Curr Microbiol (2014) 68:558–567 DOI 10.1007/s00284-013-0507-2
Comparative Genomic and Proteomic Anatomy of Mycobacterium Ubiquitous Esx Family Proteins: Implications in Pathogenicity and Virulence Wanyan Deng • Xiaohong Xiang • Jianping Xie
Received: 15 September 2013 / Accepted: 29 October 2013 / Published online: 22 December 2013 Ó Springer Science+Business Media New York 2013
Abstract Secreted proteins are among the most important molecules involved in host—pathogen interaction of Mycobacterium tuberculosis, the etiological agent of human tuberculosis (TB). M. tuberculosis encodes five types of VII secretion systems (ESX-1 to ESX-5) responsible for the exportation of many proteins. This system mediated substrates including members of the Esx family implicated in tuberculosis pathogenesis and survival within host cells. However, the distribution and evolution of this family remain elusive. To explore the evolution and distribution of Esx family proteins, we analyzed all available Mycobacteria genomes. Interestingly, amino mutations among M. tuberculosis esx family proteins may relate to their functions. We further analyzed the differences between pathogenic Mycobacteria, the attenuated Mycobacteria and non-pathogenic Mycobacteria. The stability, the globular domains and the phosphorylation of serine/ threonine residues of M. tuberculosis esx proteins with their homologies among other Mycoabcteria were analyzed. Our comparative genomic and proteomic analysis found that the change of stability, gain or loss of globular domains and phosphorylation of serine/threonine might be Electronic supplementary material The online version of this article (doi:10.1007/s00284-013-0507-2) contains supplementary material, which is available to authorized users. W. Deng X. Xiang J. Xie (&) Institute of Modern Biopharmaceuticals, State Key Laboratory Breeding Base of Eco-Enviroment and Bio-Resource of the Three Gorges Area, Key Laboratory of Ministry of Education Eco-Environment of the Three Gorges Reservoir Region, School of Life Sciences, Southwest University, Beibei 400715, Chongqing, China e-mail: [email protected]; [email protected] W. Deng e-mail: [email protected]
123
responsible for the difference between the pathogenesis and virulence of the esx proteins and its homolog widespread among Mycobacteria and related species, which may provide clues for novel anti-tuberculosis drug targets.
Introduction Mycobacterium tuberculosis, the primary causative agent of human tuberculosis and one of the oldest pathogens known to man, remains scourge to global health, with an estimated 9.4 million new infection and over 1.3 million tuberculosisrelated deaths annually [1, 2]. Multi-genome analysis predicts that the WXG-100 proteins are widespread among G?C-rich gram-positive bacteria, but absent among gramnegative bacteria [3], presumably to be secreted via ESAT-6 secretion systems (ESX-1-ESX-5) [4], or more recently designated type VII secretion systems [5]. The core components of type VII secretion systems is the esx-1 locus, a highly conserved locus with several copies in the genomes of mycobacteria [6, 7] as well as more distantly related organisms, such as Streptomyces aureus, Listeria monocytogenes, Bacillus cereus, B. subtilis, and Carnobacterium maltaromaticum [6, 8, 9]. Figure 1 showed the the genomic organization of esx-1 locus among different strains. Five of the 11 ESX loci (ESX-1 to ESX-5) within the M. tuberculosis genome appear to encode members of the recent identified type VII secretion systems (T7SS). T7SS can export a number of proteins, including Esx protein complexes and PE/PPE proteins. ESX-1 and ESX-5 are thought to play a role in mycobacterial virulence, granuloma formation, cell-tocell spread of the Mycobacteria, and escape from the arrested phagosomes [5, 10–13]. ESX-1 was reported to be governed by multiple regulators, such as the DNA binding transcription factor EspR(Rv3849), the two-component system
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
559
Fig. 1 The genomic organization of esx gene cluster in different G?C-rich Actinobacteria and Firmicutes
regulator PhoP, and the serine protease MycP1 [14–17]. In contrast, ESX-3, which encodes the proteins EsxG and EsxH, is required for optimal growth of M. tuberculosis and has been associated with essential processes such as iron and zinc acquisition [18–20]. M. tuberculosis ESX-3 was regulated by the iron-dependent transcriptional repressor IdeR as well as the zinc-uptake regulator Zur [21, 22]. Mycobacterium tuberculosis genome encodes 23 Esx proteins, EsxA–W, characterized by their small size (*100 residues), the presence of a central WXG motif, and their organization in pairs within the genome[3] (Fig. 2) and their frequent physically interacting products, such as EsxA/EsxB (ESAT-6/CFP-10, Rv3875/Rv3874) [23, 24]. The genes encoding the Esx pairs EsxA/EsxB and EsxG/ EsxH formed small operons, which is presumably the predominant organizational format of all M. tuberculosis Esx genome pairs [23, 24]. Studies have also shown that the protein products of several Esx pairs, including EsxA/ EsxB, EsxG/EsxH, EsxR/EsxS (Rv3019c/Rv3020c), and EsxO/EsxP (Rv2346c/Rv2347c) form tight complexes, which are likely to be the functional form of these proteins [25–30]. EsxRS, a homolog of EsxGH of the ESX-3 gene cluster consists of two four-helix bundles resulting from the 3D domain swapping of the C-terminal domain of EsxS, the CFP-10 homolog. The four-helix bundles at the extremities of the complex have a similar architecture to the structure of ESAT-6.CFP-10 (EsxAB) of ESX-1, but in EsxRS a hinge loop linking the alpha-helical domains of
