the phthiocerol and phenolphthiocerol components of PDIMs and PGLs, respectively, have been predicted and progressively refined to the current model (Azad et al., 1997; Kolattukudy et al., 1997; Minnikin et al., 2002; Onwueme et al., 2005a; Trivedi et al., 2005) (Figure 4a).

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The only in vitro enzymology probing the functional predictions for components of the PpsABCDE system has been provided by the studies of Trivedi et al. (2005). These studies validated the predicted functional roles of M. tuberculosis PpsA, PpsB and PpsE in vitro using purified enzymes recombinantly produced in E. coli. Each purified enzyme was obtained in its respective holo form by coexpression with Sfp, the highly active B. subtilis phosphopantetheinyl transferase mentioned above (Quadri et al., 1998b). Trivedi and coworkers used the FAAL FadD26 (see below) to load lauric acid onto holo-PpsA, and then showed that the acylated PpsA protein can utilize malonyl-CoA and NADPH to form the expected reduced acyl-diketide product, 3-hydroxy tetradecanoic acid. Holo-PpsB was shown to capture the 3-hydroxy tetradecanoic acid product thioesterified to PpsA and to utilize malonyl-CoA and NADPH to convert the captured acyldiketide into 3,5-dihydroxy hexadecanoic acid, which after chemical hydrolysis spontaneously cyclized to form the corresponding lauroyl-triketide lactone. The capture of the 3hydroxy tetradecanoic acyl chain from PpsA is presumed to be carried out by the KS domain of PpsB (Figure 4a). Lastly, the hypothesized involvement of PpsE in the final step of (phenol)phthiocerol biosynthesis was validated by demonstrating the ability of the protein to extend the synthetic substrate Nacetyl cysteamine (NAC) thioester of 9-hydroxy decanoic acid using malonyl-CoA or methylmalonyl-CoA as extender unit donors. In this reaction set-up, 9-hydroxy decanoyl-NAC (a surrogate of the ketide chain that would be transferred from PpsD onto PpsE) was extended to the expected products; i.e. 11-hydroxy 3-keto dodecanoic acid with the malonyl-CoA cosubstrate or 11-hydroxy 2-methyl 3-keto dodecanoic acid with the methylmalonyl-CoA cosubstrate. Overall, the biochemical studies by Trivedi et al. (2005) are consistent with the prediction that PpsA is involved in extension of long-chain fatty acids to initiate the synthesis of (phenol)phthiocerols, validate the functional cooperation of PpsA and PpsB for biosynthesis of the b-diol functionality of (phenol)phthiocerols and provide support for the role of PpsE in the final acyl chain extension step of (phenol)phthiocerol biosynthesis. Interestingly, PpsE, which does not release its ketide products efficiently in vitro and appears to lack a TE domain for product release (Figure 4a) (Trivedi et al., 2005), has been shown to interact with TesA both in vivo and in vitro (Rao & Ranganathan, 2004). TesA is the type II thioesterase needed for dimycocerosate ester production and might be involved in polyketide product release (Alibaud et al., 2011; Chavadi et al., 2011a). PpsE has also been shown to interact with MmpL7 (Jain & Cox, 2005), a member of the MmpL family of RNDtype transporters required for PDIM localization to the cell envelope (Camacho et al., 2001; Cox et al., 1999). The PpsE– MmpL7 interaction study has led to the idea that PDIM (and probably PGL) biosynthesis might be coupled with lipid transport (Jain & Cox, 2005). FadD22 The work of Ferreras et al. (2008) revealed that the protein FadD22 is an unusual stand-alone, didomain initiation module comprised of a p-hydroxybenzoic acid adenylation domain and an ArCP domain. FadD22 forms the first acyl-


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S-enzyme covalent intermediate in the p-hydroxyphenylpolyketide chain assembly line leading to the formation of the phenolphthiocerol constituent of PGLs. This study, which was based on the FadD22 ortholog in M. marinum (Mmar_1761, 75% identity with M. tuberculosis FadD22), established that incorporation of the p-hydroxybenzoic acid starter unit into the PGL biosynthetic machinery does not require formation of a p-hydroxybenzoyl-CoA thioester intermediate as the donor of the p-hydroxybenzoic acid starter unit, as earlier suggested (Kolattukudy et al., 1997). Work by He et al. (2009) (described below) has shown that FadD22 presents the p-hydroxybenzoyl-S-ArCP domain primer to the iterative type I PKS Pks15/1 for formation of long-chain p-hydroxyphenylalkanoate products. Interestingly, Ferreras et al. (2008) harnessed detailed enzymological information on FadD22 to develop the first specific small-molecule inhibitor of PGL biosynthesis. The inhibitor is the p-hydroxybenzoyl-AMP analog 50 -O-[N(4-hydroxybenzoyl)sulfamoyl]-adenosine (pHB-AMS). The PGL biosynthesis inhibitory activity of pHB-AMS relies on a mechanistic principle analogous to that of the first reported inhibitor of mycobacterial siderophore biosynthesis (Ferreras et al., 2005; Quadri, 2007). The PGL biosynthesis inhibitor pHB-AMS has potent and specific activity in M. tuberculosis and several other mycobacterial pathogens. As expected, pHB-AMS does not inhibit PDIM biosynthesis (for which FadD22 is not required) or mycobacterial growth in vitro (Ferreras et al., 2008). The inhibitor work from Ferreras and coworkers established the feasibility of selectively targeting dimycocerosate ester biosynthesis and created the first chemical biology tool that could be explored for probing the biological relevance of PGLs in M. leprae and other genetic manipulation-intractable PGL producers. Pks15/1 He et al. (2009) demonstrated that Pks15/1 is a 6-domain reducing iterative type I PKS (Figure 4a) that uses malonylCoA as donor of the malonyl extender unit to extend a p-hydroxybenzoyl starter unit to produce long-chain p-hydroxyphenylalkanoate biosynthetic intermediates needed for biosynthesis of the phenolphthiocerol moiety of PGLs. Using in vitro and in vivo FadD22–Pks15/1 reconstituted systems, He and coworkers showed that Pks15/1 functionally cooperates with FadD22 (Ferreras et al., 2008), which activates p-hydroxybenzoic acid and presents the p-hydroxybenzoyl-S-ArCP domain primer to Pks15/1. The studies on Pks15/1, which were based on the Pks15/1 ortholog in M. marinum (Mmar_1762, 81% identity with M. tuberculosis Pks15/1), revealed that Pks15/1 is a PKS with a relaxed control of catalytic cycle iterations. This mechanistic property explains the origin of some of the characteristic alkyl chain length variability seen in phenolphthiocerols. Notably, one of the most prominent p-hydroxyphenylalkanoate products synthesized by the FadD22–Pks15/1 reconstituted systems corresponds to 23-(4-hydroxyphenyl)tricosanoic acid (C29H50O3). This product is predicted to arise from elongation of the p-hydroxybenzoic acid starter unit with eleven malonyl extender units, and its generation would


require a total of 68 catalytic operations by the FadD22– Pks15/1 system (He et al., 2009). Coincidently, 23-(4hydroxyphenyl)tricosanoic acid corresponds to the predicted p-hydroxyphenylalkanoate intermediate extended by the modular PpsABCDE system to form the phenolphthiocerol with the largest carbon chain reported in M. marinum (He et al., 2009). FadD26, FadD28 and FadD29 In 1997, Fitzmaurice & Kolattukudy (1997) noted the homology of the FadD28 ortholog in M. bovis BCG (99% identity with M. tuberculosis FadD28) to acyl adenylateforming enzymes, demonstrated that purified M. bovis BCG FadD28 protein catalyzes formation of CoA thioesters of fatty acids (n-C10 to n-C18), and concluded that the enzyme was an acyl-CoA synthase-like protein. More recently, Gokhale and coworkers investigated the acyl adenylate-forming activity of the FadD26, FadD28 and FadD29 proteins from M. tuberculosis H37Rv in vitro (Trivedi et al., 2004). This work revealed that these proteins are FAALs (Arora et al., 2009; Trivedi et al., 2004), and do not catalyze formation of acyl-CoA thioesters as suggested in previous studies (Camacho et al., 2001; Cole et al., 1998; Fitzmaurice & Kolattukudy, 1997, 1998). The fatty acyl adenylate-forming activity of M. tuberculosis H37Rv FadD28 has been investigated also by others (Wilson & Aldrich, 2010). Preliminary X-ray crystallographic analysis of the N-terminal domain (first 460 amino acids) of M. tuberculosis FadD28 indicates that the overall fold of the protein is similar to that of the acetyl-CoA synthetase from Salmonella enterica, the phenylalanine adenylation domain of gramicidin nonribosomal peptide synthetase, and the long-chain fatty acyl-CoA synthetase from Thermus thermophilus (Goyal et al., 2006). Inhibition of FadD28 by acylsulfamoyl (acyl-AMS) analogs has been recently reported as well (Arora et al., 2009; Grimes & Aldrich, 2011). Gokhale and coworkers have also shown that FadD26 specifically transfers the activated fatty acid to PpsA for further carbon chain extension (Trivedi et al., 2005). PapA5 In 2004, Onwueme et al. (2004) proposed that the protein PapA5 and other mycobacterial Paps belong to a new subfamily of acyltransferases. In particular, Onwueme et al. (2004) put forward the hypothesis that PapA5 of M. tuberculosis as well as PapA5 orthologs found in other mycobacteria are acyltransferases that catalyze the esterification of the b-diols in phthiocerols, phenolphthiocerols and related congeners with long-chain multimethyl-branched fatty acids to form corresponding dimycocerosate esters. Indeed, Onwueme and coworkers demonstrated the capacity of recombinant M. tuberculosis PapA5 to catalyze hydroxylester formation chemistry using surrogate alcohol and acyl-CoA thioester substrates in place of the predicted mycobacterial diol-containing aliphatic polyketide and mycocerosyl thioester substrates, respectively. More recently, other investigators have validated the ability of PapA5 to utilize diol-containing aliphatic polyketides of mycobacterial origin as acyl acceptor substrates (Chavadi et al., 2012) and to



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catalyze the direct transfer of mycocerosic acid analogs thioesterified at the ACP domain of Mas to 2-dodecanol (Trivedi et al., 2005). The structural analysis of ˚ resolution reported by M. tuberculosis PapA5 at 2.75 A Buglino et al. (2004) supports the biochemical information, including the proposed role of conserved histidine and aspartic acid active site residues critical to PapA5 acyltransferase activity (Onwueme et al., 2004). The crystal structure of PapA5 revealed a two-domain structure that shares unexpected similarity to structures of chloramphenicol acetyltransferase, dihydrolipoyl transacetylase, carnitine acetyltransferase and the condensation domain of the nonribosomal peptide synthesis enzyme VibH. Interestingly, an additional a-helix not observed in other acyltransferases or VibH blocks the putative entrance to the active site cavity of PapA5, indicating that conformational changes may be associated with PapA5 activity.

and play an essential role in the architecture and permeability barrier of the mycobacterial cell envelope (Figure 1). In M. tuberculosis and other mycobacterial pathogens, mycolic acids are also critical virulence determinants and modulators of the host–pathogen interaction and the host’s immune response. The structure, biosynthesis and physiological roles of mycolic acids have been investigated for several decades. Many excellent reviews on these subjects have been published (Asselineau et al., 2002; Barry et al., 1998; Brennan, 2003; Brennan & Nikaido, 1995; Dover et al., 2004; Kolattukudy et al., 1997; Takayama et al., 2005; Verschoor et al., 2012; Veyron-Churlet et al., 2005). The sections below focus on key genetic and enzymology studies linking the fadD32–pks13–accD4 locus to mycolic acid synthesis (Figure 2d).

LppX

pks13

The protein LppX has been shown to be a lipoprotein (Sulzenbacher et al., 2006; Tschumi et al., 2009), as predicted by sequence analysis (Lefevre et al., 2000). Interestingly, Lefevre et al. (2000) reported that M. bovis BCG LppX and M. tuberculosis H37Rv LppX (98% identity) can be found in the culture medium filtrate, at the cell surface, and in membrane and cell-wall fractions, thus suggesting that LppX can be exported to the cell envelope. Based on the structure of LppX, Sulzenbacher et al. (2006) proposed that the lipoprotein carries PDIM molecules across the mycobacterial cell envelope. The crystal structure of M. tuberculosis LppX ˚ resolution has revealed a conserved fold with a Uat 2 A shaped b-half-barrel with a large hydrophobic cavity that could accommodate one PDIM molecule (Sulzenbacher et al., 2006). LppX has a fold shared with the outer membrane lipoprotein LolB and the periplasmic molecular chaperone LolA, which are two proteins involved in the localization of lipoproteins to the outer membrane of gram-negative bacteria. Experimental evidence of the proposed function of LppX in PDIM transport is currently lacking.

Mycolic acid biosynthesis has been investigated for decades, yet the key enzyme catalyzing the condensation of the two fatty acyl components that give rise to the meromycolate chain and the shorter aliphatic a-branch of mycolic acids (Figure 4b), respectively, remained elusive until recently. An early proposal for the mechanism of this terminal condensation step put forward by Gastambide-Odier and Lederer included a Claisen-type condensation similar to that involved in the formation of long-chain, multimethyl-branched fatty acids in M. tuberculosis (Gastambide-Odier & Lederer, 1960). Building on this early proposal and the subsequent discovery that formation of mycobacterial multimethylbranched fatty acids involves iterative type I PKSs (Dubey et al., 2002; Mathur & Kolattukudy, 1992), Portevin et al. (2004) utilized in silico analyses of genomes from various members of the Corynebacterineae suborder to identify the PKS encoded by the gene pks13 in the fadD32-pks13-accD4 gene cluster as the candidate enzyme for catalyzing the terminal condensation step of mycolic acid biosynthesis. Portevin et al. (2004, 2005) conducted mutational studies to address the function of the pks13 orthologs found in Corynebacterium glutamicum and M. smegmatis. These studies revealed that deletion of pks13 in C. glutamicum renders a mutant with cell envelope alterations, reduced growth and mycolic acid deficiency, but nonetheless capable of producing the fatty acid precursors (Portevin et al., 2004). Introduction of a wild-type copy of pks13 into the C. glutamicum pks13 knockout mutant partially restored the wild-type phenotype. Gande et al. (2004) also generated a pks13 deletion mutant of C. glutamicum and reported the same mutant phenotypes. The work with M. smegmatis showed that pks13 is an essential gene in this species, and that a conditional M. smegmatis pks13 mutant with its only copy of pks13 expressed from a thermosensitive plasmid displays impaired mycolic acid biosynthesis at the nonpermissive condition (Portevin et al., 2005). Overall, the work by Portevin, Gande and their coworkers conclusively established the need of a functional copy of pks13 during the final condensation step of mycolic acid synthesis.

