Bacterial surface appendages as targets for novel antibacterial therapeutics.

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Bacterial surface appendages as targets for novel antibacterial therapeutics

David Steadman‡,1, Alvin Lo‡,2, Gabriel Waksman*,1 & Han Remaut*,2,3

ABSTRACT: The rise of multidrug resistant bacteria is a major worldwide health concern. There is currently an unmet need for the development of new and selective antibacterial drugs. Therapies that target and disarm the crucial virulence factors of pathogenic bacteria, while not actually killing the cells themselves, could prove to be vital for the treatment of numerous diseases. This article discusses the main surface architectures of pathogenic Gram-negative bacteria and the small molecules that have been discovered, which target their specific biogenesis pathways and/or actively block their virulence. The future perspective for the use of antivirulence compounds is also assessed. Antibiotic resistance The unbridled use of available antibacterials have brought a broad-spectrum selective pressure on bacterial communities, which has resulted in the selection of multidrug resistance into clinical strains. This is becoming an alarming worldwide concern as infectious diseases become increasingly difficult to treat. A more specific, targeted approach to antimicrobial therapy is to disarm pathogens, thus reducing the emergence and spread of drug resistance. By focusing on blocking bacterial virulence factors and finding ways to prevent their formation, a new generation of targeted antimicrobial drugs could be developed, which could have a worldwide medical, social and economical impact.

KEYWORDS

• antibacterial • antibiotic • antibiotic resistance • antivirulence • curli • pili • pilicides

Surface-associated virulence factors of Gram-negative bacteria Gram-negative bacteria present a variety of different organelles on their cell surfaces (Figure 1) , which serve many different functions, including host cell adhesion and invasion, cell motility, DNA and protein secretion or transfer into host cells, and bacterial conjugation [1] . Chaperone-usher (CU) pili are adhesive filamentous appendages that are involved in host recognition and attachment, as well as bacterial biofilm formation. They are typified by Type 1 pili (Fim) (Figure 1A) and P pili (Pap), which are critical for the adhesion of uropathogenic Escherichia coli (UPEC) to bladder and kidney cells, respectively [2] . UPEC is the main cause of urinary tract infections (UTIs), which are estimated to affect 150 million individuals worldwide each year [3] . Curli (Figure 1B) are extracellular amyloid fibers produced by many different types of bacteria, known to contribute to the formation of biofilms [4] . Gram-negative bacteria may harbor Type IV pili ( Figure 1C ; not to be confused by the Type IV secretion system [T4SS] pili; see below), which mediate twitching motility and play important roles in the pathogenicity of bacterial pathogens such as Neisseria meningitis. Type III secretion needles (Figure 1D) are hollow needle-like structures, which are produced by the Institute of Structural & Molecular Biology, Birkbeck & University College London, Malet Street, London, WC1E 7HX, UK Structural & Molecular Microbiology, Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussels, Belgium 3 Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium *Authors for correspondence: [email protected] and [email protected] ‡ Authors contributed equally 1 2

10.2217/FMB.14.46 © 2014 Future Medicine Ltd

Future Microbiol. (2014) 9(7), 887–900

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Review Steadman, Lo, Waksman & Remaut

Effector molecules EspB/EspD

Host cell membrane

VirB2/ B5

EspA

FimH FimG

EscF

FimF

E

CsgA

FimA CsgB

OM

FimD

CsgG

CsgF

EscC

PilQ

VirB8/ B9/B10

CsgE PilE

P

EscD

FimC:FimA IM

VirB7

EscJ

PilG

PilD

SecYEG

PilF

VirB3/B4/ B6/B8, VirD4

SepQ, EscN, R, S, T, U and V

PilT

C

VirB11 FimA

CsgA

PilE ADP + Pi

ATP

ADP + Pi

ATP

ADP + Pi

ATP

Figure 1. Pilus assemblies on the surface of Gram-negative bacteria. (A) Type 1 pilus assembly in Escherichia coli. (B) Curli assembly in E. coli. (C) Type IV pilus assembly in Neisseria meningitidis. (D) Type III secretion needle assembly in E. coli. (E) Type IV secretion pilus assembly in Agrobacterium tumefaciens. C: Cytoplasm; E: Extracellular: IM: Inner membrane; OM: Outer membrane; P: Periplasm.