EsxS undergoes a loop-to-helix transition that creates the domain swapped EsxRS tetramer [25]. These suggest that higher order ESX oligomers may increase the avidity of ESX binding to host receptor molecules or, alternatively, the conformational change that creates the domain swapped structure might be the basis of ESX complex dissociation to free ESAT-6 for its cytotoxic effect. Mycobacterium tuberculosis ESX systems are differentially regulated and serve diverse roles in infection. Core components of type VII secretion systems include FtsK/ SpoIIIE-like ATPases (Rv3870 and Rv3871 in ESX-1), transmembrane proteins (Rv3877 in ESX-1), and a subtilisin-like serine protease (MycP1 in ESX-1) [5, 6]. The best characterized of these systems is ESX-1 (rv3868/eccA to rv3883c/mycP), which is known to secrete the EsxA/EsxB protein complex, as well as at least seven other mycobacterial proteins, including EspA (Rv3616c), EspB (Rv3881c), EspC (Rv3615c), EspE (Rv3864), EspF (Rv3865), PE35 (Rv3872), and EspR (Rv3849) [5, 14, 31–34]. Two welldocumented esx family proteins CFP-10 and ESAT-6 have been shown to be coordinately regulated [23] and both proteins are secreted despite the lack of a classic signal sequence [35]. The protein secretion is an active process involving a membrane protein complex formed by the products of several flanking genes [6, 36–39]. Several other esx family members are also known to be secreted, including the products of esxG/esxH and esxR/esxS [40–42], which formed tight complexes similar to esxA and esxB [27, 30].
123
560
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
Fig. 2 Esx gene clusters are arranged in operons in the M. tuberculosis genome. a Schematic representation of the 11 operons with esx genes only. The intergenic distance between the genes is given in base pairs (bp). b The structure of esxA(Rv3875) and esxB(Rv3874) complex
Mycobacterium tuberculosis Esx family proteins EsxA [43–46] and esxB [38, 42, 47] are two dominant targets for T cells in the early phases of infection. In addition, some other members of esx family proteins such as Rv0288(EsxH) [42, 48], Rv1037c(EsxI), Rv1198(EsxL), Rv2346c(EsxO), Rv3619c(EsxV) and Rv179(EsxN) [40], and Rv3017c(EsxQ) and Rv3019c(EsxR) [48, 49] are reported to induce T cells responce. Given the importance of esx proteins in the pathogenic processes and virulence of M. tuberculosis, further analysis of the distribution, evolution and difference between different Mycobacteria might be worthwhile and provide novel clues for the pathogenesis and virulence of M. tuberculosis. Our comparative genomic and proteomic analysis indicate the presence of several differences of esx proteins such as the stability, the gain/lose global domains, and the phosphorylation of serine/threonine residues of esx proteins may influence the function of corresponding proteins in attenuated M. bovis BCG and M. tuberculosis and non-pathogenic M. smegmatis.
Materials and Methods Search for Corresponding Esx Genes of Mycobcteria All esx genes analyzed in our study, were based on search of the genes from gene database of National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/gene). FASTA sequences of the genes were used to search for corresponding genes in other Mycobacteria in the European
123
Nucleotide Archive sequence database (www.ebi.ac.uk/ ena). Furthermore, BLAST was used to find the differences between different Mycobacterium in terms of single nucleotide variations, insertions, and deletions in the esx genes. Computational Analysis of Esx Proteomes of Mycobacteria A list of Primary accession numbers in UniProtKB (Universal Protein Resource) of esx genes in M. tuberculosis H37Rv and the corresponding genes in other Mycobacteria was prepared. Vector align program was used to compare the amino acid sequences of these genes among Mycobacteria. Genes having substitutions, insertions or deletions in amino acid sequence were selected for further analysis. Prediction of Protein Stability Comparison of various physical and chemical parameters of proteins coded by the Esx genes in both strains was carried out via ProtParam tool from ExPASy portal (http:// web.expasy.org/protparam/). The computed parameters include the instability index, aliphatic index, and grand average of hydrophobicity (GRAVY). Protein Structure Prediction GlobPlot (http://globplot.embl.de/cgiDict.py) was used to predict globularity in the proteins with difference between M. tuberculosis and M. smegmatis.
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
561
Fig. 3 The esx family protein quantity among various Mycobacteria
Prediction of Phosphorylation Sites NetPhosBac (http://www.cbs.dtu.dk/services/NetPhosBac1.0/) was used to predict serine and threonine phosphorylation sites in the Esx proteins displaying difference between M. tuberculosis and M. smegmatis.