Biosynthesis of mycolic acids Mycolic acids at a glance Mycolic acids are a family of structurally related 2-alkyl 3-hydroxyl fatty acids uniquely found in the cell envelope of the Corynebacterineae suborder, a taxonomic group that includes the genera Mycobacterium, Corynebacterium, Nocardia and Rhodococcus (Figure 5b). Mycolic acids are primarily found covalently linked to the bacterial cell wall via an ester bond between their carboxylic acid group and a hydroxyl group of a cell wall sugar residue (e.g. pentaarabinose tetramycolates). Noncovalently bound, solvent-extractable free and esterified mycolic acids (e.g. trehalose mono/dimycolates, glucose monomycolate and glycerol monomycolate) are also common. The structural complexity and molecular weight range of mycolic acids are, for the most part, genus/species-specific, with the largest and most complex mycolic acids found in the Mycobacterium genus. Mycolic acids are major constituents of the cell wall

Genetic studies linking the fadD32–pks13–accD4 locus to mycolic acid synthesis

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fadD32 and accD4 Portevin et al. (2004, 2005) proposed that the FAAL gene fadD32 and the acyl-CoA carboxylase subunit gene accD4 of the fadD32–pks13–accD4 locus encode the enzymes responsible for the activation of the two fatty acyl substrates for the condensation step catalyzed by Pks13 during mycolic acid biosynthesis. FadD32 was predicted to catalyze the activation (via adenylation) and subsequent transfer of the meromycolate chain onto Pks13 by the mechanism of acyl activation and transfer shown for the FadD32–Pks13 pair by Trivedi et al. (2004). AccD4 was postulated to be involved in the activation of an acyl-CoA biosynthetic intermediate through a C-2 carboxylation, with consequent formation of 2-carboxyacylCoA, the expected extender unit substrate of the condensation reaction catalyzed by Pks13. Consistent with these hypotheses, mutational analysis by Portevin et al. (2005) revealed that C. glutamicum strains with an insertion/deletion within either fadD32 or accD4 are mycolic acid deficient and display the colony morphology characteristic of mutants of corynebacteria lacking mycolic acid. Gande et al. (2004) reported similar results with their C. glutamicum mutant strain, which has a deletion of accD4 (previously accD3). The fadD32 and accD4 mutations were complemented by introduction of the corresponding wild-type gene, thus conclusively establishing the requirement of both fadD32 and accD4 for mycolic acid production. Both fadD32 and accD4 are predicted to be essential genes in mycobacteria due to their requirement for mycolic acid biosynthesis. While the essentiality of each of these genes has not been formally investigated, both fadD32 and accD4 have been predicted to be essential genes based on high density transposon mutagenesis studies (Griffin et al., 2011; Sassetti et al., 2003). Enzymological studies supporting predictions derived from analyses of the fadD32–pks13–accD4 locus FadD32 Trivedi et al. (2004) validated the predicted acyl adenylateforming activity of the FadD32 protein from M. tuberculosis H37Rv using recombinant FadD32 produced in E. coli and dodecanoic acid as a model fatty acid substrate. FadD32 adenylates the fatty acid, but it is not competent for formation of the corresponding fatty acyl-CoA thioester, thus indicating that the protein is a FAAL and not a fatty acyl-CoA ligase. Strikingly, however, replacement of only two phenylalanine residues with alanines converts FadD32 into a fatty acyl-CoA ligase (Goyal et al., 2012). Leger et al. (2009) further characterized the acyl-AMP ligase activity of FadD32. They showed that the enzyme displays a Michaelis–Menten profile and prefers long-chain fatty acid substrates over medium- and short-chain ones. In line with the acyl-AMP ligase activity of FadD32, known inhibitors of fatty acyl-adenylating enzymes block the adenylation activity of the enzyme in vitro (Leger et al., 2009). FadD32 and Pks13 interplay Trivedi et al. (2004) and, independently, Leger et al. (2009) demonstrated that FadD32 can transfer the activated fatty acyl chain to recombinant Pks13. More recently, Gavalda et al.


(2009) provided further support for the FadD32-dependent transfer of dodecanoic acid onto Pks13 and established that the fatty acyl chain is specifically transferred onto the phosphopantetheinyl group of the N-terminal ACP domain of the PKS (Figure 4b), thus formally exposing the fatty acyl-ACP synthetase activity of FadD32. Moreover, they established that the dodecanoyl chain loaded by FadD32 onto the N-terminal ACP domain is subsequently transferred onto the adjacent KS domain of Pks13 (Figure 4b). Overall, the functional characterization of FadD32 indicates that the protein belongs to the FAAL family (Goyal et al., 2012; Trivedi et al., 2004). Interestingly, Hung and coworkers recently reported a 4,6-diaryl-5,7-dimethyl coumarin series that blocks mycolic acid biosynthesis by inhibiting the acylACP synthetase activity of FadD32 (Kawate et al., 2013; Stanley et al., 2013). The compounds inhibit M. tuberculosis replication both in vitro and in animal models of tuberculosis. This excellent work validated FadD32 as a novel target in the mycolic acid biosynthetic pathway for antituberculosis drug development. Pks13 Gavalda et al. (2009) investigated the self-loading of 2-carboxyacyl chains onto Pks13. As noted above, a 2-carboxyacyl chain [arising from action of the AccD4 and AccD5 carboxylases on a fatty acyl-CoA substrate (Gande et al., 2004, 2007)] is the predicted extender unit in the terminal condensation reaction catalyzed by Pks13 during mycolic acid biosynthesis (Portevin et al., 2004, 2005). The loading was investigated using various 2carboxyacyl-CoA thioesters. These in vitro studies revealed that Pks13 self-loads with a carboxyacyl chain from a carboxyacyl-CoA thioester substrate onto its AT domain and then transfers the carboxyacyl chain from the AT domain to the phosphopantetheinyl group of the adjacent C-terminal ACP domain (Figure 4b). The studies also established that the 2-carboxyl group is a critical structural determinant for substrate competency and that the loading efficiency increases with the carboxyacyl chain length. Recent biochemical and structural studies on the AT domain of Pks13 conducted by Bergeret et al. (2012) provided further insights into the loading of carboxyacyl chains onto Pks13. The biochemical studies revealed that a 52-kDa fragment containing the AT-ACP didomain fragment of the synthase retains the ability to load unusually long acyl extender units, with a preference for 2-carboxylated acyl chains. More importantly, the analysis of the high resolution crystal structures of the apo, palmitoylated and carboxypalmitoylated forms of the AT-ACP fragment of Pks13 revealed how the very unusual substrates (C24–C26 carboxyacyl-CoA thioesters) of the AT domain can be accommodated in the domain core via a dedicated hydrophobic channel (Bergeret et al., 2012). Notably, Alland and coworkers recently identified a novel class of thiophenes that inhibit fatty acyl-AMP loading onto Pks13, block mycolic acid biosynthesis, and have bactericidal activity against M. tuberculosis (Wilson et al., 2013). This excellent work validated Pks13 as a new druggable target in the mycolic acid biosynthetic pathway.


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Biosynthesis of sulfolipids


Sulfolipids at a glance Sulfolipids form a family of structurally related sulfated metabolites, and they are one of the most abundant organic solvent-extractable glycolipids thought to be part of the outer leaflet of the outer membrane of the cell envelope of M. tuberculosis (Figure 1) (Goren, 1970a,b; Goren et al., 1971, 1976a; Middlebrook et al., 1959; Schelle & Bertozzi, 2006). Sulfolipid-1 (SL-1), the most abundant and best characterized sulfolipid, consists of a sulfated trehalose acylated with four long-chain fatty acyl groups (Figure 5c). One of these groups is derived from palmitic or stearic acids (synthesized by fatty acid synthase I). The other groups are derived from unusual long chain multimethyl-branched fatty acids known as phthioceranic and hydroxyphthioceranic acids. Sulfolipids are not critical contributors to the structural integrity or the permeability barrier of the mycobacterial cell envelope, and their biological role remains obscure (Gilmore et al., 2012; Passemar et al., 2014). A variety of activities, at times inconsistent with each other, have been attributed to these metabolites based on numerous in vitro studies. Sulfolipids have been proposed to prevent phagosome– lysosome fusion (Goren et al., 1976b), increase phagosome acidification (Brodin et al., 2010), block monocyte priming (Brozna et al., 1991; Pabst et al., 1988), prime activated monocytes for an enhanced oxidative response (Zhang et al., 1988), augment IL-1 and TNF-a secretion and alter protein phosphorylation in monocytes (Brozna et al., 1991), stimulate neutrophil superoxide production and prime neutrophil response to metabolic agonists (Zhang et al., 1991), induce mitochondrial swelling, disrupt mitochondrial membranes and inhibit mitochondrial oxidative phosphorylation (Kato & Goren, 1974), induce a transcriptional response in leukocytes that includes genes distinct from those induced by wellcharacterized proinflammatory mediators (Gilmore et al., 2012), play a role in mediating bacterial susceptibility to human cationic antimicrobial peptides (Gilmore et al., 2012), and negatively regulate the intracellular growth of M. tuberculosis in a host species-specific manner (Gilmore et al., 2012). Unexpectedly, due to the plethora of biological activities proposed for sulfolipids based on in vitro experiments, recent studies with defined SL-1 deficient mutants in animal models of tuberculosis indicate that the metabolite appears to be fully dispensable for infection in mice and guinea pigs (Chesne-Seck et al., 2008; Converse et al., 2003; Gilmore et al., 2012; Kumar et al., 2007; Rousseau et al., 2003c). These results appear to contrast with early observations suggesting a correlation between the amount of sulfolipids produced and virulence (Gangadharam et al., 1963; Goren et al., 1974). Interestingly, however, mutants deficient in SL-1 but accumulating its diacyl sulfolipid biosynthetic precursor (SL1278) display some level of attenuation in mice (Converse et al., 2003; Domenech et al., 2004). Recent work by Astarie-Dequeker and coworkers indicates that the contribution of the sulfolipids to virulence is minimal and masked by PDIMs, which are dominant virulence factors that mask the functions of other cellenvelope lipid families (Passemar et al., 2014). Additional studies are needed to clarify the role of sulfolipids

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in mycobacterial biology. The ensuing sections summarize recent genetic and biochemical studies linking the Pks2 protein and other enzymes to SL-1 production. Genetic studies linking pks2 and nearby genes to sulfolipid production pks2 In 2001, Sirakova and coworkers probed the suspected involvement of the mas-like pks2 gene (Figure 3c) in production of methyl-branched fatty acids and found that the gene is involved in biosynthesis of sulfolipids in M. tuberculosis H37Rv (Figure 3c) (Sirakova et al., 2001). A phage-mediated delivery system was used alongside homologous recombination to replace an internal 1.2-kb segment of the coding region for the DH and ER domains of Pks2 with a 1.8-kb hyg gene cassette. The effect of the pks2 knockout on production of methyl-branched lipids was investigated. The mutant was shown to lack both phthioceranic and hydroxyphthioceranic acids, the major multimethylbranched fatty acids found esterified to the trehalose-2-sulfate moiety of sulfolipids. More recently, others have shown that disruption of pks2 leads to SL-1 deficiency and verified that reintroduction of the pks2 gene in the pks2 knockout mutant restores the ability of the strain to synthesize SL-1 (Converse et al., 2003; Domenech et al., 2004). The work of Astarie-Dequeker and coworkers also supports the involvements of pks2 in sulfolipid production (Passemar et al., 2014). fadD23 The fadD23 gene, located next to pks2 (Figure 3c), is predicted to encode a FAAL involved in activating and loading fatty acyl primers onto Pks2 for acyl chain extension (Trivedi et al., 2004). In line with this prediction, Lynett and Stokes (2007) found that transposon-based disruption of fadD23 leads to sulfolipid deficiency. Genetic complementation of the fadD23 mutant with the wild-type gene restored the wild-type phenotype, thus unambiguously establishing the requirement of fadD23 for sulfolipid biosynthesis. papA1, papA2 and chp1 Guided by the PapA5 precedent (Buglino et al., 2004; Chavadi et al., 2012; Onwueme et al., 2004; Trivedi et al., 2005), Bhatt et al. (2007) and Kumar et al. (2007) hypothesized the involvement of genes papA1 and papA2 (encoding Paps, located in proximity to pks2 Figure 3c) in acylation of the trehalose-2-sulfate core of sulfolipids. More recently, Seeliger et al. (2012) hypothesized, on the basis of sequence homology analysis, that the gene chp1 (rv3822) of the sulfolipid biosynthetic gene locus encodes a third acyltransferase involved in the acylation of the trehalose moiety of sulfolipids (Figure 3c). Sequence analysis of Chp1 indicates that the protein is unrelated to PapA1 or PapA2 and has a predicted N-terminal transmembrane domain followed by C-terminal a/b-hydrolase domain with a putative Ser–Asp–His catalytic triad. Mutational analysis probing the involvement of papA1, papA2 and chp1 in sulfolipid