Type III secretion system (T3SS) and are utilized by the causative agents of plague, pneumonia and gastroenteritis, among others, to inject effector proteins from the bacteria into host cells [5] . Finally, T4SS pili (Figure 1E) are hollow pili produced by T4SS, and are capable of transferring DNA and protein substrates into host cells. T4SS pili are utilized by many human and plant pathogens including Helicobacter pylori, Agrobacterium tumefaciens and Bordetella pertussis, among others [6] . As such, all of these surfaceassociated organelles serve as critical virulence factors for a wide range of pathogenic bacteria, which are responsible for numerous diseases affecting many different species of plant and animal life. The rise of multidrug-resistant bacteria and a simultaneous drop in development of new antibiotics creates a renewed challenge in targeting these pathogens [7] . Selective drugs that specifically target critical bacterial virulence factors without killing the bacterial cell, so-called antivirulence drugs, promise to offer

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many advantages over traditional antibiotics and are rapidly gaining attention as a future direction in antibacterial treatment [7,8] . Herein, we describe the work that has been carried out in targeting the surface-associated virulence factors of various Gram-negative bacteria with novel antivirulence compounds. CU pathway-assembled pili (CU pili) Bacteria CU pili play a crucial role in specific bacteria–host interactions by mediating attachment of bacterial cells to host cell glycolipid and glycoprotein receptors via specific adhesive pilus subunits (termed ‘adhesins’). CU pili (Figure 1A) are adhesive surface organelles found on the surface of Gram-negative bacteria and serve as adherence and colonization factors primarily of the gastrointestinal and urinary tracts. Type 1 and P pili from UPEC are prototypical CU pili and have been the most extensively studied bacterial cell surface appendages to date, in terms of their structural and functional aspects [9–12] .

future science group

Bacterial surface appendages as targets for novel antibacterial therapeutics Type 1 pili promote bladder cell attachment and invasion, as well as biofilm formation [13,14] during cystitis, whereas P pili target cells of the upper urinary tract and are associated with the development of acute pyelonephritis [15] . The CU pili assembly system consists of three different protein components; the outer-membrane bound usher, the periplasmic chaperone and the pilus subunits [16,17] . The usher is responsible for the polymerization of subunits into the growing pilus fiber [18] , while the chaperone assists in the folding of subunits and targets them to the usher where they can be incorporated into a growing pilus [19] . The pilus itself grows in a ‘top-down’ fashion, with the adhesin subunit the first to be incorporated [18] . Type 1 pilus and P pili have very similar architecture, but vary in their protein components. P pili consist of six different subunits, composing the flexible tip fibrillum and the rigid pilus rod. The adhesin PapG is found at the distal end of the tip fibrillum [20] , followed by one copy of the adaptor subunit PapF and 5–10 PapE subunits [20,21] . The fibrillum is then connected to the main pilus rod by the PapK adaptor subunit [20] . The pilus rod itself is approximately 6.8 nm wide and is composed of over 1000 copies of the PapA subunit, and terminated within the cell wall by one copy of the PapH subunit [22] . Type 1 pili display a slightly simplified architecture (Figure 1A) , with a shorter tip fibrillum comprised of one copy of the adhesin FimH, attached to two linker subunits FimG and FimF [23] . The pilus rod is comprised of the main subunit, FimA [24] .