Result and Discusion The Distribution and Evolution of Esx Proteins Genome analyses suggest that a related secretion system is found in other Actinobacteria and in some Firmicutes [3]. The protein sequences of M. tuberculosis 23 Esx proteins were download from Tuberculist (http://tuberculist.epfl.ch/ index.html) except for esxM (there is no sequence information about this protein in M. tuberculosis H37Rv), and search the corresponding homologous genes of other Mycobacteria through blast in NCBI (http://www.ncbi.nlm. nih.gov/). Comparative genomic analysis found that esx proteins were largely limited to pathogenic Mycobacteria, such as M. tuberculosis, M. bovis, M. ulcerans, and M.marinum (Fig. 3). Evolutionary studies found that
various esx orthologues are deleted in avirulent mycobacterial strains, like M. tuberculosis H37Ra, M. bovis BCG, M. tuberculosis F11 Mycobacterium microti, another avirulent strain such as M. smegmatis and one that is attenuated like M. bovis BCG carries deletions of almost all these genes except esxE(Rv3904c), esxU(Rv3445c) and esxV(Rv3619c), and M. tuberculosis F11 carries only esxC (Supplementary Table 1). The distribution of esx proteins suggests their unique roles in the virulence, pathogenesis, and persistence of Mycobacteria. Analysis of the genome sequence of M. smegmatis revealed three ESAT-6 (esx) gene cluster regions (regions 4, 1, and 3), with absence of regions 2 and 5 [6]. Regions 2 and 5 might be deleted from the genome of this organism, which might be occurred after the divergence of M. smegmatis, as evidenced by the last two duplicates of the ESAT-6 (esx) gene cluster evolution [6]. This point is supported by comparative genomics analyses of the genomes of closely-related fast-growing Mycobacteria such as M. flavescens, M. vanbaalenii, M. sp. MCS, and M. sp. JLS in which ESAT-6 (esx) gene cluster. Regions 2 and 5 were also found to be absent, as well as M. sp KMS in which ESAT-6 (esx) gene cluster region 2 was present, but region 5 was absent (Supplementary Table 2). The fact that the genome of
123
562
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
M. smegmatis is *1.7 times larger than that of M. tuberculosis [50], and thus does not display the same reductive properties to that observed in the genome of, for example, M. leprae was confirmed to have lost ESAT-6 (esx) gene cluster region 2 and 4 by deletion [51]. Strikingly, of the 11 pairs of CFP-10/ESAT-6 family proteins found in M. tuberculosis, only orthologues of CFP-10 and ESAT-6 are conserved in M. leprae (ML0050c and ML0049c, respectively), which further emphasizes their importance in the lifecycle of Mycobacterial pathogens [30]. Comparative Genomics Yield Insights into the Extent of Sequence Variation To further examine the relationships between, and evolutionary history of, the members of the esx family, to determine the extent of esx sequence similarity and variation, orthologues in the fully sequenced and annotated genomes of Mycobacteria were analyzed by comparative genomics. During this analysis, a complete investigation of the presence and absence of genes, gene sizes, deletions, and conservative substitutions was performed. We found that the difference of some of esx family members among Mycobacteria originate from the deletion of C terminal sequences. The deletion of about 45 amino in its C terminal of M. bovis esxF when compare with M. tuberculosis esxF. Moreover, we observed that the extension of M. bovis esxC in its C-terminal sequence whereas approximately deletion of 41 amino in C-terminal of M. tuberculosis esxC. The amino sequence of some members of M. tuberculosis esx family proteins are highly homologous sequence. There are several amino mutations between esxH and esxR or esxG and esxS (Supplementary Table 3). Besides, there is only one or two mutation between some members of esx family protein, such as esxJ and esxK, esxN and esxO, esxI and esxL, and esxP and esxW (Supplementary Table 3). The redundancy between esx family proteins might imply their functional overlapping. Analyses of Aliphatic Index and GRAVY The aliphatic index of a protein signifies the relative volume occupied by aliphatic side chains. Aliphatic hydrophobicity increases with increase in temperature and hence it is a positive factor for increase in thermal stability of globular proteins [52]. GRAVY is an indication of protein solubility where a positive value correlates with hydrophobicity and negative with hydrophilicity. More hydrophilic the protein greater will be the extent of hydrogen boding with water molecules and higher will be the solubility. The analysis of GRAVY by ProtParam (Fig. 4) shows that the esx protein esxG shared by M. tuberculsosis and M. smagmatis is hydrophobicity, while some esx
123
proteins such as esxH, esxR,esxB,esxT, and esxU are hydrophilicity. Among these five members, the GRAVY value of esxB in M. tuberculosis H37Rv (-0.703) is much lower than its homolog MSMEG_0065 in M. smegmatis (-0.063). Interestingly, some members of esx proteins are hydrophobic in M. tuberculosis H37Rv (esxE and esxA), while their homologous proteins in M. smegmatis (MSMEG_1871 and MSMEG_0066) are hydrophilic. The GRAVY value of M. tuberculosis H37Rv esxE is 0.004, while the homolog MSMEG_1871 is -0.823. On the contrary, the GRAVY value of well-documented virulence factor esxA in M. tuberculosis H37Rv is negative with hydrophilicity, but MSMEG_0066 in M. smagmatis is positive value correlates with hydrophobicity. Together, these data suggest that the variations in protein sequence of esx proteins between M. tuberculosis H37Rv and M. smagmatis manifest in changes in GRAVY values, resulting in the thermal stability of proteins, which might underlie the differential virulence characteristic of the shared esxA(ESAT6) and esxB(CFP10) between M. tuberculosis and non-pathogenic pathogen M. smagmatis, while as virulence factor in the former. Analyses of Instability Index The instability index is indicative of the stability of the protein under in vitro conditions. Instability index [40 is a sign of unstable protein and \40 is an indication of stable protein. The in vivo instability of proteins is possibly determined by the order of certain amino acids in its sequence, some dipeptides occurring differently in unstable and stable proteins. Presence of such dipeptides facilitates the analysis of protein stability [53]. Examples of instability index analyses of esx family proteins in M. tuberculosis H37Rv and their homologs in M. smegmatis are shown (Fig. 5). We found that some members of esx proteins are stable (esxE/Rv3904c/MSMEG_1871) or unstable (esxA/ Rv3875, MSMEG_0066, esxT/Rv3444c, MSMEG_1539 and esxU/Rv1538, MSMEG_1538) in both M. tuberculosis H37Rv and M. smegmatis. A perusal of the same clearly reveals that a few of the esx proteins are stable in H37Rv (esxG/Rv0287, esxH/Rv0288, esxR/Rv3019c, and esxB/ Rv3874), but the corresponding protein in M. smegmatis (MSMEG_0629, MSMEG-0621, and MSMEG_0065) are unstable. These differences between pathogenic M. tuberculosis H37Rv and non-pathogenic M. smegmatis might partly be responsible for the cytotoxic effect of esx proteins in M. tuberculosis H37Rv. Analyses of Globular Domains of Esx Proteins Globular domains in protein molecules confer special functions to a protein. Thus, addition or deletion of globular
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
563
Fig. 4 Protein sequence variations between M. tuberculosis and M. smegmatis manifest in changes in GRAVY values
Fig. 5 Variations in protein sequence variations between M. tuberculosis H37Rv and M. smegmatis affect the stability of esx proteins
domains in a protein might leads to the loss or gain of function. In M. tuberculosis H37Rv, esxF(Rv3905c) has two globular domains, the deletion of a stretch of amino acids in C terminal of M. bovis esxF(Mb3935c) results in deletion of the globular domain, probably leading to a loss of function in M. bovis (Fig. 6a). Similarly, the extension and variation of C terminal of M. bovis esxC, leading to the deficiency of one globular domains (Fig. 6b). In addition, the substitutions of amino between M. tuberculosis esxB(Rv3874) and M. smegmatis esxB(MSMEG_0065) are responsible for the lose of globular domains in N and C terminal of M. smegmatis esxB(MSMEG_0065). Finally, we found that the lose of globular domains M. tuberculosis esxA(Rv3875) and its homolog M. smegmatis esxA(MSMEG_0066) in C and N terminal might be originated in mutation of some special amino in its N and C terminal, respectively (Supplementary Table 4). These suggest that the deletion of globular domains
of esx proteins in M. bovis and M. smegmatis might result in a loss of their function. Analyses of Protein Phosphorylation Sites in Esx Proteins The role of phosphorylation of serine/threonine residues in regulating cell signaling and host–pathogen interaction is well documented. Thus, differences in these sites, as a consequence of variations in nucleotide sequences between M. tuberculosis H37Rv and M. smegmatis, will highlight the likely differences in protein–protein interactions and binding of different domains consequently leading to altered downstream effects. Analysis of potential serine/threonine phosphorylation sites in two typical esx family protein ESAT6 and CFP10, which all exit in M. tuberculosis and M. smagmatis with high homology, revealed a number of
123
564
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
Fig. 6 Gain or loss of globular domain is a function of variation
123
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
565
Fig. 7 Nucleotide variations among Mycobacteria result in gain or loss of serine/threonine phosphorylation sites
differences between these two strains. Gain or loss in serine/ threonine phosphorylation sites mostly due to base substitution was observed in esxA and esxB (Fig. 7). Interestingly, gain of serine/threonine phosphorylation sites was noted in M. tuberculosis esxF(Rv3905c) and esxC(Rv3890c) due to C-terminal extension and variation in amino sequence, respectively. The results presented earlier clearly highlight the important consequence of amino sequence changes between Mycobacteria in esx genes. Such changes manifest in major structural and physicochemical alterations in the corresponding proteomes thereby likely impacting their ability to bring about protein–protein interactions that are very important for host pathogen cross talks and consequent virulence and pathogenesis.
Conclusion In summary, through a comparative genomic analysis, we identified several key changes in the amino sequence between esx homologies of Mycobacteria. These changes mainly included duplications and deletions, which altered the ORFs of the gene. These alterations in the genes quite often resulted in major physicochemical changes in the
encoded proteins. For example, the instability values of M. tuberculosis esxB is lower than its homolog in M. smegmatis as some substitution of few amino sequence of this protein. In addition, the gain or loss global domains may affect the function of the protein. The duplication, deletion, and variation of C terminal amino sequences are responsible for the loss of global domains of the proteins. As shown in our data, M. bovis esxF(Mb3935c) loss the globular domain as the deletion of a stretch of amino acids in its C terminal when compared with M. tuberculosis H37Rv esxF(Rv3905c). In addition, the extension and variation of C terminal of M. bovis esxC also lead to the deficiency of globular domains. Therefore, search of the literature established correlation of many such changes to in vivo function including virulence and pathogenesis. Further biochemical and functional studies are required to establish the role of such changes in amino acid sequence in the attenuation of M. tubeculosis H37Ra, M. bovis BCG, and non-pathogenic M. smegmatis. Such studies along with the combination of clinical data will improve our understanding of the mechanisms of virulence, pathogenesis, and latency of tuberculosis caused by M. tuberculosis and inform the design of better new interventions.