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production supports the predicted functions of these genes. Disruption of papA1, papA2 or chp1 with a hyg gene cassette introduced by allelic exchange rendered mutants of M. tuberculosis (strains H37Rv or Erdman) lacking SL-1 (Bhatt et al., 2007; Kumar et al., 2007). Genetic complementation controls with wild-type papA1, papA2 or chp1 alleles indicated that the SL-1 deficiency is indeed due to the lack of the deleted genes. Accumulation of partially acylated biosynthetic intermediates was not detected in the studies with the papA1 and papA2 mutants of strain H37Rv reported by Bhatt et al. (2007). However, more in-depth analysis allowed Kumar et al. (2007) to detect partially acylated intermediates in the papA1 and papA2 mutants of strain Erdman. The intermediate accumulation data have led to the proposal that trehalose-2-sulfate is first 20 -acylated with a palmitoyl (or stearoyl) group by PapA2, and then the monoacylated intermediate is 30 -acylated with a (hydroxy)phthioceranoyl group by PapA1 to yield the diacylated intermediate known as SL1278 (Kumar et al., 2007). Analysis of partially acylated intermediates also revealed that SL1278 accumulates in the chp1 mutant (strain Erdman), where the intermediate is found in the surface lipid fraction as well as in the cell pellet fraction (Seeliger et al., 2012). mmpL8 and sap The mmpL8 gene, which is located in the sulfolipid biosynthetic gene locus (Figure 3c), encodes a predicted RND-type membrane transporter. The gene was proposed to be involved in sulfolipid transport due to its location and the demonstrated involvement of its homolog mmpL7 in transport of dimycocerosate esters (Converse et al., 2003; Domenech et al., 2004). In line with this hypothesis, Converse et al. (2003), Domenech et al. (2004), as well as Layre et al. (2011) established that disruption of mmpL8 renders a mutant of M. tuberculosis (Erdman or H37Rv strains) unable to synthesize SL-1. Complementation experiments revealed that introduction of the wild-type gene in the mmpL8 knockout mutant strain restores SL-1 production, thus establishing that the SL-1 deficiency is due to lack of a functional mmpL8. Interestingly, disruption of mmpL8 leads to intracellular accumulation of diacylated (SL1278 or SL-N) and triacylated trehalose-2-sulfate intermediates, some of which are present in minor quantities in wild-type M. tuberculosis (Converse et al., 2003; Domenech et al., 2004; Layre et al., 2011; Seeliger et al., 2012). More recently, Seeliger et al. (2012) implicated a second gene in sulfolipid transport. This gene is known as sap (rv3821), and it is located in the sulfolipid biosynthetic gene locus (Figure 3c). The sap gene encodes an integral protein with sequence homology to the protein Gap of M. smegmatis, which is required for transport of glycopeptidolipids to the cell surface. Seeliger et al. (2012) proposed and probed the involvement of sap in transmembrane transport of SL-1. In line with their proposal, disruption of sap in M. tuberculosis (strain Erdman) leads to intracellular accumulation of SL1278 and reduction of SL-1 production, a mutant phenotype that is corrected by introduction of wild-type sap in the sap null strain.


stf0 Mougous et al. (2004) identified the gene stf0 (rv0295c) of M. tuberculosis as a potential sulfotransferase and proposed and probed the involvement of stf0 in synthesis of trehalose2-sulfate and SL-1. The gene is not located in proximity to genes known to be involved in sulfolipid production (Figure 3c), yet their mutational analysis revealed that inactivation of stf0 leads to a M. tuberculosis mutant strain deficient in both trehalose-2-sulfate production and SL-1 production. Genetic complementation analysis confirmed the involvement of stf0 in production of trehalose-2-sulfate and SL-1. Interestingly, the stf0 knockout mutant was lacking unsulfated SL-1 intermediates as well. This finding has led to the suggestion that Stf0-dependent trehalose sulfation is the first step in SL-1 biosynthesis and is required for trehalose acylation. The stf0 ortholog in M. smegmatis has also been shown to be essential for trehalose-2-sulfate biosynthesis (Mougous et al., 2004). Enzymological studies supporting predictions derived from analyses of the sulfolipid biosynthetic gene locus PapA1 and PapA2 Kumar et al. (2007) investigated the catalytic activity of PapA1 and PapA2 from M. tuberculosis H37Rv. In vitro, recombinant PapA2 can synthesize the trehalose-2-sulfate-20 palmitate monoacylated intermediate from trehalose-2-sulfate and palmitoyl-CoA. The enzyme does not utilize unsulfated trehalose as substrate, a finding indicating that the sulfate is essential for PapA2-dependent acylation of the sugar. The monoacylated intermediate (referred to as SL659 based on its observed mass) cannot be further acylated by PapA2. However, SL659 can be further acylated by recombinant PapA1, which is able to utilize palmitoyl-CoA as the fatty acyl chain donor. PapA1 does not acylate trehalose-2-sulfate or unsulfated trehalose-20 -palmitate. These observations led Kumar and coworkers to propose that both the palmitoyl group and the sulfate group of SL659 are essential for PapA1dependent acylation of the sugar moiety. Characterization of the dipalmitoylated trehalose-2-sulfate product of PapA1 indicated that both palmitoyl groups are on the same glucose residue (Kumar et al., 2007). While the specific position of acylation remains unknown, acylation on the 30 -position is likely, as it would be consistent with the structure of SL1278, which is 30 -acylated with a phthioceranoyl group (Domenech et al., 2004). Notably, the Km of PapA1 for palmitoyl-CoA is much higher than that observed with PapA2, suggesting that palmitoyl-CoA is not the physiological substrate of PapA1. This is in line with the idea that the physiological acyl substrate of PapA1 is the (hydroxy)phthioceranoyl product of Pks2 thioesterified to the ACP domain of the synthase (Kumar et al., 2007). Chp1 Seeliger et al. (2012) recently reported studies on the membrane topology and the catalytic activity of Chp1. The investigation of the subcellular localization of Chp1 expressed in M. smegmatis by fractionation and immunoblotting methods revealed that the protein is anchored to the cytosolic


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membrane. This observation is consistent with the predicted transmembrane helix at the N-terminus of Chp1. Analysis of the topology of the protein by assessment of the enzymatic activity of Chp1-alkaline phosphatase and Chp1-b-galactosidase fusions expressed in M. smegmatis suggests that the protein is membrane-anchored in a way that the predicted C-terminal catalytic domain is in the cytosol. In vitro studies with a recombinantly produced Chp1 catalytic domain fragment and a fluorescent fluorophosphonate activity-based probe specific for serine hydrolases have validated the relevance of the putative catalytic Ser-156 residue of the predicted Ser–Asp–His catalytic triad of the protein (Seeliger et al., 2012). Notably, probing the activity of the catalytic domain fragment of Chp1 using the synthetic diacyl sulfolipid trehalose-2-sulfate-20 -palmitate-30 -stearate (T2S-PS) as a SL1278 analog has revealed that the enzyme can utilize this analog as both an acyl donor substrate and an acyl acceptor substrate. In vitro, Chp1 is capable of catalyzing acyl transfers of one and two stearoyl chains to T2S-PS from another molecule of T2S-PS to form trehalose-2-sulfate-20 -palmitate-30 -stearate-60 -stearate (T2S-PS2) and trehalose-2-sulfate-20 -palmitate-30 -stearate-60 stearate-6-stearate (T2S-PS3). Interestingly, however, the enzyme is unable to use the acyl-CoA as the acyl donor. Overall, the in vitro enzymology indicated that Chp1 can catalyze regiospecific transfer of a fatty acyl group from the 30 -position of the acyl donor molecule to the 6-position or 60 -position of the acyl acceptor molecule. Overall, the data are consistent with the current working model recently proposed by Seeliger et al. (2012). Stf0 Mougous et al. (2004) demonstrated that stf0, which encodes a predicted sulfotransferase, is required for biosynthesis of trehalose-2-sulfate and SL-1 in M. tuberculosis. The predicted ability of the Stf0 protein to sulfate trehalose was investigated in vitro with recombinant Stf0 protein and using the sulfate donor 30 -phosphoadenosine 50 -phosphosulfate (Mougous et al., 2004). These in vitro studies confirmed that Stf0 catalyzes formation of trehalose-2-sulfate. The activity of the enzyme with trehalose analogs and modified trehalose substrates is drastically reduced, a finding indicating that the enzyme requires unmodified trehalose as substrate and it is sensitive to small structural alterations of the disaccharide. Additional insights into Stf0 come from the analysis of its crystal structure (Mougous et al., 2004). The structure of Stf0 (M. smegmatis ortholog) in complex ˚ resolution) has revealed that the Stf0 with trehalose (2.6 A monomer belongs to the sulfotransferase structure superfamily. The structural analysis also illuminated the molecular basis of trehalose specificity and binding. It showed a unique dimer configuration that binds trehalose into a bipartite active site cavity. Interestingly, a second M. tuberculosis sulfotransferase (the product of rv1373) has been recombinantly produced, purified, and shown to sulfate mycobacterial glycolipids in vitro to render corresponding sulfolipids (Rivera-Marrero et al., 2002). However, the participation of this sulfotransferase in sulfolipid biosynthesis in vivo remains to be demonstrated.


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Biosynthesis of mannosyl-b-1-phosphomycoketides Mannosyl-b-1-phosphomycoketides at a glance This family of unusual glycolipids was initially identified as a new class of lipid antigens from M. tuberculosis and M. avium presented by the CD1 system during infection (de Jong et al., 2007; Ly et al., 2013; Moody et al., 2000). Mannosyl-b-1phosphomycoketides (MPMs) represent a minor component of the cell envelope (possibly in the extractable lipid/ glycolipid layer, Figure 1) of M. tuberculosis and other slow growing pathogenic mycobacteria (e.g. M. avium, M. bovis) (Matsunaga et al., 2004). They consist of a mannosyl-b-1phosphate head group and an atypical saturated 4, 8, 12, 16, 20-pentamethylpentacosyl lipid tail (mycoketide) of variable length (Matsunaga et al., 2004) (Figure 5d). The multimethylbranched portion of the mycoketide moiety of MPMs is a key molecular determinant for the CD1 system. The recently reported crystal structure of CD1c in complex with a synthetic MPM has revealed that the unusual multimethylbranched alkyl chain of the mycoketide binds into the A0 pocket of the CD1c groove (Scharf et al., 2010). Additional studies have demonstrated that CD1c tetramers detect ex vivo T cell responses to processed phosphomycoketide antigens, a finding that further illustrates the biological significance of these mycobacterial metabolites (Ly et al., 2013). The physiological role of MPMs remains incompletely understood. Studies with pks12 deficient mutants, which lack the ability to produce mycoketides (see below), suggest that the mycoketides and/or the MPMs are virulence factors and, to some extent, affect the cell wall permeability barrier. The first study on this front was conducted by Sirakova et al. (2003b), and it showed that inactivation of pks12 by allelic exchange with the hyg gene marker produced a M. tuberculosis mutant attenuated in a murine model. Unfortunately, lack of complementation control and the unexpected loss in this pks12 null mutant of the capacity to produce dimycocerosate esters [well-known virulence factors (Onwueme et al., 2005a)] confound the results of this study. Similarly, a transposon based-disruption of the pks12 ortholog in M. marinum has shown to lead to attenuation in the goldfish (Carassius auratus) infection model, but the link between the lack of pks12 and the attenuation is not conclusive due to the lack of a genetic complementation control in the study (Ruley et al., 2004). More recently, however, the relevance of pks12 in virulence has been conclusively established in M. avium. Transposon-based disruption of the pks12 ortholog in M. avium 104 strain produced a mutant attenuated in a murine model, a defect corrected by complementation with the wild-type gene (Li et al., 2010). Additional studies have shown that mutants of the clinical isolate M. avium subsp. avium strain HMC02 (white opaque variant) with transposon-based disruptions of their corresponding pks12 ortholog have altered colony morphology and increased susceptibility to clarithromycin, ciprofloxacin and penicillin, yet a phenotype indistinguishable from wild-type for growth in vitro and in the human THP-1 cell line (Philalay et al., 2004). Interestingly, examination of the drug susceptibility of the M. tuberculosis pks12 null mutant noted above (Sirakova et al., 2003b) revealed that the mutant strain is more