Review

the chaperone-subunit acceptor complex in a process termed donor strand exchange (DSE) (Figure 2B) [29] . Both the chaperone donor strand and subunit Nte consists of a motif of alternating hydrophobic residues (termed ‘P1–P5 residues’), which fit into corresponding sites in the subunit hydrophobic groove, in type 1 and P pili, termed P1–P5 (Figure 2B) [19,30] . In the chaperone–subunit complex, the G1 strand runs opposite to its canonical topology in an Ig-fold (i.e., parallel rather than antiparallel to strand F). As a result, the chaperone G1 strand occupies the P1–P4 pockets of the subunit, whereas after DSE, the subunit hydrophobic groove is filled by the incoming subunit Nte from P5–P2 [29] . The exchange of the G1 strand of the chaperone with the Nte of the incoming chaperone–subunit complex occurs by a concerted mechanism whereby the P5 residue of the incoming subunit’s Nte initiates the process by inserting into the empty P5 pocket of the hydrophobic groove of the acceptor chaperone–subunit complex (Figure 2) [29] . This step has been shown to be crucial for successful DSE [29] . After insertion into the P5 pocket, the Nte displaces the chaperone as it fills the P4–P2 pockets [31] . In P pili, termination of pilus assembly occurs when the PapD–PapH chaperone subunit is built in at the base of the pilus rod. PapH cannot undergo further DSE as it does not have an available P5 pocket for an incoming Nte peptide [32] . The mechanism of termination for type 1 pilus assembly is currently unknown, as no analog of PapH has been found.

●●CU pilus biogenesis

Pilus subunits are synthesized in the cytoplasm, transported via the Sec machinery and taken up by their periplasmic chaperones, which are crucial for correct subunit folding [25,26] . They display an immunoglobulin (Ig)-like structural fold in which the seventh strand, strand G, is missing, leaving a deep hydrophobic groove where the seventh strand is usually located in the complete Ig-folded proteins. As a result, subunits cannot fold unassisted. In a process known as donor strand complementation [27] , chaperone proteins stabilize the pilus subunits by donating an elongated β-strand (strand G1) to the incomplete Ig-like fold of each pilus subunit, ensuring they are stabilized and correctly folded (Figure 2A) [28] . Similarly, each pilus subunit contains a 10–20 residues long N-terminal extension (Nte) peptide, which during subunit polymerization, replaces the chaperone G1 strand of


●●Receptor analogs

On the basis that by impeding UPEC adhesion, infection can be prevented, the type 1 pilus adhesin FimH has been an attractive target for receptor analog development in recent years. Insights gleaned from the structural data of FimH bound to α-d-mannose and mannose derivatives, termed mannosides [33–35] , have fueled the rational design of antiadhesive carbohydrate-based ligands. The structural data revealed that the key molecular interaction lies in the deep mannose-binding pocket of FimH with a hydrophobic entrance termed the tyrosine gate. The mannosyl unit of α-D-mannosides nestles perfectly in the deep binding pocket of FimH, while its aglycon (nonsugar) moiety protrudes outward from the binding site (Figure 3E) . It has been established that bulky aglycon moieties can form aromatic stacking or hydrogen bonding

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I

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V

FimC:FimH

DSE +GNte

O Cl

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N N O

NH

AL1

O

S

N O

FimH:FimGNTe

Figure 2. Development of type 1 pilus biogenesis inhibitors. (A) Pilin domain of FimH (shown as molecular surface in brown; residues 158–279; PDB ID 1ZE3) with FimC donor strand peptide (gray) and FimG donor strand peptide (green, taken from PDB 3JWN), P5 pocket circled. (B) Schematic of DSE with P1–P5 pockets shown as white circles and interacting residues in each donor strand labeled. (C) Chemical structure of AL1. (D) Computational docking pose of pilus biogenesis inhibitor AL1 (purple sticks) docked into FimH (shown as molecular surface in brown) with P5 pocket circled. DSE: Donor strand exchange.

interactions with the tyrosine gate leading to higher affinities for FimH [36–39] . Therefore, the aglycon can be systematically optimized to achieve optimal binding affinity, solubility and bioavailability. The biphenyl mannosides (Figure 3A) have shown great promise as therapeutics [37,40–41] . In murine models, these receptor analogs were effective in treating chronic UTIs and in preventing reinfection of UPEC when administered prophylactically [41] . In addition, they can be used to treat established [41] and UPEC-caused catheter-associated UTIs [42] and act synergistically with standard antibiotics, presumably by isolating the bacteria in the bladder lumen, where they are exposed to maximum dose of standard antibiotic [41] . A biphenyl mannoside derivative, (4´-[a-D-mannopyranosyloxy]-N,3´dimethylbiphenyl-3-carboxamide) (Figure 3B) has also recently been shown to be effective in preventing acute infection and treating chronic cystitis caused by multidrug-resistant E. coli ST131 in a murine model [43] . The development and use of mannosides as antiadhesives for UPEC has been extensively reviewed elsewhere [2,44–46] . Adherent-invasive E. coli have been shown to induce gut inflammation in patients with Crohn’s disease [47] . It has been reported that thiazolylaminomannosides (Figure 3C) can efficiently prevent the type 1 pili-mediated attachment of adherent-invasive E. coli to intestinal cells in vitro. Thiazolylaminomannosides were effective at approximately 10,000-fold lower concentration than α-D-mannose and their antiadhesive properties were validated in an ex vivo