123
566
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
Acknowledgments This work was funded by National Natural Science Foundation (Grant Nos. 81071316, 81271882, 81371), National Megaprojects for Key Infectious Diseases (No. 2008ZX10003-006), New Century Excellent Talents in Universities (NCET-11-0703), Excellent PhD thesis fellowship of southwest university (Grant Nos. kb2009010 and ky2011003), the Fundamental Research Funds for the Central Universities (Grant Nos. XDJK2012D011, XDJK2013D003), Natural Science Foundation Project of CQ CSTC (Grant Nos. CSTC, 2010BB5002). The Chongqing Municipal Committee of Education for postgraduates excellence program (No. YJG123104, No.), The Undergraduates Teaching Reform Program (No. 2011JY052).
References 1. Oxlade O, Schwartzman K, Behr MA, Benedetti A, Pai M, Heymann J, Menzies D (2009) Global tuberculosis trends: a reflection of changes in tuberculosis control or in population health? Int J Tuberc Lung Dis 13:1238–1246 2. Vashishtha VM (2009) WHO global tuberculosis control report 2009: tuberculosis elimination is a distant dream. Indian Pediatr 46:401–402 3. Pallen MJ (2002) The ESAT-6/WXG100 superfamily—and a new Gram-positive secretion system? Trends Microbiol 10:209–212 4. Brodin P, Rosenkrands I, Andersen P, Cole ST, Brosch R (2004) ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol 12:500–508 5. Abdallah AM, Gey van Pittius NC, Champion PA, Cox J, Luirink J, Vandenbroucke-Grauls CM, Appelmelk BJ, Bitter W (2007) Type VII secretion—mycobacteria show the way. Nat Rev Microbiol 5:883–891 6. Gey Van Pittius NC, Gamieldien J, Hide W, Brown GD, Siezen RJ, Beyers AD (2001) The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G?C Gram-positive bacteria. Genome Biol 2:RESEARCH0044 7. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 8. Akpe San Roman S, Facey PD, Fernandez-Martinez L, Rodriguez C, Vallin C, Del Sol R, Dyson P (2010) A heterodimer of EsxA and EsxB is involved in sporulation and is secreted by a type VII secretion system in Streptomyces coelicolor. Microbiology 156:1719–1729 9. Stinear TP, Seemann T, Harrison PF, Jenkin GA, Davies JK, Johnson PD, Abdellah Z, Arrowsmith C, Chillingworth T, Churcher C, Clarke K, Cronin A, Davis P, Goodhead I, Holroyd N, Jagels K, Lord A, Moule S, Mungall K, Norbertczak H, Quail MA, Rabbinowitsch E, Walker D, White B, Whitehead S, Small PL, Brosch R, Ramakrishnan L, Fischbach MA, Parkhill J, Cole ST (2008) Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Res 18:729–741 10. Abdallah AM, Savage ND, van Zon M, Wilson L, Vandenbroucke-Grauls CM, van der Wel NN, Ottenhoff TH, Bitter W (2008) The ESX-5 secretion system of Mycobacterium marinum modulates the macrophage response. J Immunol 181:7166–7175 11. Davis JM, Ramakrishnan L (2009) The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell 136:37–49 12. van der Wel N, Hava D, Houben D, Fluitsma D, van Zon M, Pierson J, Brenner M, Peters PJ (2007) M. tuberculosis and M.
123
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129:1287–1298 Volkman HE, Clay H, Beery D, Chang JC, Sherman DR, Ramakrishnan L (2004) Tuberculous granuloma formation is enhanced by a Mycobacterium virulence determinant. PLoS Biol 2:e367 Raghavan S, Manzanillo P, Chan K, Dovey C, Cox JS (2008) Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature 454:717–721 Frigui W, Bottai D, Majlessi L, Monot M, Josselin E, Brodin P, Garnier T, Gicquel B, Martin C, Leclerc C, Cole ST, Brosch R (2008) Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP. PLoS Pathog 4:e33 Gonzalo-Asensio J, Mostowy S, Harders-Westerveen J, Huygen K, Hernandez-Pando R, Thole J, Behr M, Gicquel B, Martin C (2008) PhoP: a missing piece in the intricate puzzle of Mycobacterium tuberculosis virulence. PLoS ONE 3:e3496 Ohol YM, Goetz DH, Chan K, Shiloh MU, Craik CS, Cox JS (2010) Mycobacterium tuberculosis MycP1 protease plays a dual role in regulation of ESX-1 secretion and virulence. Cell Host Microbe 7:210–220 Serafini A, Boldrin F, Palu G, Manganelli R (2009) Characterization of a Mycobacterium tuberculosis ESX-3 conditional mutant: essentiality and rescue by iron and zinc. J Bacteriol 191:6340–6344 Siegrist MS, Unnikrishnan M, McConnell MJ, Borowsky M, Cheng TY, Siddiqi N, Fortune SM, Moody DB, Rubin EJ (2009) Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition. Proc Natl Acad Sci USA 106:18792–18797 Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84 Maciag A, Dainese E, Rodriguez GM, Milano A, Provvedi