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susceptible to clarithromycin than the parental strain, but it retains wild-type susceptibility to ciprofloxacin and penicillin (Philalay et al., 2004). Unfortunately, genetic complementation controls have not been reported in these studies. Studies with clinical isolates of M. intracellulare have revealed a positive correlation between expression of its Pks12 ortholog, production of MPMs, barrier to ethidium bromide accumulation, and intrinsic drug resistance (Matsunaga et al., 2012). Thus, the overall picture suggests that MPMs might contribute to strengthening the permeability barrier of the mycobacterial cell envelope. The recent genetic and biochemical studies linking Pks12 to production of MPMs are reviewed below. Genetic studies linking pks12 to mannosyl-b-1-phosphomycoketide synthesis The first mutational analysis conclusively linking pks12 (Figure 2) and production of MPMs was provided by Matsunaga et al. (2004, 2012). They proposed a model involving Pks12 in the biosynthesis of mycoketides and demonstrated that mutants of M. tuberculosis (H37Rv or CDC1551 strains) and M. bovis (strain BCG) in which pks12 was disrupted with a hyg resistance marker cassette introduced by allelic exchange lacked mycoketides and MPMs. Complementation experiments verified that the mutant phenotype was due to the lack of a functional copy of pks12. Interestingly, pks12 appears to form an operon with rv2047c and rv2049c, which are two non-essential genes encoding conserved hypothetical proteins of unknown function (Figure 3d). Sirakova et al. (2003b) had reported earlier that the disruption of pks12 in M. tuberculosis H37Rv leads to dimycocerosate ester deficiency. The study by Sirakova and coworkers did not provide a complementation control experiment, nor did it investigate the effect of the mutation in MPMs or mycoketides, which were not known at the time. The dimycocerosate ester deficiency observed by Sirakova and coworkers contrasts with the data of Matsunaga et al. (2004), which clearly demonstrate that pks12 is not required for production of dimycocerosate esters. The dimycocerosate ester deficiency of the M. tuberculosis H37Rv mutant constructed by Sirakova and coworkers is likely due to spontaneous mutation(s) that emerged during the culturing of the strain in vitro (Andreu & Gibert, 2008; Domenech & Reed, 2009; Kirksey et al., 2011; Zheng et al., 2008). Enzymological studies supporting the proposed role of Pks12 in mycoketide synthesis Matsunaga et al. (2004) proposed that Pks12, the largest protein encoded in the genome of M. tuberculosis, would assemble mycoketides by extending a fatty acyl starter unit via repetitive condensations of alternating malonate and methylmalonate extender units. Pks12 has two modules, each containing a set of fatty acid synthase-like domains that would confer to the module the capacity to add a 2-carbon ketide unit and reduce it to a saturated alkyl chain (Figure 4d). Malonate and methylmalonate specificities have been predicted for the AT domains of the N- and C-terminal modules, respectively, of Pks12. Recently, Chopra et al. (2008) have confirmed that Pks12 has the capacity to synthesize


mycoketides by reconstituting the mycoketide synthase activity in vitro using recombinant Pks12. In this mycoketide synthase reconstitution study, the phosphopantetheinylated form of Pks12 was generated by coexpression of the protein with the B. subtilis phosphopantetheinyl transferase Sfp (Quadri et al., 1998b). Importantly, the work of Chopra and coworkers afforded an exciting mechanistic insight into the biosynthetic strategy utilized by Pks12. They provided compelling evidence derived from a combination of biochemical, computational, mutational, analytical ultracentrifugation and atomic force microscopy studies supporting the idea that Pks12 monomers are assembled into a supramolecular complex through specific interactions between the C- and N-terminus linker domains to generate a unique modular array that catalyzes repetitive extender unit condensations in an iterative fashion (Chopra et al., 2008). The data have led Chopra and coworkers to propose that Pks12 perform repetitive cycles of iterations via a novel ‘‘modularly iterative’’ mechanism of polyketide biosynthesis.

Biosynthesis of polyacyltrehalose Polyacyltrehalose at a glance Polyacyltrehalose (PAT) is a penta-acylated trehalose ester uniquely found as a component of the outer leaflet of the outer membrane of the cell envelope of virulent M. tuberculosis strains. PAT has a trehalose head esterified with four fatty acyl chains typically derived from mycolipenic (phthienoic) or mycolipanoic acids (C27) at the 3, 6, 20 and 60 positions and one fully saturated acyl chain derived from stearic or palmitic acids at the 2 position (Figure 5e) (Jackson et al., 2007; Minnikin et al., 2002). The biological function of PAT is poorly understood. PAT is an immunoreactive glycolipid, and recent studies have indicated that a mutation leading to lack of PAT and a related acylated trehalose compound (i.e. DAT) produces a mutant of M. tuberculosis H37Rv with hyperaggregation properties in liquid culture, altered cell envelope properties, and improved ability to bind and enter phagocytic and non-phagocytic host cells (Dubey et al., 2002; Rousseau et al., 2003a). Interestingly, PAT-deficient mutant strains have wild-type like levels of replication and persistence in the lung, spleen and liver of mice infected via the respiratory (aerosol or nasal exposure) or intravenous routes, thus indicating that PAT is not critical for virulence (Chesne-Seck et al., 2008; Passemar et al., 2014; Rousseau et al., 2003a). Interestingly, however, a minor contribution to virulence of PATs/DATs has been recently established by investigating a PAT/DAT-deficient mutant generated in a strain deficient for PDIMs (Passemar et al., 2014). The following text describes genetic and biochemical studies linking pks3/4 and several genes in close proximity to this gene locus to PAT biosynthesis. Genetic studies linking the pks3/4 locus and nearby genes to polyacyltrehalose synthesis pks3/4 Dubey et al. (2002) established the link between the pks3/4 locus (Figure 3e) and PAT biosynthesis. They noted that the originally annotated genes pks3 and pks4 of M. tuberculosis


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H37Rv Pasteur strain appeared to correspond to a single gene in M. bovis BCG, and that a single base change eliminating the stop codon of pks3 would fuse the gene to pks4. As per the annotation in M. tuberculosis H37Rv Pasteur, pks3 would encode a protein comprised of a KS domain. In contrast, pks4 would encode a multidomain-containing protein with an N- to C-terminus domain arrangement consisting of AT, DH, ER, KR and ACP domains. A pks3 + pks4 fusion will generate a single gene encoding a protein with a multidomain set equivalent to that found in Mas (Figure 4e), the iterative type I PKS involved in biosynthesis of the multimethyl-branched mycocerosic acids needed for biosynthesis of dimycocerosate esters (Figure 5a) (Onwueme et al., 2005a). These observations prompted Dubey et al. (2002) to resequence the pks3pks4 region of M. tuberculosis. Their sequencing of the M. tuberculosis H37Rv ATCC #25618 strain revealed that the annotated stop codon of pks3 in H37Rv Pasteur was indeed a Tyr codon in H37Rv ATCC #25618, thus indicating that pks3 and pks4 are a single gene (named msl3, but most often and herein referred to as pks3/4). More recently, the nucleotide sequence of pks3/4 has been confirmed in the Erdman strain of M. tuberculosis (Hatzios et al., 2009). The similarity between pks3/4 and mas led Dubey et al. (2002) to postulate and probe the involvement of pks3/4 in the biosynthesis of multimethyl-branched fatty acids in M. tuberculosis. They constructed and characterized a M. tuberculosis H37Rv ATCC #25618 mutant in which pks3/4 was disrupted by a hyg cassette introduced by allelic exchange. The analysis of the lipids and glycolipids produced by the M. tuberculosis mutant strain revealed that pks3/4 inactivation abolished production of mycolipanoic, mycolipenic and mycolipodienoic acids, which are the three trimethyl-branched fatty acids in the bacterium. Consistent with this finding, the mutation also produced PAT deficiency. The pks3/4 knockout, however, did not affect production of other methyl-branched fatty acids such as mycocerosic, phthioceranic, mycolipanolic and hydroxyphthioceranic acids. A more recent mutational analysis providing an appropriate complementation control has conclusively established the involvement of pks3/4 in PAT and DAT production (Passemar et al., 2014). papA3 Onwueme et al. (2004) proposed that papA3, a gene located immediately downstream of pks3/4 (Figure 3e), encodes an acyltransferase of the Pap family involved in polyketide biosynthesis. More recently, guided by the proximity of papA3 to pks3/4 and the resemblance of the PAT biosynthetic gene locus to the SL-1 biosynthetic gene locus, Hatzios et al. (2009) postulated and investigated the involvement of papA3 in PAT synthesis. They constructed a papA3 deletion mutant in the Erdman strain of M. tuberculosis using a specialized transduction phage methodology to achieve an allelic exchange that replaced papA3 with a hyg marker cassette. The lipid analysis of the papA3 deletion mutant clearly demonstrated that deletion of papA3 leads to loss of PAT. Production of SL-1, another acylated trehalose-based glycolipid, was not affected in the mutant strain. The defect was corrected by introduction of a plasmid expressing papA3 into


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the mutant strain, and therefore the requirement of papA3 for PAT production has been conclusively established. chp2, fadD21 and mmpL10 These genes are clustered with pks3/4 (Figure 3e) and they are likely to be involved in the production of PAT. The gene chp2 (rv1184c) is a homolog of chp1, which encodes a membraneanchored acyltransferase involved in acylation of trehalose-2sulfate during SL-1 biosynthesis (see above). It is likely that chp2 is involved in at least one of the acylation steps needed to form PAT. The gene fadD21 is predicted to encode a FAAL likely to be involved in activating and loading fatty acyl primers onto Pks3/4 for acyl chain extension (Trivedi et al., 2004). The mmpL10 gene [encoding a predicted RND-type membrane transporter (Domenech et al., 2005)] has been suggested, by analogy to the involvement of mmpL8 in the SL-1 pathway, to be involved in transport of PAT or its precursor to the cell surface (Hatzios et al., 2009). The involvement of chp2, fadD21 and mmpL10 in PAT production remains to be investigated. Enzymological studies supporting the role of PapA3 in polyacyltrehalose production PapA3 is the only protein of those encoded by the genes noted above from the PAT biosynthetic gene locus that has been probed for enzymatic activity in vitro. Hatzios et al. (2009) have proposed that PapA3 is an acyltransferase that catalyzes trehalose acylation. This idea, which was primarily born from analogy to the documented functions of PapA5 and PapA1, was interrogated experimentally with recombinant PapA3 produced in E. coli (Hatzios et al., 2009). The recombinant protein was shown to have acyltransferase activity using palmitoyl-CoA or docosanoyl-CoA as an acyl donor and trehalose or trehalose 2-palmitate as an acyl acceptor. However, PapA3 failed to utilize butyryl-CoA or crotonylCoA as acyl donors and trehalose 3-palmitate, trehalose 2-sulfate, b,a-trehalose or maltose as acyl acceptors. Detailed characterization of the in vitro activity of PapA3 has revealed that the protein can acylate first the 2-position and then the 3-position of a single glucose residue of trehalose to form 2,3-dipalmitoylated trehalose. Based on these studies, Hatzios et al. (2009) have suggested that PapA3 might be responsible for the addition of the palmitoyl acyl chain and the mycolipenoyl acyl chain at the 2-position and 3-position, respectively, in the trehalose nucleolus of PAT.

Biosynthesis of lipooligosaccharides Lipooligosaccharides at a glance Lipooligosaccharides (LOSs) are among the non-covalently bound, extractable glycolipids believed to be associated with the mycolic acid layer of the mycobacterial cell envelope (Figure 5f) (Ortalo-Magne et al., 1996). LOSs have been found in several Mycobacterium species, including M. canettii (a member of the M. tuberculosis complex) (Daffe´ et al., 1991), opportunistic mycobacterial pathogens such as M. marinum and M. kansasii (Burguiere et al., 2005; Gilleron et al., 1993; Hunter et al., 1983; Rombouts et al., 2011), and the saprophytic M. smegmatis (Kamisango et al.,


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1985; Saadat & Ballou, 1983). LOSs are a structurally diverse group of trehalose-based glycolipids with a primary core corresponding to a a,a0 -trehalose nucleolus that is acylated and glycosylated in a species-/strain-specific manner. The trehalose nucleolus is typically acylated with one to four methyl-branched fatty acids and linear fatty acids. The glycosylation pattern is mostly based on D-glucose, but it might include L-rhamnose, L-xylose or other sugar residues. Decoration of sugar residues by O-methylation is also observed. There is limited information on the precise structures of LOSs produced by different mycobacteria. The best characterized structures correspond to LOSs of M. marinum (Burguiere et al., 2005; Rombouts et al., 2009, 2010, 2011) and M. kansasii (Hunter et al., 1983, 1984). LOSs are highly antigenic molecules and their biological properties are poorly understood. Clear alterations in colony morphology are observed in M. kansasii and M. marinum upon loss or reduction of LOS biosynthesis capacity (Belisle & Brennan, 1989; Burguiere et al., 2005; Ren et al., 2007; Sarkar et al., 2011; van der Woude et al., 2012). However, LOSs do not appear to be as critical to colony morphology in species of the M. tuberculosis complex (Lemassu et al., 1992) or M. gastri (Gilleron et al., 1993), where the rough versus smooth colony morphology has no clear correlation with LOS production. LOSs have also been implicated in the formation of biofilms and sliding motility in M. marinum and M. kansasii (Ren et al., 2007), a fact consistent with the idea that LOSs help define the surface properties of the bacterium. However, LOSs do not appear to play a significant role in biofilm formation or sliding motility in M. smegmatis (Etienne et al., 2009). Overall, the data support the idea that the biological role of these glycolipids may be species specific. Importantly, LOSs have been connected to host– pathogen interaction and virulence. Early studies with M. kansasii led to the idea that M. kansasii LOSs function as ‘‘antivirulence factors’’, by, somehow, masking other cell envelope-associated components that contribute to virulence. These early studies showed that rough variants of M. kansasii lacking LOSs produce chronic systemic infections in mice, a property contrasting the rapid clearance of the LOS-producing smooth variant of M. kansasii from the tissues of infected mice (Belisle & Brennan, 1989; Collins & Cunningham, 1981). More recent studies with M. marinum have revealed that LOSs could play a role in infection of host macrophages and have immunomodulatory properties. M. marinum mutants with defects in LOS biosynthesis are taken up by macrophages less efficiently than the wild-type strain (Burguiere et al., 2005; Ren et al., 2007). M. marinum LOSs inhibit TNF-a secretion in LOS-stimulated human macrophages, suggesting that LOSs could interfere with the pro-inflammatory response of the host immune system (Rombouts et al., 2009). More importantly, recent studies indicate that a transposon-insertion mutant of M. marinum that is deficient for LOS-IV production and accumulates LOS-III (a LOS-IV precursor) displays a significant increase in virulence in the zebrafish (Danio rerio) embryo infection model relative to the parental wild-type strain (van der Woude et al., 2012). This observation is consistent with early studies suggesting that LOSs attenuate M. kansasii virulence in a mouse model (Belisle &