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assay performed on colonic tissue of a transgenic mouse model of Crohn’s disease [47] . Receptor analogs that target the P pilus adhesin PapG have also been identified through employment of a similar principle [48–50] . PapG adhesins mediate adherence of UPEC to host kidney epithelium by binding to host globoside (GbO4) receptors (Figure 3F) . A galabiose derivative, 2-[(S)-2methoxycarbonyl-2-acetamido-ethylthio] ethyl(3-O-3-[2-(methoxycarbonylphenylthio) propyl]-alpha-D-galactopyranosyl)-(1–4)-alphaD-galactopyranoside (Figure 3D) was shown to inhibit human erythrocyte hemagglutination by PapGII, an isoform that is most relevant to human pyelonephritis, with an IC50 value of 68 μM [49] . ●●Pilus polymerization inhibitors

An alternative to chemically blocking the adhesion of bacteria to cells is to target the biogenesis of pili in order to prevent the formation of adhesive organelles on the surface of bacteria. For type 1 pili, the adhesive subunit FimH is the first subunit to be incorporated into the growing pilus rod and is crucial for successful pilus assembly [16] . It has also been shown that a lack of FimG or FimF, the subunits that connect the adhesin to the pilus rod, results in an inhibition of pilus polymerization [51] . Hence, compounds that directly target the DSE reaction between FimH and FimG could prevent pilus polymerization and thus be useful antivirulence therapies. We have recently described an in silico compound screening approach to filter compounds libraries for putative binders of the FimH P5 pocket and adjacent hydrophobic


Bacterial surface appendages as targets for novel antibacterial therapeutics groove [52] . This allowed the identification a novel pilus biogenesis inhibitor that directly targets FimH/FimG DSE (Figure 2) . In order to target DSE, compounds would have to initially bind in the empty P5 pocket of the FimC–FimH complex (Figure 2) . Twenty compounds were identified that gave a >50% reduction in the formation of the

complex between FimH and the Nte of FimG (FimGNte) at 500 μM concentration. Of these, the most potent compound, N-(4-chloro-phenyl)2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4] oxadiazol-2-yl sulfanyl}-acetamide (AL1) (Figure 2C) , was chosen for further testing, and in a FimC:FimH FimGNte DSE assay, AL1 was

H OH

H OH

H OH OH O

HO HO

H H

OH O

HO HO

H

H O

H H

H

H

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O OH

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Thiazolylaminomannosides

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2-[(S)-2-methoxycarbonyl-2-acetamido-ethylthio]ethyl (3-O-3-[2-(methoxycarbonylphenylthio)propyl]-α-D-galactopyranosyl)(1–4)-α-D-galactopyranoside

Figure 3. Chemical structures of antiadhesive carbohydrate ligands. (A–C) Ligand for the FimH adhesin and (D) PapG adhesin. (E) Close up image of oligomannose-3 (shown in sticks in green) bound to FimH adhesin (shown ribbons and molecular surface in brown; PDB ID 2VCO). (F) PapGII lectin domain (shown in ribbons and molecular surface in brown) bound to globoside (GbO4) receptor (shown in sticks in green; PDB ID: 1j8r).