R, Pasca MR, Smith I, Palu G, Riccardi G, Manganelli R (2007) Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon. J Bacteriol 189:730–740 Rodriguez GM, Voskuil MI, Gold B, Schoolnik GK, Smith I (2002) ideR, An essential gene in Mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response. Infect Immun 70:3371–3381 Berthet FX, Rasmussen PB, Rosenkrands I, Andersen P, Gicquel B (1998) A Mycobacterium tuberculosis operon encoding ESAT6 and a novel low-molecular-mass culture filtrate protein (CFP10). Microbiology 144:3195–3203 Okkels LM, Andersen P (2004) Protein-protein interactions of proteins from the ESAT-6 family of Mycobacterium tuberculosis. J Bacteriol 186:2487–2491 Arbing MA, Kaufmann M, Phan T, Chan S, Cascio D, Eisenberg D (2010) The crystal structure of the Mycobacterium tuberculosis Rv3019c-Rv3020c ESX complex reveals a domain-swapped heterotetramer. Protein Sci 19:1692–1703 Lightbody KL, Ilghari D, Waters LC, Carey G, Bailey MA, Williamson RA, Renshaw PS, Carr MD (2008) Molecular features governing the stability and specificity of functional complex formation by Mycobacterium tuberculosis CFP-10/ESAT-6 family proteins. J Biol Chem 283:17681–17690 Lightbody KL, Renshaw PS, Collins ML, Wright RL, Hunt DM, Gordon SV, Hewinson RG, Buxton RS, Williamson RA, Carr MD (2004) Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility. FEMS Microbiol Lett 238:255–262 Meher AK, Bal NC, Chary KV, Arora A (2006) Mycobacterium tuberculosis H37Rv ESAT-6-CFP-10 complex formation confers thermodynamic and biochemical stability. FEBS J 273:1445–1462 Renshaw PS, Lightbody KL, Veverka V, Muskett FW, Kelly G, Frenkiel TA, Gordon SV, Hewinson RG, Burke B, Norman J,
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
Williamson RA, Carr MD (2005) Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6. EMBO J 24:2491–2498 Renshaw PS, Panagiotidou P, Whelan A, Gordon SV, Hewinson RG, Williamson RA, Carr MD (2002) Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6*CFP-10 complex. Implications for pathogenesis and virulence. J Biol Chem 277:21598–21603 Bitter W, Houben EN, Bottai D, Brodin P, Brown EJ, Cox JS, Derbyshire K, Fortune SM, Gao LY, Liu J, Gey van Pittius NC, Pym AS, Rubin EJ, Sherman DR, Cole ST, Brosch R (2009) Systematic genetic nomenclature for type VII secretion systems. PLoS Pathog 5:e1000507 Fortune SM, Jaeger A, Sarracino DA, Chase MR, Sassetti CM, Sherman DR, Bloom BR, Rubin EJ (2005) Mutually dependent secretion of proteins required for mycobacterial virulence. Proc Natl Acad Sci USA 102:10676–10681 McLaughlin B, Chon JS, MacGurn JA, Carlsson F, Cheng TL, Cox JS, Brown EJ (2007) A mycobacterium ESX-1-secreted virulence factor with unique requirements for export. PLoS Pathog 3:e105 Xu J, Laine O, Masciocchi M, Manoranjan J, Smith J, Du SJ, Edwards N, Zhu X, Fenselau C, Gao LY (2007) A unique Mycobacterium ESX-1 protein co-secretes with CFP-10/ESAT-6 and is necessary for inhibiting phagosome maturation. Mol Microbiol 66:787–800 Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, Griffiths KE, Marchal G, Leclerc C, Cole ST (2003) Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat Med 9:533–539 Hsu T, Hingley-Wilson SM, Chen B, Chen M, Dai AZ, Morin PM, Marks CB, Padiyar J, Goulding C, Gingery M, Eisenberg D, Russell RG, Derrick SC, Collins FM, Morris SL, King CH, Jacobs WR Jr (2003) The primary mechanism of attenuation of bacillus Calmette–Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc Natl Acad Sci USA 100:12420–12425 Sassetti CM, Rubin EJ (2003) Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci USA 100:12989–12994 Stanley SA, Raghavan S, Hwang WW, Cox JS (2003) Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc Natl Acad Sci USA 100:13001–13006 Guinn KM, Hickey MJ, Mathur SK, Zakel KL, Grotzke JE, Lewinsohn DM, Smith S, Sherman DR (2004) Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol Microbiol 51:359–370 Alderson MR, Bement T, Day CH, Zhu L, Molesh D, Skeiky YA, Coler R, Lewinsohn DM, Reed SG, Dillon DC (2000) Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(?) T cells. J Exp Med 191:551–560
567