Brennan, 1989; Collins & Cunningham, 1981). Notably, these findings contrast with a study showing that a M. marinum strain isolated from Chaetodon fasciatus (butterflyfish) and lacking LOS-IV production [isolate Mma7 (Rombouts et al., 2009)] is attenuated in the zebrafish model (van der Sar et al., 2004). It should be noted, however, that the Mma7 isolate utilized in this study is not fully characterized and its attenuation may be due to mutations outside the LOS biosynthetic gene locus. Finally, additional studies have shown that defects in LOS biosynthesis lead to an alteration in the release of cell surface proteins in M. marinum (van der Woude et al., 2012), suggesting that LOS deficiency might lead to a pleiotropic effect. Additional studies are needed to clearly and unambiguously define the role of LOSs in mycobacterial biology. The genetic analyses of the lipooligosaccharide production gene locus are discussed in the following section. Genetic studies linking the pks5 locus and nearby genes to lipooligosaccharide synthesis pks5 The gene pks5 and its orthologs are predicted to encode a Mas-like PKS (Figure 4f). Etienne et al. (2009) investigated the relevance of the ortholog of M. tuberculosis pks5 found in M. smegmatis (msmeg_4727) in the production of LOSs and multimethyl-branched fatty acids. The investigators hypothesized that msmeg_4727 would be involved in production of LOSs in M. smegmatis. By analogy with Mas, the product of pks5 or its orthologs have been predicted to be involved in synthesis of multimethyl-branched fatty acids required for LOS biosynthesis. Etienne and coworkers also suggested that the cluster of fourteen genes (msmeg_4727 through msmeg_4741) encompassing msmeg_4727 is involved in LOS production in M. smegmatis. This chromosomal region has comparable counterparts in M. marinum, M. canettii and even in the naturally LOS-deficient M. tuberculosis H37Rv (Figure 3f), in which genetic rearrangements have been proposed to be responsible for the lack of LOSs (Rombouts et al., 2011). To probe the involvement of msmeg_4727 (pks5) in LOS biosynthesis, Etienne et al. (2009) used an allelic exchange approach to engineer a null mutant of a LOS-producing M. smegmatis strain. The mutant had a large segment of the gene replaced by a hyg resistance marker cassette. Lipid analysis indicated that the mutant was specifically missing LOSs and 2,4-dimethyl-2-eicosenoic acid, a constituent of the LOSs of M. smegmatis. Complementation of the mutant with wild-type msmeg_4727 restored the wild-type phenotype, thus clearly establishing the role of msmeg_4727 in LOS biosynthesis. More recently, and in line with the results generated with M. smegmatis, van der Woude et al. (2012) reported that the disruption of the ortholog of M. tuberculosis pks5 found in M. marinum (mmar_2340) leads to complete deficiency in LOS-I, LOS-II, LOS-III and LOS-IV production. Intriguingly, the pks5 null mutant of the naturally LOSdeficient strain H37Rv of M. tuberculosis has been reported to have no appreciable lipid/glycolipid deficiency compared with wild-type H37Rv (Passemar et al., 2014; Rousseau et al., 2003b), and yet it displays a severe growth defect in mice


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(Rousseau et al., 2003b). The role of pks5 in M. tuberculosis H37Rv remains unknown.


losA Aside from the pks5 (msmeg_4727) study described above, the genetic studies of LOS biosynthetic genes have been conducted exclusively with M. marinum, in which a 30-kb chromosomal region is probably involved in production of LOSs (Burguiere et al., 2005; Ren et al., 2007). In fact, using M. marinum, Burguiere et al. (2005) provided the first genetic analysis leading to the identification of both a gene required for LOS biosynthesis and a gene cluster likely to be involved in LOS biosynthesis. This genetic analysis established that the gene losA (mmar_2313), encoding a putative glycosyltransferase (M. tuberculosis rv1500 ortholog), is required for the final assembly of M. marinum LOS-IV (Burguiere et al., 2005). The studies by Burguiere and coworkers demonstrated that M. marinum MRS2521, a mutant with a transposon insertion disrupting losA (Alexander et al., 2004), lacks LOS-IV production and accumulates LOS-III. This defect is corrected by complementation with a plasmid expressing losA. On the basis of their mutational analysis and detailed characterization of the LOSs from wild-type and mutant strains, Burguiere and coworkers proposed that LosA is a glycosyltransferase involved in transferring sugars to LOS-III to form LOS-IV. A recent report by van der Woude et al. (2012) supports this view. udgL and ilvB1_3 Ren et al. (2007) reported mutant analysis studies linking two additional genes to LOS production in M. marinum. These genes are both in the 30-kb gene locus containing losA and other genes with probable involvement in LOS biosynthesis (Burguiere et al., 2005; Ren et al., 2007). These studies characterized the M. marinum mutants MRS1271 and MRS1178, which were originally isolated by Alexander et al. (2004). The MRS1271 mutant was shown to have a transposon insertion-base disruption of udgL (mmar_2309), a gene encoding a predicted member of the UDP-glucose (UDP-Glc)/GDP-mannose dehydrogenase family. The MRS1178 mutant had the insertion in ilvB1_3 (mmar_2332), predicted to encode a decarboxylase. Both mutants were shown to have a defect in LOS biosynthesis. A recent independent study by van der Woude et al. (2012) also supports the proposed involvement of ilvB1_3 in LOS biosynthesis. Detailed mutant analysis has revealed that MRS1271 lacks LOS-II, LOS-III and LOS-IV, but it accumulates LOS-I. On the other hand, MRS1178 is deficient for biosynthesis of LOS-II, LOS-III and LOS-IV, but it accumulates an intermediate (LOS-II*) between LOS-I and LOS-II. Lipid and biochemical analysis of MRS1271 suggest that the mutant is defective in synthesis of D-xylose and that udgL encodes a UDP-glucose dehydrogenase required for D-xylose biosynthesis. MRS1178 produces only the intermediate LOS-II*, which contains D-xylose but lacks a sugar residue known today to be caryophyllose (Rombouts et al., 2009). Thus, MRS1178 appears to be defective in the synthesis or transfer of caryophyllose. Complementation of each of these mutant

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strains with the corresponding wild-type gene has been shown to reasonably restore the wild-type phenotype. Based on the lack of higher-molecular weight LOSs (i.e. LOS-III and LOS-IV) in the MRS1271 and MRS1178 mutants, it has been suggested that LOS synthesis proceeds via a pathway of sequential additions of sugar units to the existing precursors; LOS-I to LOS-II to LOS-III to LOS-IV (Ren et al., 2007). wcaA Sarkar et al. (2011) studied the involvement of wcaA (mmar_2333) in the production of LOSs in M. marinum. The gene wcaA is located in the genetic locus associated with LOS production and encodes a putative glycosyltransferase (CAZy GT2 family) with two predicted transmembrane domains in its C-terminal region. Specialized transduction was utilized to engineer a M. marinum wcaA null mutant in which allelic exchange replaced the gene with a hyg resistance cassette. Lipid analysis of the resulting M. marinum mutant strain, which displayed rough colony morphology, indicated that the strain was lacking LOS-II, LOS-III and LOS-IV, but still produced LOS-I and accumulated LOS-II*, the LOS-II precursor mentioned above as accumulating in the ilvB1_3 null mutant (Ren et al., 2007). Genetic complementation analysis confirmed that the mutant phenotype was due to the loss of wcaA. Based on the sequence homology analysis of wcaA and the results of the mutational analysis, Sarkar et al. (2011) have proposed that wcaA is likely to be involved in the transfer of a nucleotide-bound caryophyllose residue (or its precursor) to a polyprenol phosphate or other lipid-based unit for subsequent use as a sugar donor by another glycosyltransferase to extend LOS-II* to LOS-II. papA4 Rombouts et al. (2011) have shown that a M. marinum mutant carrying a transposon insertion disrupting papA4 (mmar_2343), a gene located in the LOS biosynthesis gene locus of M. marinum, has a dramatic decrease in production of LOSs I through IV relative to the wild-type control strain. By analogy to the role of PapA5 and other Pap enzymes in lipid and/or glycolipid biosynthesis (Buglino et al., 2004; Chavadi et al., 2012; Onwueme et al., 2004; Trivedi et al., 2005), M. marinum papA4 has been proposed to be involved in acylation of the trehalose core of LOSs. Altogether, these observations strongly suggest a role of M. marinum papA4 in LOS biosynthesis. Other M. marinum genes More recently, van der Woude et al. (2012) reported a study that led to the identification of ten additional genes (added to the six noted above) with confirmed (or tentative due to lack of complementation controls) roles in LOS biosynthesis in M. marinum. The investigated M. marinum mutants were derived from a Himar1-based transposon library constructed using the Mycobacterium-specific phage ’MycoMarT7. The mutants with altered LOS production had insertions in fadD25 (mmar_2341, predicted FAAL), papA3 (mmar_2355, predicted Pap), galE6 (mmar_2336, predicted UDP-glucose 4-epimerase), wecE (mmar_2320, predicted sugar


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aminotransferase), mmar_2319 (predicted transmembrane protein), mmar_2327 (predicted transmembrane protein), mmar_2307 (predicted transmembrane protein), cphB (mmar_2405, possible cyanophycinase homolog) and whiB4 (mmar_5170, WhiB-like transcriptional regulator). The analysis identified mutants completely deficient in LOS-I, LOSII, LOS-III and LOS-IV production (fadD25, papA3 and cphB), a mutant with no LOS-II, LOS-III, and LOS-IV, yet accumulating LOS-I (mmar_2307), mutants deficient for LOS-IV and accumulating LOS-II and LOS-III (mmar_2319) or LOS-III (wecE), a mutant with no LOS-III and LOS-IV, but accumulating LOS-II (mmar_2327), a mutant deficient in LOS-II, LOS-III and LOS-IV, but displaying LOS-II* accumulation (galE6) as seen in the ilvB1_3 mutant, and a mutant with an overall reduction of LOS production and a concurrent reduction in the expression of several genes involved in LOS biosynthesis (whiB4). Unfortunately, the study included complementation controls for only the papA3 and whiB4 mutant strains. Based on their results, van der Woude and coworkers have proposed that the WecE protein is likely to be involved in the synthesis of the unusual sugar residue of LOS-IV and WhiB4 is likely to regulate expression of several LOS biosynthesis genes. The gene papA3 was proposed to encode an acyltransferase (PapA3) with a function comparable to that reported for PapA4 (Mmar_2343) (Rombouts et al., 2011). It is noteworthy that the M. tuberculosis gene encoding PapA3 is in a different genetic context than M. marinum papA3, and the former has been shown to be involved in PAT biosynthesis (Hatzios et al., 2009). The product of fadD25 has been proposed to be involved in activation and loading of a fatty acyl starter unit onto Pks5 for acyl chain extension (Rombouts et al., 2011). Interestingly, some of these genes (i.e. whiB4, cphB and papA3) shown by van der Woude and coworkers to be involved in LOS production lie clearly beyond the previously predicted boundaries of the chromosomal locus assigned to LOS synthesis (mmar_2307 to mmar_2344), and they would have not been predicted to have roles in LOS synthesis based on bioinformatics. These genes extend the previously known genomic locus involved in LOS production in M. marinum (Ren et al., 2007). Enzymology studies will be needed to validate the hypothesized roles of the predicted translational products of the genes involved in LOS biosynthesis.

Biosynthesis of Pks6-derived polar lipids Biology of pks6 at a glance The biological function of M. tuberculosis pks6 (rv0405, Figure 2) remains obscure. The gene has been identified in various screens of M. tuberculosis mutant libraries as a gene implicated in virulence or host–pathogen interaction. Signature-tagged mutagenesis studies with M. tuberculosis have shown that the pks6 mutant strain is impaired for growth in the lungs of mice (Camacho et al., 1999) and attenuated in a human macrophage (THP-1 derived) infection model (Rosas-Magallanes et al., 2007). A pks6 null strain has also been identified in a transposon mutant library as having greater affinity for human macrophages (THP-1) than the


parental wild-type strain (Lynett & Stokes, 2007). Another differential signature-tagged transposon mutagenesis study has suggested that pks6 is involved in a pathway that permits M. tuberculosis to counter IFN-g-dependent immune mechanisms (Hisert et al., 2004). Thus, based on the phenotype of the pks6 transposon mutants, it appears that pks6 is a relevant player in M. tuberculosis virulence. It is important to note, however, that genetic complementation controls are needed to conclusively link the phenotypes observed for the pks6 mutants to the lack of the Pks6 protein. Genetic studies linking pks6 and nearby genes to production of novel lipids pks6 The inactivation of pks6 by a transposon insertion has been shown to lead to the selective loss of uncharacterized complex polar lipids from the cell envelope of M. tuberculosis, suggesting that Pks6, the predicted PKS encoded by this gene, is involved in the biosynthesis of novel lipids (Waddell et al., 2005). fadD30 The pks6 gene appears to form an operon with fadD30 (Figure 3g), a gene encoding a FAAL that would activate and load fatty acyl primers onto Pks6 for acyl chain extension for production of polyketide products (Trivedi et al., 2004) (see below). Interestingly, a M. tuberculosis mutant with a transposon insertion disrupting fadD30 has been shown to be attenuated for survival in a nonhuman primate infection (aerosol) model of tuberculosis (Dutta et al., 2010). The gene fadD30 has also been indentified in a genome-wide screen of loss-of-function mutants for genes that modulate host immune responses (Beaulieu et al., 2010). mmpL1 and mmpS1 A predicted operon encoding MmpL1 (RND-type membrane transporter) and MmpS1 (MmpS protein family member; membrane protein) is located directly upstream of the fadD30–pks6 gene array (Figure 3g). Mycobacterial MmpL proteins have been shown to be involved in the export of cell wall lipids (Camacho et al., 2001; Converse et al., 2003; Cox et al., 1999; Domenech et al., 2004; Layre et al., 2011; Seeliger et al., 2012). Thus, it has been speculated that MmpL1 (and perhaps MmpS1) has a role in the export of the novel polyketide product synthesized by the FadD30–Pks6 system. However, unlike the pks6 mutant, the mmpL1 mutant does not appear to be significantly attenuated in murine models of tuberculosis (Domenech et al., 2005; Lamichhane et al., 2005). Enzymological studies supporting predictions derived from analysis of the fadD30–pks6 locus FadD30 Trivedi et al. (2004) demonstrated the expected long-chain fatty acyl adenylation activity of the FadD30 protein from M. tuberculosis (strain H37Rv) using recombinant protein


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generated in E. coli and lauric acid as a fatty acid substrate. FadD30 was competent for lauric acid adenylation in the presence of ATP, but not capable of catalyzing formation of the corresponding fatty acyl CoA thioester, therefore indicating that the protein belongs to the FAAL family (Arora et al., 2009; Trivedi et al., 2004).

lipids. The physiological role of these genes is currently unknown. Enzymological studies supporting predictions derived from analyses of the rv1371–pks18–rv1373 locus Pks18

Biosynthesis of Pks18-derived a-pyrone metabolites

Saxena et al. (2003) and Sankaranarayanan et al. (2004) probed the predicted synthase activity of Pks18 using recombinant protein expressed in and purified from E. coli. In vitro assays demonstrated that Pks18 is a novel type III PKS with remarkably broad specificity for the starter unit substrate. Pks18 has the capacity to utilize long-chain aliphatic-CoA thioesters (C6–C20) as starter unit donors and malonyl-CoA as an extender unit donor to synthesize triketide and tetraketide a-pyrones in vitro (Figure 5g). It is worth mentioning that long-chain a-pyrone metabolites have not yet been shown to be produced by mycobacteria. Sankaranarayanan et al. (2004) also reported the crystal structure of Pks18. The structural analysis revealed an ˚ long, unusual substrate binding tunnel. This tunnel is 20 A and it extends from the surface of the enzyme to the active site. This substrate binding tunnel represented a structural feature not seen in the superfamily of plant and bacterial type III polyketide synthases before this study.