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Review Steadman, Lo, Waksman & Remaut shown to inhibit DSE product formation with an IC50 value of 286 μM. Computational docking of this compound showed that AL1 bound in a similar fashion to the FimGNte, partially filling the P5 pocket (Figure 2D) . To confirm that this inhibition of DSE translated to an in vivo inhibition of pilus formation, AL1 was resynthesized and a pilusdependent biofilm formation assay was carried out. AL1 did indeed suppress biofilm formation with an IC50 value of 37 μM. Interestingly, AL1 was shown not only to prevent formation of type 1 pili, but it also removed preformed pili from the surface of bacteria, possibly through destabilization of the basal subunit:chaperone complex leading to dissociation of the pili [52] . AL1 therefore represents the first small molecule to successfully target DSE, providing an exciting basis for further design and development of antivirulence drugs.

relationship data have been obtained to enable the development of a pilicide with an EC50 value of 400 nM [63] . The effectiveness of pilicides in animal models awaits future studies. Some of these 2-pyridones have also been shown to be effective against curli formation [64] . Curli Curli are the main protein component of the extracellular matrix in E. coli pellicle biofilms, as well as other bacterial cells, and consist of extracellular proteinaceous fibers that contribute to immune system activation, host coloniztion and cell invasion [65] . In contrast to structurally similar disease-forming amyloids, curli formation relies on a distinct biogenesis pathway (Figure 1B) rather than protein misfolding [4] . ●●Curli biogenesis

●●Pilicides

A series of small molecules, termed pilicides, have been developed comprising a bicyclic 2-pyridone scaffold (Figure 4C) , designed to disrupt chaperone–subunit interactions [53] . These pilicides were initially designed to be dipeptidomimetics, which would target CU pilus chaperone proteins, which are highly conserved across bacterial species [54] , and inhibit the formation of chaperone–subunit complexes [53] . A range of these molecules have been synthesized by a variety of methods, and binding of molecules to chaperones PapD and FimC has been assessed by surface plasmon resonance [53,55,56] and nuclear magnetic resonance [57–59] . It was thought that these compounds would bind at the chaperone–subunit interface, preventing the formation of chaperone–subunit complexes; however, a crystal structure of the PapD inhibitor (pilicide 2C; Figure 4C ) complex showed that the compounds bound to the PapD chaperone on a conserved hydrophobic patch on the rear of the chaperone, a region known to interact with the N-terminal periplasmic domain of the usher (Figure 4A & B) [60] . Disruption of this interaction would be expected to have a negative effect on the targeting of subunits for polymerization at the usher. Indeed, binding studies showed that pilicide impeded chaperone–subunit recruitment to the usher [60] . Through further development of the bicyclic 2-pyridone scaffold, compounds have been identified that show improved potency against type 1 pilus-mediated biofilm formation [61,62] , and comprehensive structure–activity

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The Csg proteins required for curli biogenesis (CsgA, CsgB, CsgC, CsgE, CsgF and CsgG) are secreted into the periplasm by the Sec transport system [4] . CsgG is then transported to the outer membrane where it forms a pore-like structure, through which the curli-forming subunits can be secreted. The major and minor curli subunits, CsgA and CsgB, respectively, localize at the cell surface where CsgA polymerizes to form the growing fiber [66] . The other three proteins, CsgC, CsgE and CsgF, mediate the secretion and localization of the curli subunit proteins [65] . Several roles have been proposed for each of these proteins with CsgE displaying characteristics of a periplasmic chaperone for CsgA [67] , and CsgF suggested as a potential chaperone for CsgB, ensuring that CsgB is surface-exposed to allow CsgA polymerization [68] . It has been suggested that CsgC may be responsible for modulating CsgG activity via modification of cysteine residues within the outer membrane pore. However, the full mechanistic details of CsgC, CsgE and CsgF, in curli biogenesis are currently not well understood [65] . ●●Curlicides

A range of cyclic-2-pyridones have been found to have an enhancing or inhibitory effects on amyloid formation [69,70] , making them potential drug candidates for the treatment of Alzheimer’s disease [71] . Development of bicyclic 2-pyridone scaffolds has also been carried out to create compounds that show inhibitory effects on curli biogenesis [72] . This has been attributed to the compounds affecting the polymerisation of the main curli subunit CsgA,