41. Rosenkrands I, Weldingh K, Jacobsen S, Hansen CV, Florio W, Gianetri I, Andersen P (2000) Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection. Electrophoresis 21:935–948 42. Skjot RL, Oettinger T, Rosenkrands I, Ravn P, Brock I, Jacobsen S, Andersen P (2000) Comparative evaluation of low-molecularmass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens. Infect Immun 68:214–220 43. Andersen P, Andersen AB, Sorensen AL, Nagai S (1995) Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J Immunol 154:3359–3372 44. Pollock JM, Andersen P (1997) Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle. Infect Immun 65:2587–2592 45. Ravn P, Demissie A, Eguale T, Wondwosson H, Lein D, Amoudy HA, Mustafa AS, Jensen AK, Holm A, Rosenkrands I, Oftung F, Olobo J, von Reyn F, Andersen P (1999) Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis. J Infect Dis 179:637–645 46. Elhay MJ, Oettinger T, Andersen P (1998) Delayed-type hypersensitivity responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the guinea pig. Infect Immun 66:3454–3456 47. van Pinxteren LA, Ravn P, Agger EM, Pollock J, Andersen P (2000) Diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10. Clin Diagn Lab Immunol 7:155–160 48. Majlessi L, Rojas MJ, Brodin P, Leclerc C (2003) CD8?-T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex class I-restricted epitope shared by proteins of the ESAT-6 family. Infect Immun 71:7173–7177 49. Skjot RL, Brock I, Arend SM, Munk ME, Theisen M, Ottenhoff TH, Andersen P (2002) Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat-6 gene family. Infect Immun 70:5446–5453 50. Reyrat JM, Kahn D (2001) Mycobacterium smegmatis: an absurd model for tuberculosis? Trends Microbiol 9:472–474 51. Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson NR, Wheeler PR, Honore N, Garnier T, Churcher C, Harris D, Mungall K, Basham D, Brown D, Chillingworth T, Connor R, Davies RM, Devlin K, Duthoy S, Feltwell T, Fraser A, Hamlin N, Holroyd S, Hornsby T, Jagels K, Lacroix C, Maclean J, Moule S, Murphy L, Oliver K, Quail MA, Rajandream MA, Rutherford KM, Rutter S, Seeger K, Simon S, Simmonds M, Skelton J, Squares R, Squares S, Stevens K, Taylor K, Whitehead S, Woodward JR, Barrell BG (2001) Massive gene decay in the leprosy bacillus. Nature 409:1007–1011 52. Ikai A (1980) Thermostability and aliphatic index of globular proteins. J Biochem 88:1895–1898 53. Guruprasad K, Reddy BV, Pandit MW (1990) Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng 4:155–161
123
Comparative Genomic and Proteomic Anatomy of Mycobacterium Ubiquitous Esx Family Proteins: Implications in Pathogenicity and Virulence Wanyan Deng • Xiaohong Xiang • Jianping Xie
Received: 15 September 2013 / Accepted: 29 October 2013 / Published online: 22 December 2013 Ó Springer Science+Business Media New York 2013
Abstract Secreted proteins are among the most important molecules involved in host—pathogen interaction of Mycobacterium tuberculosis, the etiological agent of human tuberculosis (TB). M. tuberculosis encodes five types of VII secretion systems (ESX-1 to ESX-5) responsible for the exportation of many proteins. This system mediated substrates including members of the Esx family implicated in tuberculosis pathogenesis and survival within host cells. However, the distribution and evolution of this family remain elusive. To explore the evolution and distribution of Esx family proteins, we analyzed all available Mycobacteria genomes. Interestingly, amino mutations among M. tuberculosis esx family proteins may relate to their functions. We further analyzed the differences between pathogenic Mycobacteria, the attenuated Mycobacteria and non-pathogenic Mycobacteria. The stability, the globular domains and the phosphorylation of serine/ threonine residues of M. tuberculosis esx proteins with their homologies among other Mycoabcteria were analyzed. Our comparative genomic and proteomic analysis found that the change of stability, gain or loss of globular domains and phosphorylation of serine/threonine might be Electronic supplementary material The online version of this article (doi:10.1007/s00284-013-0507-2) contains supplementary material, which is available to authorized users. W. Deng X. Xiang J. Xie (&) Institute of Modern Biopharmaceuticals, State Key Laboratory Breeding Base of Eco-Enviroment and Bio-Resource of the Three Gorges Area, Key Laboratory of Ministry of Education Eco-Environment of the Three Gorges Reservoir Region, School of Life Sciences, Southwest University, Beibei 400715, Chongqing, China e-mail: [email protected]; [email protected] W. Deng e-mail: [email protected]
123
responsible for the difference between the pathogenesis and virulence of the esx proteins and its homolog widespread among Mycobacteria and related species, which may provide clues for novel anti-tuberculosis drug targets.