Genetic analysis of pks18 and adjacent genes

Rv1373

The initial sequence analysis of M. tuberculosis pks18 (rv1372, Figure 2) indicated that the gene encodes a predicted type III PKS that belongs to the chalcone/stilbene synthase superfamily (Figure 4h) (Cole et al., 1998). Currently, there is no reported mutational analysis linking pks18 to production of mycobacterial metabolites, and the physiological function of pks18 remains undefined. Himar1based transposon mutagenesis in M. tuberculosis (strain H37Rv) has demonstrated that this orphan pks gene is not essential for growth in vitro (Sassetti et al., 2003). Interestingly, however, the PKS encoded by pks18 has been detected by mass spectrometry in lung tissues of M. tuberculosis-infected guinea pigs (Kruh et al., 2010), a finding indicating that the gene is expressed in vivo. The gene pks18 appears to form an operon with genes rv1371 and rv1373 (Figure 3h). As per gene annotation, rv1371 encodes a probable membrane protein with similarity to D5-fatty acid desaturases. Transposon site hybridization (TraSH) analysis has revealed that rv1371 is required for growth of M. tuberculosis (strain H37Rv) in mouse spleen (Sassetti & Rubin, 2003) and proteomic studies have detected the predicted translation product of rv1371 in both culture filtrates and cell membrane fractions of M. tuberculosis H37Rv propagated ex vivo (Malen et al., 2007; Mawuenyega et al., 2005). The initial sequence analysis of rv1373 revealed that the gene encodes a protein with sequence similarity to sulfotransferases (Cole et al., 1998). The gene is predicted to be non-essential in vitro by Himar1-based transposon mutagenesis in M. tuberculosis H37Rv (Griffin et al., 2011; Sassetti et al., 2003). There are no reports of mutational analysis studies probing the possible involvement of rv1371 and rv1373 in biosynthesis of mycobacterial

Rivera-Marrero et al. (2002) validated the predicted sulfotransferase activity of Rv1373 using recombinant protein in in vitro sulfation assays. The purified Rv1373 was shown competent for sulfation of typical ceramide glycolipids and mycobacterial glycolipids. The participation of Rv1373 in sulfolipid biosynthesis in vivo remains to be investigated.

FadD30 and Pks6 interplay Critical Reviews in Biochemistry and Molecular Biology Downloaded from informahealthcare.com by Memorial University of Newfoundland on 08/01/14 For personal use only.

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Studies by Trivedi et al. (2004) have shown that FadD30 can transfer the lauroyl acyl chain from the activated intermediate lauroyl-AMP to Pks6. These studies were conducted with proteins from M. tuberculosis (strain H37Rv) recombinantly produced in E. coli. To obtain Pks6 with phosphopantetheinylated ACP domains, the synthase was coexpressed with the phosphopantetheinyl transferase Sfp (Quadri et al., 1998b). Pks6 has two predicted ACP domains (Figure 4g), and the loading of the lauroyl acyl chain has been proposed to take place at the N-terminal domain, which shows homology to peptidyl carrier protein domains and would be the position for fatty acyl loading prior to its extension for formation of the polyketide product of Pks6 (Trivedi et al., 2004).

Involvement of the pks10–pks7–pks8–pks17–pks9– pks11 locus in lipid synthesis Biology of the pks10–pks7–pks8–pks17–pks9–pks11 gene cluster The biological role of the pks10–pks7–pks8–pks17–pks9– pks11 gene cluster (Figure 3i) remains poorly understood. Proteome analysis has validated the expression of the proteins Pks7, Pks10, Pks11 and Pks17 in M. tuberculosis grown in vitro (de Souza et al., 2011a,b; Kelkar et al., 2011; Malen et al., 2010; Mawuenyega et al., 2005). Expression of the proteins Pks7, Pks8, Pks9, Pks10 and Pks17 has been demonstrated in M. tuberculosis-infected guinea pig lungs (Kruh et al., 2010). Overall, these results have shown that each of the PKSs encoded in the gene cluster is expressed in at least one condition (in vivo or in vitro). To date, pks8 and pks17 are the only genes in the cluster that have been convincingly shown to participate in biosynthesis of a lipid product in M. tuberculosis, and yet their deletion leads to a mutant with no significant growth phenotype in vitro or in vivo (Dubey et al., 2003). The genes pks7, pks10 and pks11 have been proposed to be required for dimycocerosate ester production (Rousseau et al., 2003b; Sirakova et al., 2003a; Waddell et al., 2005); however, as discussed below, this idea remains controversial and it is likely to be incorrect.

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Genetic studies linking the pks10–pks7–pks8–pks17– pks9–pks11 cluster to lipid production


pks7, pks10 and pks11 Dimycocerosate ester deficiency has been observed in the three corresponding null mutants engineered in M. tuberculosis H37Rv (Rousseau et al., 2003b; Sirakova et al., 2003a; Waddell et al., 2005). As noted earlier in this review, unidentified spontaneous mutations in legitimate dimycocerosate ester biosynthetic genes in the ppsABCDE– mas–pks15/1 chromosomal region are likely to be the underlying cause of the dimycocerosate ester deficiency of the pks7, pks10 and pks11 mutant strains. The pks7 and pks10 mutant strains have been shown to be attenuated in murine models of tuberculosis (Rousseau et al., 2003b; Sirakova et al., 2003a), an expected phenotype for mutant strains deficient for dimycocerosate ester production (Onwueme et al., 2005a). The pks11 mutant strain remains to be examined in the murine model, but one would expect to be attenuated due to its dimycocerosate ester deficiency. pks8 and pks17 The protein encoded by pks8 plus the protein encoded by pks17 provide a multidomain arrangement (KS-AT-DHER + KR-ACP) paralleling that of the Mas protein from the dimycocerosate ester biosynthetic pathway (Figure 4i). Guided by this fact, Dubey et al. (2003) investigated the possible involvement of the pks8–pks17 locus (which they referred to as msl5) in the biosynthesis of methyl-branched fatty acids in M. tuberculosis H37Rv. Disruption of the pks8– pks17 locus by allelic exchange with a hyg-resistance marker cassette was shown to correlate with loss of capacity to produce 2-methyl, unsaturated C16–C20 fatty acids, which are found as minor fatty acyl components of sulfolipids and acyltrehaloses. It is unclear whether one or both genes is required for fatty acid production. Despite the lack of these fatty acids, the pks8–pks17 null mutant did not display significant changes in overall glycolipid composition, growth in vitro, growth in macrophages, or virulence in a murine model of tuberculosis when compared with the parental M. tuberculosis wild-type strain. Biochemical studies supporting functional predictions for pks11 Saxena and coworkers investigated the predicted synthase activity of Pks11, which is the only protein encoded in the pks10–pks7–pks8–pks17–pks9–pks11 gene cluster that has been probed for enzymatic activity in vitro. Pks11 recombinantly expressed in and purified from E. coli was used in in vitro assays to validate the predicted type III PKS activity of the protein (Saxena et al., 2003). Pks11 was shown to be a condensing enzyme capable of synthesizing long-chain a-pyrones (Figure 5g). The synthase displayed preference for long-chain fatty acyl-CoA (C12–C20) starter unit donors in combination with malonyl-CoA as the extender unit donor. The long-chain fatty acyl-CoA preference is a property shared with Pks18 (see above). More recently, Gokulan et al. (2013) solved the crystal structure of M. tuberculosis Pks11 and conducted an additional exploration of the activity of the


enzyme in vitro. Notably, the structural analysis revealed that Pks11 has an unusual hydrophobic substrate-binding tunnel comparable to that seen in Pks18 (25% amino acid identity to Pks11). The exploration of the synthase activity conducted by Gokhale and coworkers demonstrated that Pks11 can assemble unique cyclic methyl-branched alkylpyrones using palmitoyl-CoA in combination with methylmalonyl-CoA and malonyl-CoA as sequential extender units. These results enrich the previously established biosynthetic capacity of Pks11 in vitro; i.e. assembly of alkylpyrones from fatty acylCoA and malonyl-CoA (Saxena et al., 2003). It is noteworthy that mycobacteria have not yet been shown to produce longchain a-pyrone metabolites.

Biosynthesis of glycopeptidolipids Glycopeptidolipids at a glance Glycopeptidolipids (GPLs) are a major family of free glycolipids present in the capsule layer (and possibly also in the outer leaflet of the outer membrane) of the cell envelope of several Mycobacterium species (Chatterjee & Khoo, 2001; Mukherjee & Chatterji, 2012; Schorey & Sweet, 2008). The GPL-producing species include saprophytic mycobacteria of no clinical significance, such as M. smegmatis, and many nontuberculous mycobacteria of clinical relevance. Species of the M. avium–intracellulare complex (MAC) and the M. chelonae–abscessus complex are among the GPL producers associated with human disease. Notably, GPLs are not found in the M. tuberculosis complex. The structures of the GPLs from several mycobacteria have been investigated and characterized. Essentially, GPLs are composed of an N-acylated lipopeptide core decorated with a variable pattern of glycosylation that is built from O-methylated and O-acetylated sugar units. The archetypal peptide moiety is the tripeptide-amino alcohol D-phenylalanine-Dallothreonine-D-alanine-L-alaninol (D-Phe-D-alloThr-D-Ala-Lalaninol), but other variations in the peptide portion of the molecule have been documented (Chatterjee & Khoo, 2001; Ichimura & Kasama, 2012) (Figure 5h). The most commonly found lipid substituents are saturated or unsaturated long fatty acyl chains (e.g. C26–C34) with a hydroxy or a methoxy functionality. The structures of GPLs have been thoroughly reviewed elsewhere (Chatterjee & Khoo, 2001; Hsu et al., 2012; Mukherjee & Chatterji, 2012; Schorey & Sweet, 2008). GPLs have been implicated in many aspects of mycobacterial biology, including biofilm formation (Kocincova et al., 2008; Recht et al., 2000; Tatham et al., 2012), sliding motility (Etienne et al., 2002; Recht et al., 2000; Tatham et al., 2012), and host–pathogen interaction and pathogenesis (Barrow et al., 1995; Kano et al., 2005; Rhoades et al., 2009; Shimada et al., 2006; Sweet et al., 2010; Villeneuve et al., 2003, 2005). Notably, alterations in the GPL profile have been observed in drug-resistant clinical isolates of MAC (Khoo et al., 1999). This finding suggests that GPL production might have an impact on drug susceptibility as well. Recent studies have revealed that loss of GPL production leads to an increase in antimicrobial drug susceptibility in M. smegmatis, an observation that is in line with the idea that GPLs contribute to strengthening the permeability barrier


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of the mycobacterial cell envelope (Tatham et al., 2012). The following sections present the reported genetic and biochemical studies on GPL biosynthesis. Genetic studies of the glycopeptidolipid biosynthetic gene locus


pks Sonden et al. (2005) reported that disruption of the gene pks (Figure 3j) in M. smegmatis leads to a GPL-deficient mutant. The pks gene is predicted to encode a type I PKS, and it is located in a region of the M. smegmatis (strain mc2155) genome encompassing multiple genes that have been implicated in GPL production (Pang et al., 2013; Ripoll et al., 2007; Sonden et al., 2005). The M. smegmatis pks null mutant, which carries a Tn611 transposon insertion in pks, also displayed the pleiotropic phenotype characteristic of GPL-deficient mutants. The mutational analysis reported by Sonden and coworkers did not include a complementation control, but the essential requirement of pks for GPL biosynthesis was recently established unambiguously by complementing the pks null mutant with the wild-type gene expressed from a mycobacterial shuttle vector (Vats et al., 2012). Overall, the phenotype of the pks null mutant is consistent with the idea that pks participates in the biosynthesis of the long-chain fatty acyl moiety of the lipopeptide core of GPLs. The N-acylation of the peptide moiety of GPLs with the lipid product synthesized by the Pks protein (encoded by pks) has been proposed to require papA3 (Ripoll et al., 2007), which is a gene located adjacent to pks and encoding a member of the Pap family of acyltransferases (Buglino et al., 2004; Onwueme et al., 2004). Corresponding pks and papA3 orthologs have been identified in the GPL biosynthetic gene clusters of other GPL producers (Ripoll et al., 2007). faal28 The M. smegmatis faal28 (fadD23, Figure 3j) gene is predicted to encode a member of the FAAL protein family (Trivedi et al., 2004). Vats et al. (2012) proposed the involvement of this gene in activation and loading of the fatty acyl starter unit onto Pks for its extension to the long-chain fatty acid found in the lipopeptide core of GPLs. In line with this prediction, Vats et al. (2012) demonstrated that disruption of faal28 by the recombineering method results in GPL deficiency. The requirement of faal28 for GPL production was verified by complementing the faal28 null mutant strain with wild-type faal28 expressed from a mycobacterial shuttle vector. mps1 and mps2 Billman-Jacobe et al. (1999) conducted the first mutational analysis that demonstrated the requirement of a nonribosomal peptide synthetase (NRPS)-encoding locus (mps, Figure 3j) for the production of GPLs. This was conducted in M. smegmatis (strain mc2155), and it involved the characterization of transposon insertion mutants in the mps locus and a mps null mutant generated by a targeted allelic replacement approach that disrupted mps by the