Bacterial surface appendages as targets for novel antibacterial therapeutics

Review

S N O

N

CO2Li

O Pilicide 2C

Figure 4. Bicyclic-2-pyridone pilicides that bind to chaperone proteins to inhibit type 1 and p pilus biogenesis. (A) FimD usher (blue ribbons) and FimC chaperone:FimF subunit complex (brown ribbons and black ribbons, respectively, from PDB ID 3BUW) with pilicide 2C (green sticks) superimposed (from 2J7L). The pilicide blocks recruitment of the chaperone–subunit complex to the usher N-terminal domain. (B) Close-up image of pilicide 2C bound to the hydrophobic patch of PapD (shown as molecular surface in brown, from PDB ID 2J7L). (C) Chemical structure of pilicide 2C.

either by affecting the stability of the CsgA monomer, or by directly affecting the polymerization step through reduction of the amount of cell-associated CsgB [73] . These compounds (Figure 5A) have also been shown to be effective inhibitors of type 1 pili, thereby exhibiting a dual mode of action as curlicides and pilicides, and drastically reducing the number of intracellular bacterial communities in a mouse bladder model [73] . This dual mode of action has been further studied in the development of fluorescent compounds, which inhibit both pili and curli formation [64] . Structure–activity data was used to modify previously synthesized pilicides and curlicides with either coumarin or boron-dipyrromethene fluorescent ligands and the resulting fluorescent compounds were still potent inhibitors of pili and curli [64] . These compounds could then be imaged by fluorescent


microscopy and showed that compounds with similar activities appeared to localize differently within different bacterial cells, although the implications of this are currently not fully understood [64] . Type III secretion needle Type III secretion needles are expressed by a large number of Gram-negative bacteria, and span the inner and outer membranes of cells where they are responsible for the injection of bacterial proteins into host cells, as well as the assembly of flagella [1] . Nonflagella T3SS consist of over 20 proteins and contain three main structural components: a basal body, which spans both the inner and outer membranes, the needle, and the translocon, a pore-forming complex that is inserted into the membrane of the host cell (Figure 1D) [74] .


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CF3

Cl

O H N

O Cl

S N O

O

O MBX 1641

CO2Li

OH N

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NH N

Compound 14 Br

Dual pilicide/curlicide FN075

O

O2N

H3CO

HO N H

N

Br

INP0007

N

S

F

O

OH OCH3

N O TTS29

Figure 5. Small molecules inhibitors of various pilus assemblies. (A) Dual pilicide/curlicide FN075. (B) A phenoxyacetamide, MBX 1641, which inhibits the T3SS of Psuedomonas aeuriginosa. (C) A benzimidazole, which inhibits the T3SS of Yersinia pseudotuberculosis. (D) INP0007, a salicilydene acylhydrazide T3SS inhibitor. (E) TTS29, a thiazolidinone that inhibits the Type III secretion system of various Gram-negative bacteria.

●●T3SS biogenesis

T3SS assembly consists of three sequential steps: formation of the basal body; assembly of the export apparatus and needle; secretion of the translocon-forming proteins and localization of these within the host cell membrane [75] . In enteropathogenic E. coli, the basal body of T3SS machinery is composed of three distinct proteins: EscC (in the outer membrane), EscD and EscJ (in the inner membrane), while the export apparatus is formed by SepQ, EscN, EscR, EscS, EscT, EscU and EscV. The needle consists of a single protein (EscF), which polymerizes on the cell surface [75] . All of the T3SS proteins secreted by the apparatus are present in the cytoplasm in complex with dedicated chaperones. Class I chaperones recognize effector proteins, class II chaperones interact with translocators to neutralize their toxicity [76] , and class III chaperones bind the needle subunit proteins to prevent polymerization within the cytoplasm [77] . ●●Inhibition of T3SS

Many diverse classes of molecules have been investigated as inhibitors of the T3SS [78,79] . Several phenoxyacetamides (Figure 5B) and benzimidazoles (Figure 5C) have been shown to inhibit the T3SSs from Pseudomonas aeruginosa and Yersinia pseudotuberculosis, respectively [80,81] . Perhaps the most widely studied family of T3SS inhibitors are the salicilydene acylhydrazides

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(Figure 5D) ,

with inhibitory activities noted against several Gram-negative pathogens including: Chlamydia trachomatis, Salmonella typhimurium and Y. pseudotuberculosis [82–86] . Crucially, however, the exact mechanism of action for this family of compounds is currently unknown, preventing the development of compounds with increased activity [78] . Thiazolidinones (Figure 5E) have been shown to reduce the amount of T3SS needle complex proteins in S. typhimurium, as well as affecting the type II secretion system of Pseudomonas sp., suggesting the target may be the secretin, a common outer membrane component [87] . Through design of more hydrophilicsubstituted thiazolidinones and thiazolidinone dimers, it was possible to increase the activity of the initial hit compound by tenfold, although no follow-up studies have been carried out [88,89] . Other diverse classes of compounds have been identified as potential T3SS inhibitors through various high-throughput screens [90,91] and from natural sources [92–94] , but the mechanisms by which these compounds act have yet to be elucidated. The main difficulty in targeting the T3SS is that nearly all of the inhibitors discovered through cell-based high-throughput screens lack potency and a clear mode of action. In an effort to address this problem, the ATPase YscN, which is essential for T3SS activity in Yersinia pestis, has been targeted [95] . In total, 20,000 compounds were initially screened in silico, of which


Bacterial surface appendages as targets for novel antibacterial therapeutics 37 were assayed for ATPase inhibitory activity. Six of these compounds showed 50% inhibition or more of YscN at 40 μM, which was reflected in a reduction of Y. pestis toxicity [95] , validating the ATPases of T3SS as potential targets for antivirulence compounds. T4SS pili T4SS pili are an important virulence factor in many Gram-negative and Gram-positive bacteria. They are capable of secreting DNA and protein substrates and have important roles in bacterial conjugation and pathogenesis [96,97] . The T4SS from the plant pathogen A. tumefaciens is the most widely studied due to its ability to transform plant cells through its virulence T4SS (Figure 1E) [98] . The virulence T4SS consists of 12 proteins: VirB1–11 and VirD4. VirB6–10 make up the translocation complex that spans both membranes [99–101] . VirB4, VirB11 and VirD4 are ATPases that provide the energy required to secrete substrates through the T4SS [102] . VirB2 and VirB5 make up the pilus rod and tip, respectively [103] . The function of VirB3 is not fully understood, but it is known to be associated with the inner membrane [104] . ●●Small molecule inhibitors of T4SS

Mutagenesis studies have shown that VirB8 dimerization (Figure 6B) is crucial for the formation of a functional T4SS in Brucella suis [105] . This dimerization event has been used as a target

N OH

OH

Review

for chemical inhibition and a library of approximately 30,000 compounds has been screened to identify 48 compounds that specifically target VirB8 dimerization in Brucella abortis [106] . One of these compounds, B81–2 (Figure 6A) was shown to affect VirB8 dimerization and reduce the intracellular growth of B. abortus in macrophages, while demonstrating negligible toxicity in mammalian cell lines [106] . Using a combination of x-ray crystallography and in silico docking studies, the binding site of another active compound, B81–1 (Figure 6A), was identified (Figure 6B & C) [6] . B81–1 bound in a deep groove opposite to the dimerisation interface of VirB8, making hydrogen bonds with K182 and hydrophobic interactions with W198 (Figure 6C) . An in silico docking approach predicted the same binding site for B81–2 and other compounds, which had been identified as VirB8 dimerization inhibitors [6] . The docking also provided structural information for structure–activity relationship studies to be carried out, which largely supported the proposed binding model. Proteins with mutations made at the putative binding site showed increased resistance to both B81–1 and B81–2, supporting the structural data [6] . Conclusion & future perspective The growing prevalence of antibiotic-resistant bacteria, coupled with the lack of new antibiotics in the antibiotic development pipeline, poses a serious threat to the healthcare system

NH

B81-1

O N N H B81-2

O O2N

Figure 6. Inhibitors of VirB8 dimerization. (A) Chemical structures of inhibitors B81–1 and B81–2. (B) X-ray crystal structure (PDB ID 4AKY) of the VirB8 dimer (shown in ribbons in brown) in complex with inhibitor B81–1 (shown in sticks in yellow). (C) Close-up of inhibitor B81–1 bound to VirB8. Residues E115 and K182 are shown as sticks and polar contacts shown in yellow dashes (from PDB ID 4AKY).