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insertion of a streptomycin resistance marker cassette. The involvement of this locus in GPL production was subsequently confirmed by Recht et al. (2000) and Sonden et al. (2005) by showing that transposon insertions in mps1 or mps2, the two NRPS-encoding genes in the locus, rendered a GPL-deficient mutant. The genetic analyses coupled with the predicted nonribosomal peptide biosynthetic capacity of the mps1–mps2 locus derived from scrutiny of the modular/domain organization of the NRPSs permit the conclusion that both these genes are required for the synthesis of the peptide moiety of the lipopeptide core of GPLs. Well conserved orthologs of mps1 and mps2 have been identified in the corresponding GPL biosynthetic gene clusters of other species (Ripoll et al., 2007; Tatham et al., 2012). gplH Tatham and coworkers reported that every GPL biosynthetic gene cluster known to date contains a gene encoding a member of the MbtH-like protein family (NCBI CDD pfam 03621). Members of this family have been linked to siderophore or antibiotic production pathways involving NRPSs. The family’s founding member is the MbtH (Rv2377c) protein encoded in the mycobactin siderophore biosynthetic gene cluster (see below) (Figure 3k) (Chavadi et al., 2011b; Quadri et al., 1998a). The mbtH-like gene (msmeg_0399) located in the GPL biosynthetic gene cluster has been named gplH (Figure 3j), a designation that arises from glycopeptidolipid and mbtH and prevents gene name overlap with mbtH (Tatham et al., 2012). Tatham et al. (2012) conducted a mutational analysis (with a corresponding complementation control) that unequivocally established that gplH is essential for GPL production in M. smegmatis (strain mc2155). It has been proposed that the GplH protein is required for the synthesis of the peptide moiety of GPL by acting as an essential activator of the amino acid adenylation activity of one or more of the four amino acid adenylation domains predicted by sequence analysis of the Mps1-Mps2 NRPS system (Tatham et al., 2012). Notably, gplH is the first mbtH-like gene shown to be required for biosynthesis of a cell wall component. The work of Tatham et al. (2012) provides the first genetic analysis conclusively demonstrating the functional role of a mbtH-like gene in a member of the Mycobacterium genus. mmpS4, mmpL4a, mmpL4b and gap Recht and coworkers and Sonden and coworkers established that a transposon insertion disrupting mmpL4a (previously known as tmptB) (Sonden et al., 2005) or mmpL4b (previously known as tmptC) (Recht et al., 2000) abrogates GPL biosynthesis in M. smegmatis (strain mc2155) (Figure 3j). These genes appear to be cotranscribed and encode homologs of the MmpL family of protein transporters. The fact that mutations in mmpL4a and mmpL4b eliminate GPL production suggests that biosynthesis is coupled to transport via essential interactions between the biosynthetic and transport proteins (Recht et al., 2000; Sonden et al., 2005). The genes mmpL4a and mmpL4b


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form a putative operon with mmpS4 (Figure 3j). The gene mmpS4 encodes a member of the MmpS family of mycobacterial membrane proteins. Deshayes et al. (2010) reported a mutational analysis (with a corresponding complementation control) that has conclusively shown that mmpS4 is required for wild-type level production of GPLs, but not involved in GPL subcellular localization in M. smegmatis. Interestingly, Sonden and coworkers also found that a M. smegmatis (strain mc2155) mutant with a transposon insertion within the gene gap, which is located downstream of mps2 (Figure 3j) and encodes a protein predicted to have six transmembrane segments, was able to synthesize GPLs, yet failed to localize GPLs to the cell surface. Complementation analysis demonstrated the restoration of the wild-type phenotype by the gap gene (Sonden et al., 2005). Overall, the genes mmpL4a and mmpL4b are believed to be involved in GPL transport, whereas mmpS4 has been suggested to facilitate GPL synthesis by encoding a protein that acts as a scaffold for coupling the biosynthesis and transport machineries, and gap is proposed to be required for proper localization of GPLs to the cell surface. Other genes Various glycosyltransferases, methyltransferases and acetyltransferases have been implicated in the building of the glycosyl portion of GPLs produced by M. smegmatis and/or other species by studies reviewed elsewhere (Fujiwara et al., 2008; Jeevarajah et al., 2002; Ripoll et al., 2007; Schorey & Sweet, 2008).

Biochemical studies supporting functional predictions for Pks Vats et al. (2012) predicted that the protein Pks encoded in the GPL biosynthetic gene locus would be involved in biosynthesis of the fatty acyl component of the lipopeptide moiety of GPLs. Based on canonical PKS enzymology, the bimodular Pks protein (Figure 4j) was predicted to extend the fatty acyl starter unit by four carbons to form a d-hydroxy fatty acid. To probe this idea, the investigators characterized the activity of recombinant Pks purified from an E. coli host. The synthase was purified in holo-form by coexpression with sfp, the gene encoding the phosphopantetheinyl transferase Sfp from B. subtilis (Quadri et al., 1998b). The in vitro characterization of Pks revealed absolute specificity for the malonate extender unit (over methylmalonate). It also demonstrated the ability of the Pks protein to form d-hydroxy fatty acyl products using acyl-N-acetylcysteamine thioesters (surrogates for fatty acyl-CoA) starter units in conjunction with the malonylCoA and NADPH cosubstrates. The formation of this product is consistent with the protein’s linear arrangement of functional domains predicted by sequence analysis. Importantly, the work clearly established that the activity of Pks leads to hydroxylation at the C-5 position in the fatty acyl group of the lipopeptide moiety of GPLs (Vats et al., 2012).


Biosynthesis of mycobactin and carboxymycobactin siderophores The mycobactin–carboxymycobactin siderophore system at a glance Mycobacterium tuberculosis, most opportunistic mycobacterial pathogens of clinical significance (e.g. M. avium), and several nonpathogenic saprophytic mycobacteria (e.g. M. smegmatis) have a siderophore system that relies on two structurally-related siderophore variants referred to as mycobactins (MBTs) and carboxymycobactins (cMBTs) (Figure 5i). It is worth noting that the name exochelin (rather than cMBT) was initially utilized to refer to some of the MBT-like siderophores extracted from the supernatant of mycobacterial cultures, but cMBT is the preferred and most commonly used name in recent literature (Quadri & Ratledge, 2005). MBTs and cMBTs share a hydroxyphenyl-capped peptide–polyketide scaffold (De Voss et al., 1999; Quadri, 2000; Quadri & Ratledge, 2005; Ratledge, 2004). This common scaffold is synthesized by a dedicated NRPS–PKS hybrid system encoded in a 25-kb gene cluster (Figure 3k) (Chavadi et al., 2011b; Quadri et al., 1998a). The hydroxyphenyl cap of the scaffold arises from salicylic acid, a biosynthetic precursor of several other siderophores (Quadri, 2000). Salicylic acid is synthesized in M. tuberculosis by the salicylate synthase MbtI (Harrison et al., 2006; Zwahlen et al., 2007). MBTs are associated with the cell envelope and have a scaffold acylated on the internal hydroxylysine residue by a fatty acyl group of variable chain length. This acyl substituent is believed to dictate the association of MBTs with the cell envelope (possibly at the outer leaflet of the outer membrane, Figure 1). cMBTs are structural variants in which the fatty acyl substituent is shorter than the one in MBTs and has a carboxylate or its methyl ester functionality at its distal end. This more hydrophilic substituent gives cMBTs a better solubility in extracellular environments than that of MBTs, and thus cMBTs are routinely isolated from the culture supernatant. The hydroxyphenyl-capped peptide–polyketide scaffold of the MBT/cMBT siderophores is surprisingly similar to the scaffolds of several secondary metabolites isolated from Nocardia species (Chavadi et al., 2011b). Studies in cellular and animal models of mycobacterial infection have demonstrated the critical relevance of the MBT/cMBT siderophore system in M. tuberculosis (De Voss et al., 2000; Farhana et al., 2008; Reddy et al., 2013; Rodriguez & Smith, 2006; Wells et al., 2013). These studies underscore MBT/cMBT biosynthesis as a target candidate for drug development (Ferreras et al., 2005; Quadri, 2007). Notably, the first antibacterial targeting MBT/cMBT synthesis in M. tuberculosis was initially reported in 2005 by Ferreras et al. (2005). This report provided the first proof-of-concept for the druggability of the pathway. The antimicrobial compound (known as salicyl-AMS) targets the salicyl-AMP ligase/salicyl-S-ArCP synthetase MbtA (Quadri et al., 1998a), which is essential for MBT/cMBT synthesis (Chavadi et al., 2011b). Salicyl-AMS inhibits MbtA, blocks siderophore synthesis and, as expected, restricts M. tuberculosis multiplication with greater potency under iron-limiting conditions (Ferreras et al., 2005). More recently, a study on the pharmacokinetics and therapeutic efficacy of salicyl-AMS


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in a mouse model provided the first in vivo proof-of-concept for the use of siderophore biosynthesis inhibitors as antibacterials (Lun et al., 2013). Additional studies by Aldrich and coworkers confirming the in vitro activity of salicyl-AMS and providing novel structure–activity relationship data for salicyl-AMS analogs have been recently reviewed (Cisar & Tan, 2008; Neres et al., 2008). Antimicrobial activity against M. tuberculosis has also been documented for potential ironuptake inhibitors with structural features resembling the hydroxyphenyl-oxazoline-containing section of MBTs (Ferreras et al., 2011; Stirrett et al., 2008) and for MBT analogs (Juarez-Hernandez et al., 2012). The genetic and biochemical studies of the MBT/cMBT biosynthetic pathway are outlined below. Genetic studies of the mycobactin–carboxymycobactin biosynthetic gene loci Mutagenesis of the mbt-1 gene cluster Over a decade ago, several genes located in a 25-kb region of the M. tuberculosis chromosome were proposed to be required for MBT/cMBT biosynthesis based on bioinformatic analysis and in vitro enzymology studies (Quadri et al., 1998a). These genes (mbtA-J) form the so-called mbt-1 gene cluster (Figure 3k), which is conserved in MBT/cMBT producers (Chavadi et al., 2011b). Several of these mbt genes encode NRPSs or PKSs that were proposed to synthesize the salicylcapped peptide–polyketide backbone of MBTs and cMBTs from salicylic acid, amino acids, and small carboxylic acids (Quadri et al., 1998a). Soon after the identification of the mbt-1 gene cluster, De Voss et al. (2000) reported that disruption of the NRPS-encoding gene mbtB (rv2383c) from the cluster renders a M. tuberculosis mutant unable to produce siderophores. More recently, Chavadi et al. (2011b) reported a systematic mutational analysis of the mbt-1 gene cluster ortholog found in M. smegmatis. The investigators constructed and analyzed mutant strains with gene-specific, unmarked in-frame deletions. This detailed mutational analysis included corresponding genetic complementation controls. Mutants were engineered for the salicyl-AMP ligase/salicyl-S-ArCP synthetase gene mbtA (msmeg_4516), the NRPS genes mbtB (msmeg_4515), mbtE (msmeg_4511) and mbtF (msmeg_4510), the PKS genes mbtC (msmeg_4513) and mbtD (msmeg_4512), the thioesterase gene mbtT (msmeg_4514), the lysine hydroxylase gene mbtG (msmeg_4509) and the gene mbtH (msmeg_4508). The analysis unambiguously demonstrated the requirement of mbtA, -B, -C, -D, -E, -F, -G and -T for siderophore production. Unexpectedly, mbtH is not essential for MBT synthesis in M. smegmatis (Chavadi et al., 2011b). It remains to be determined whether nonspecific action of any of the two MbtH paralogs encoded in the M. smegmatis genome supports the MBT/cMBT production observed in the absence of MbtH. Overall, the findings of this systematic mutational analysis are in agreement with the conclusions derived from analysis of other engineered mutants (LaMarca et al., 2004; Madigan et al., 2012; Siegrist et al., 2009; Tullius et al., 2008; Wells et al., 2013). The results are also in line with the reported conservation of mbt genes (Chavadi et al., 2011b).