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Review Steadman, Lo, Waksman & Remaut and signals a pressing need for a new generation of antimicrobial agents. Over recent years, there has been an increasing focus in antimicrobial drug research on the discovery of anti-infective agents that selectively disrupt pathways that mediate virulence without affecting bacterial growth, although such therapies await validation in the clinic. Pili are crucial virulent determinants of many bacterial pathogens in establishing infection, but are dispensable for growth, thus making them ideal targets for chemical intervention. As discussed, the development of small molecule compounds that interfere with biogenesis and function of pili as antivirulence

agents has gained traction in the last decade. The promising classes of CU pili inhibitors discovered through structure-guided searches highlight the need to better understand the structural build-up and molecular processes that drive the biogenesis of these nano-machineries, as this is key to effective development of pili inhibitors. It is anticipated that in the coming years, the increasing wealth of knowledge on the structural, functional and molecular mechanisms of the assembly of other pili nano-machineries will further fuel the discovery of novel targets for chemical inhibition and aid the design of new antivirulence therapies. For example, it may

EXECUTIVE SUMMARY Antiobiotic resistance ●●

The increasing occurrence of infections associated with multidrug-resistant bacteria presents a serious threat to public health. Development of new classes of antibiotics is lagging behind.

●●

The focus of recent antimicrobial drug research has included more emphasis on discovering anti-infective agents that selectively disrupt dispensable pathways that mediate virulence without affecting bacteria growth.

Surface-associated virulence factors of Gram-negative bacteria ●●

Although dispensable for growth, the essentiality of Gram-negative bacteria pili and their secretion pathways in mediating virulence renders them key targets for chemical intervention.

Chaperone-usher pathway-assembled pili ●●

Chaperone-usher pili serve as adherence and colonization factors primarily of the gastrointestinal and urinary tracts.

●●

Receptor analogs have been developed, which inhibit binding of the adhesin FimH.

●●

Pilus polmerization inhibitors have been developed, which prevent the incorporation of pilus subunits into growing pilus rods by binding to the pilin domain of FimH and preventing donor strand exchange.

●●

A range of molecules, termed pilicides, have been developed, which disrupt chaperone–subunit interactions and prevent biofilm formation.

Curli ●●

Curli are the main protein component of the extracellular matrix in Escherichia coli pellicle biofilms, as well as other bacterial cells.

●●

A range of compounds have been developed that prevent CsgA subunit polymerization, leading to a reduction in the number of intracellular biofilm communities in a mouse model.

Type III secretion needle ●●

Type III secretion needles are responsible for the injection of bacterial proteins into cells and the assembly of flagella.

●●

Many diverse classes of compounds have been investigated as inhibitors of Type III secretion system, but the lack of a clear mode of action for these compounds has prevented any major developments.

Type IV secretion system pili ●●

Type IV secretion system pili secrete DNA and protein substrates and have important roles in bacterial conjugation and pathogenesis.

●●

VirB8 dimerization inhibitors have been developed, which inhibit the intracellular growth of Brucella abortis in macrophages.

896



Bacterial surface appendages as targets for novel antibacterial therapeutics also be possible to target enzymes such as DsbA, which has been shown to introduce essential disulfide bonds into several key proteins that are crucial for the assembly of extracellular bacterial virulence factors [107,108] . To achieve major advancements, a concerted effort from diverse fields, including microbiology, biochemistry, structural biology, medicinal chemistry and pharmaceutical science, is pivotal. We envisage that in a decade from now, antivirulence drugs could potentially be coadministered with available antibiotics to decrease virulence and promote bacterial clearance. References 1

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Financial & competing interests disclosure A Lo and H Remaut acknowledge financial support by Flanders Institute for Biotechnology (VIB) through grant PRJ9 and by the Fonds Wetenschappelijk OnderzoekVlaanderen (FWO) through Odysseus grant G.0902.09. This work was funded in part by MRC grant 018434 to G Waksman. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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