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Mutagenesis of the mbt-2 gene cluster The M. tuberculosis mbt-2 locus consists of mbtL (rv1344, ACP gene), mbtM (fadD33; FAAL gene), mbtN (fadE14, acylACP dehydrogenase gene) and mbtK (rv1347c, lysine Nacyltransferase gene) (data not shown) (Krithika et al., 2006; LaMarca et al., 2004). LaMarca et al. (2004) established the first link between the mbt-2 locus and MBT/cMBT production. The investigators found that disruption of the gene msmeg_2131 (ortholog of M. tuberculosis mbtM found in the mbt-2 gene cluster of M. smegmatis) led to a mutant lacking wild-type MBTs, but producing small quantities of a MBT analog with an altered side chain. Based on this finding and supporting mutant complementation analysis, they hypothesized that the mbtL–mbtM–mbtN operon was involved in biosynthesis of the aliphatic side chain attached to the siderophore scaffold. Subsequent bioinformatics and enzymology studies based on M. tuberculosis proteins by Krithika et al. (2006) consolidated this hypothesis, expanded the locus to include mbtK and introduced the current name for the locus and its genes. Biochemical studies supporting predictions derived from analyses of mbt loci Proteins encoded in mbt-1 The first in vitro enzymology studies probing and supporting functional predictions for proteins encoded in the mbt-1 locus were reported by the Walsh laboratory. The research group demonstrated that the conserved M. tuberculosis protein MbtA (Rv2384) is a bifunctional salicyl-AMP ligase/salicylS-ArCP domain synthetase that activates and loads the salicylic acid starter unit onto the phosphopantetheinylated ArCP domain of MbtB (Rv2383c) (Quadri et al., 1998a). The ArCP domain of MbtB was shown to be phosphopantetheinylated by M. tuberculosis PptT (Rv2794c), a M. tuberculosis Sfp-like phosphopantetheinyl transferase characterized in the Walsh laboratory (Quadri et al., 1998a). The proposed activity of the cyclization domain–adenylation domain– peptidyl carrier protein domain module predicted for MbtB (Quadri et al., 1998a) was initially validated in vitro with a hybrid protein constructed by fusing the MbtB module to domains from bacitracin and tyrocidine synthetases. This hybrid synthetase (BacA1 Ile adenylation domain–PCP domain + MbtB cyclization domain–Thr/Ser adenylation domain–PCP domain + TycC TE domain) formed the predicted heterocyclic dipeptide products (Ile-Throxazoline and IleSeroxazoline) in vitro (Duerfahrt et al., 2004). More recently, an elegant study by McMahon et al. (2012) showed that soluble forms of recombinant MbtB, MbtE (Rv2380c) and MbtF (Rv2379c) proteins from M. tuberculosis are obtainable by coproduction with MbtH (Rv2377c) and determined the amino acid specificity of each adenylation domain of the synthetases in vitro. MbtB was found to prefer L-Thr over LSer, whereas MbtE and MbtF were shown to be specific for N6-acyl-N6-hydroxy-L-Lys and N6-hydroxy-L-Lys, respectively. These results provide new insights into the possible sequence of events during siderophore assembly (McMahon et al., 2012). Finally, the predicted lysine-N-oxygenase activity of M. tuberculosis MbtG (Rv2378c), which is


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proposed to be required for hydroxylation of the lysine residues of MBTs and cMBTs (Quadri et al., 1998a), has been demonstrated using N6-lauroyl Z-lysine-OMe, acetylated lysine and lysine as substrates (Krithika et al., 2006). Importantly, MbtG has preference for acetylated lysine (Krithika et al., 2006). This observation together with the characterization of scaffolds lacking N-hydroxylation suggests that hydroxylation may take place after peptide–polyketide scaffold assembly. The timing of the hydroxylation steps remains controversial (Krithika et al., 2006; Madigan et al., 2012; McMahon et al., 2012). Proteins encoded in mbt-2 Studies by the Gokhale laboratory provided the initial in vitro validation for the predicted activities of the enzymes encoded in the mbt-2 gene cluster (Krithika et al., 2006). Recombinantly generated MbtM was shown to be a FAAL with the capacity to adenylate a variety of medium-/longchain fatty acids. MbtM was also proven to be able to acylate post-translationally phosphopantetheinylated MbtL (ACP). For these studies, recombinant MbtL was post-translationally modified using the promiscuous surfactin phosphopantetheinyl transferases Sfp (Quadri et al., 1998b) and lauric acid (dodecanoic acid) was used as a substrate, thus leading to the formation of a lauroyl-S-MbtL thioester product. Furthermore, the predicted acyl-CoA dehydrogenase MbtN was recombinantly generated and shown to catalyze FAD-dependent introduction of a double bond on the MbtL-tethered lauroyl chain. Finally, the predicted lysine N-acyltransferase role of MbtK was also probed. The enzyme was shown to catalyze acylation of the "-amino group of Z-Lys-OMe (a model substrate that mimics a deacylated MBT acyl acceptor) with the lauroyl chain from lauroyl-CoA or lauroyl-S-MbtL, or with the a,b-unsaturated acyl chain generated from lauroyl-S-MbtL by action of MbtN. As suspected, MbtK showed a marked preference for the acylprotein thioester acyl donor. Blanchard and coworkers recently provided further insight into the catalytic mechanism of MbtK and established that the activity of MbtM (M. smegmatis ortholog) is regulated by post-translational acetylation (Frankel & Blanchard, 2008; Vergnolle et al., 2013). Overall, the in vitro studies with proteins encoded in the mbt-2 cluster are in line with the proposed role of the MbtK–MbtL–MbtM–MbtN system in transfer of the fatty acyl substituent from an ACP thioester intermediate to the internal lysine residue in the MBT/cMBT skeleton (Krithika et al., 2006).

Biosynthesis of mycolactone toxins Mycolactones at a glance Mycolactones are secreted macrocyclic polyketides that were first isolated from M. ulcerans, a major human pathogen responsible for Buruli ulcer disease (George et al., 1999). Mycolactones have also been found in M. liflandii, M. pseudoshottsii, and some isolates classified as M. marinum strains (Hong et al., 2007; Kim et al., 2009; Mve-Obiang et al., 2005; Nakanaga et al., 2013; Ranger et al., 2006). These species are aquatic mycobacteria closely related


to M. ulcerans and capable of producing disease in frogs and fish (Doig et al., 2012; Stinear et al., 2008; Yip et al., 2007). It is worth mentioning that the taxonomy of mycolactoneproducing mycobacteria is currently under debate, and it has been proposed that M. ulcerans and other mycolactoneproducing mycobacteria should be considered a single species (Pidot et al., 2010). The common scaffold of mycolactones consists of a 12-membered lactone ring that is decorated by an unsaturated fatty acyl chain of variable oxidation state and length (Fidanze et al., 2001; Scherr et al., 2013) (Figure 5j). The results of numerous in vivo and in vitro studies indicate that mycolactones are major virulence factors with immunomodulatory and cytotoxic properties. The biological properties of mycolactones and their relevance in the context of the ability of M. ulcerans to infect the host and produce the pathology of Buruli ulcer disease have been reviewed in detail elsewhere (Hong et al., 2008). Genetic studies linking the mlsA1–mlsA2–mlsB locus to mycolactone biosynthesis mlsA1, mlsA2 and mlsB The production of mycolactones in M. ulcerans (strain Agy99) has been linked to the large, low-copy-number plasmid pMUM001 (170 kb) (Stinear et al., 2004). Approximately 120 kb of pMUM001 is devoted to production of the polyketide scaffold of mycolactones via three genes encoding large type I modular PKSs. These genes are known as mlsA1 (50 kb), mlsA2 (7 kb) and mlsB (40 kb). Notably, the mls genes have a surprisingly high level of internal sequence repetition, making the locus prone to genetic instability (Porter et al., 2009). The involvement of mlsA1 (required for synthesis of the lactone core) and mlsB (required for synthesis of the fatty acyl side chain) in mycolactone production predicted by sequence analysis has been supported by transposon mutagenesis studies (Stinear et al., 2004). Inactivation of mlsA1 leads to the loss of mycolactones, the lactone core and the fatty acid originating the side chain, which is unstable and not detected in the absence of the lactone core. Disruption of mlsB renders a mutant strain capable of producing the lactone core, but missing the fatty acyl side chain. The giant MlsA1–MlsA2– MlsB PKS system encoded by mlsA1–mlsA2–mlsB consists of over 30 000 amino acids, and its domain and module organization has been extensively scrutinized by sequence analysis (Figure 4l) (Pidot et al., 2008; Stinear et al., 2004). Comparable plasmids are responsible for mycolactone production in other mycobacteria (Pidot et al., 2008; Stinear et al., 2004; Tobias et al., 2013). To investigate whether the genetic information in pMUM001 was sufficient to confer mycolactone production capacity, Porter et al. (2009) utilized two independent bacterial artificial chromosome shuttle vectors to transfer the entire 174-kb pMUM001 plasmid to a non-mycolactoneproducing M. marinum (strain M) and to its isogenic recA null mutant derivative. Southern hybridization and RT-PCR confirmed, respectively, the presence and transcription of the mls genes in both M. marinum hosts (wild-type and recA). However, production of mycolactones (or its known biosynthetic intermediates) was not detected in either strain,


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a finding suggesting that additional functions encoded in the chromosome of M. ulcerans might be required for mycolactone production. It should be noted, however, that heterologous protein expression was not verified in the M. marinum transformants and the possibility that mutations inactivating genes needed for mycolactone production emerged postintroduction of the artificial chromosomes in M. marinum was not ruled out. Interestingly, Porter et al. (2013) showed that expression in the homologous M. ulcerans host of an engineered 3-module (loading module + module 8 + module 9) protein derived from the 18-module MlsA1–MlsA2–MlsB PKS system leads to production of the predicted triketide lactone product. This study represents the first example of the use of the enzymatic components of the mycolactone biosynthetic machinery for the production of small molecules through genetic engineering. mup038, mup045 and mup053 Three additional genes (mup038, mup045 and mup053) located in pMUM001 have been proposed to be involved in mycolactone production. The gene mup038 encodes a predicted type II thioesterase that might be required for the removal of aberrant polyketide extension products thioesterified onto the ACP domains of the PKSs, whereas mup045 encodes a putative b-ketoacyl transferase proposed to catalyze formation of the ester bond between the lactone core and the fatty acyl side chain, and mup053 encodes a cytochrome P450 hydroxylase believed to catalyze the C-120 hydroxylation of the fatty acyl side chain (Hong et al., 2007; Stinear et al., 2004, 2005; Yip et al., 2007). Enzymological studies supporting predictions derived from analyses of the mlsA1–mlsA2–mlsB system KR domains Bali & Weissman (2006) probed and validated the activity of the KR domains of the MlsA1–MlsA2–MlsB system in vitro (Figure 4l). Based on sequence analysis, the system has 18 predicted KR domains. Module 5 of MlsA1 and modules 1 and 2 of MlsB have each a proposed A-type KR domain. Each of the rest of the modules (excluding the loading module) contains a predicted B-type KR domain. Remarkably, all the A-type domains have 100% sequence identity, and the same is true for the B-type domains. The sequence identity between the A-type and B-type domains, however, is 40%. Despite their 100% identity, the three A-type domains are predicted to act on biosynthetic intermediates with significant differences in acyl chain length and functionality. The 15 identical B-type domains also would need to act on structurally different extended intermediates. To characterize the KR domains, Bali and coworkers expressed in E. coli and purified the discrete A-type and B-type domain fragments. The catalytic competence of the recombinant KR domains and their steady-state kinetic constants were determined in vitro using NADPH and a variety of surrogate substrates. The two KR domains displayed some differences in substrate preferences, yet their activity was overall comparable to that of KR domains of other PKS systems. Overall, Bali & Weissman (2006) concluded that the in vitro substrate specificity of the domains


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resembles that of KR domains from other PKS systems and strengthened the view that substrate tethering contributes to stereochemical control in modular PKSs by impacting ketoreduction energetics. MlsA2-TE domain The C-terminal TE domain of MlsA2 (Figure 4l) is predicted to catalyze the macrocyclization of the fully extended, linear acyl intermediate bound to the PKS with consequent lactone core release. Meier et al. (2008) cloned, expressed and characterized the activity of both a TE domain fragment and an ACP-TE didomain fragment. The activity of the TE domain was characterized using a continuous spectrophotometric assay based on p-nitrophenyl propionate hydrolysis. In all, the kinetic characterization, substrate specificity and active site labeling studies validated the catalytic proficiency of the TE domain and revealed intriguing properties that led Meier et al. (2008) to suggest that the domain might utilize a non-canonical mode of macrocyclization.

Conclusions and future perspectives Fifteen years ago, the analysis of the M. tuberculosis genome revealed a surprisingly large number of genes encoding predicted PKSs. The majority of these PKS-encoding genes have today been linked to specific biosynthetic pathways required for production of unique lipid metabolites that are components of the extraordinarily complex mycobacterial cell envelope, and in many cases critical for virulence and host–pathogen interaction. Targeted and random mutational analyses have demonstrated the connection between individual PKS genes (or gene clusters) and the production of unique lipid metabolites in M. tuberculosis. Comparable genetic approaches have established the involvement of some PKS genes of other mycobacteria in the production of their characteristic metabolites. On the other hand, in vitro enzymology studies have validated predicted catalytic properties of several mycobacterial PKSs and contributed to the consolidation of working models for the participation of PKSs in compound-specific biosynthetic pathways. Despite this remarkable progress, the genomes of M. tuberculosis and other Mycobacterium species still contain several orphan PKS genes, and the mechanistic enzymology of most mycobacterial PKSs remains unexplored. Additional research efforts are needed to continue defining and refining mechanistic models for the biosyntheses of the unique lipid metabolites found in mycobacteria. A better understanding of these biosynthetic pathways may illuminate potential target candidates for exploring the development of innovative therapeutic approaches against mycobacterial pathogens. Another aspect of the mycobacterial PKSs in need of further exploration is the prevalence of polymorphisms in PKS gene loci with phenotypic consequences in mycobacterial clinical isolates. Polymorphisms in PKS genes can produce changes in lipid composition that may affect virulence, host– pathogen interaction, or drug susceptibility if the permeability barrier of the cell envelope is compromised. Genetic polymorphisms outside the PKS genes (e.g. promoter regions) affecting PKS gene expression might also lead to lipid composition changes with phenotypic consequences.

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Ultimately, one would like to know whether there are links between strain-specific polymorphisms and epidemiological profiles, drug susceptibility patterns or clinical outcomes.

Acknowledgements


The author thanks the endowment support from Carol and Larry Zicklin.

Declaration of interest The author reports no conflicts of interest. This study was supported in part by National Institutes of Health grant AI105884-01A1 to LENQ.

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