Introduction Mycobacterium tuberculosis, the primary causative agent of human tuberculosis and one of the oldest pathogens known to man, remains scourge to global health, with an estimated 9.4 million new infection and over 1.3 million tuberculosisrelated deaths annually [1, 2]. Multi-genome analysis predicts that the WXG-100 proteins are widespread among G?C-rich gram-positive bacteria, but absent among gramnegative bacteria [3], presumably to be secreted via ESAT-6 secretion systems (ESX-1-ESX-5) [4], or more recently designated type VII secretion systems [5]. The core components of type VII secretion systems is the esx-1 locus, a highly conserved locus with several copies in the genomes of mycobacteria [6, 7] as well as more distantly related organisms, such as Streptomyces aureus, Listeria monocytogenes, Bacillus cereus, B. subtilis, and Carnobacterium maltaromaticum [6, 8, 9]. Figure 1 showed the the genomic organization of esx-1 locus among different strains. Five of the 11 ESX loci (ESX-1 to ESX-5) within the M. tuberculosis genome appear to encode members of the recent identified type VII secretion systems (T7SS). T7SS can export a number of proteins, including Esx protein complexes and PE/PPE proteins. ESX-1 and ESX-5 are thought to play a role in mycobacterial virulence, granuloma formation, cell-tocell spread of the Mycobacteria, and escape from the arrested phagosomes [5, 10–13]. ESX-1 was reported to be governed by multiple regulators, such as the DNA binding transcription factor EspR(Rv3849), the two-component system
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
559
Fig. 1 The genomic organization of esx gene cluster in different G?C-rich Actinobacteria and Firmicutes
regulator PhoP, and the serine protease MycP1 [14–17]. In contrast, ESX-3, which encodes the proteins EsxG and EsxH, is required for optimal growth of M. tuberculosis and has been associated with essential processes such as iron and zinc acquisition [18–20]. M. tuberculosis ESX-3 was regulated by the iron-dependent transcriptional repressor IdeR as well as the zinc-uptake regulator Zur [21, 22]. Mycobacterium tuberculosis genome encodes 23 Esx proteins, EsxA–W, characterized by their small size (*100 residues), the presence of a central WXG motif, and their organization in pairs within the genome[3] (Fig. 2) and their frequent physically interacting products, such as EsxA/EsxB (ESAT-6/CFP-10, Rv3875/Rv3874) [23, 24]. The genes encoding the Esx pairs EsxA/EsxB and EsxG/ EsxH formed small operons, which is presumably the predominant organizational format of all M. tuberculosis Esx genome pairs [23, 24]. Studies have also shown that the protein products of several Esx pairs, including EsxA/ EsxB, EsxG/EsxH, EsxR/EsxS (Rv3019c/Rv3020c), and EsxO/EsxP (Rv2346c/Rv2347c) form tight complexes, which are likely to be the functional form of these proteins [25–30]. EsxRS, a homolog of EsxGH of the ESX-3 gene cluster consists of two four-helix bundles resulting from the 3D domain swapping of the C-terminal domain of EsxS, the CFP-10 homolog. The four-helix bundles at the extremities of the complex have a similar architecture to the structure of ESAT-6.CFP-10 (EsxAB) of ESX-1, but in EsxRS a hinge loop linking the alpha-helical domains of
EsxS undergoes a loop-to-helix transition that creates the domain swapped EsxRS tetramer [25]. These suggest that higher order ESX oligomers may increase the avidity of ESX binding to host receptor molecules or, alternatively, the conformational change that creates the domain swapped structure might be the basis of ESX complex dissociation to free ESAT-6 for its cytotoxic effect. Mycobacterium tuberculosis ESX systems are differentially regulated and serve diverse roles in infection. Core components of type VII secretion systems include FtsK/ SpoIIIE-like ATPases (Rv3870 and Rv3871 in ESX-1), transmembrane proteins (Rv3877 in ESX-1), and a subtilisin-like serine protease (MycP1 in ESX-1) [5, 6]. The best characterized of these systems is ESX-1 (rv3868/eccA to rv3883c/mycP), which is known to secrete the EsxA/EsxB protein complex, as well as at least seven other mycobacterial proteins, including EspA (Rv3616c), EspB (Rv3881c), EspC (Rv3615c), EspE (Rv3864), EspF (Rv3865), PE35 (Rv3872), and EspR (Rv3849) [5, 14, 31–34]. Two welldocumented esx family proteins CFP-10 and ESAT-6 have been shown to be coordinately regulated [23] and both proteins are secreted despite the lack of a classic signal sequence [35]. The protein secretion is an active process involving a membrane protein complex formed by the products of several flanking genes [6, 36–39]. Several other esx family members are also known to be secreted, including the products of esxG/esxH and esxR/esxS [40–42], which formed tight complexes similar to esxA and esxB [27, 30].
123
560
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
Fig. 2 Esx gene clusters are arranged in operons in the M. tuberculosis genome. a Schematic representation of the 11 operons with esx genes only. The intergenic distance between the genes is given in base pairs (bp). b The structure of esxA(Rv3875) and esxB(Rv3874) complex
Mycobacterium tuberculosis Esx family proteins EsxA [43–46] and esxB [38, 42, 47] are two dominant targets for T cells in the early phases of infection. In addition, some other members of esx family proteins such as Rv0288(EsxH) [42, 48], Rv1037c(EsxI), Rv1198(EsxL), Rv2346c(EsxO), Rv3619c(EsxV) and Rv179(EsxN) [40], and Rv3017c(EsxQ) and Rv3019c(EsxR) [48, 49] are reported to induce T cells responce. Given the importance of esx proteins in the pathogenic processes and virulence of M. tuberculosis, further analysis of the distribution, evolution and difference between different Mycobacteria might be worthwhile and provide novel clues for the pathogenesis and virulence of M. tuberculosis. Our comparative genomic and proteomic analysis indicate the presence of several differences of esx proteins such as the stability, the gain/lose global domains, and the phosphorylation of serine/threonine residues of esx proteins may influence the function of corresponding proteins in attenuated M. bovis BCG and M. tuberculosis and non-pathogenic M. smegmatis.
Materials and Methods Search for Corresponding Esx Genes of Mycobcteria All esx genes analyzed in our study, were based on search of the genes from gene database of National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/gene). FASTA sequences of the genes were used to search for corresponding genes in other Mycobacteria in the European
123
Nucleotide Archive sequence database (www.ebi.ac.uk/ ena). Furthermore, BLAST was used to find the differences between different Mycobacterium in terms of single nucleotide variations, insertions, and deletions in the esx genes. Computational Analysis of Esx Proteomes of Mycobacteria A list of Primary accession numbers in UniProtKB (Universal Protein Resource) of esx genes in M. tuberculosis H37Rv and the corresponding genes in other Mycobacteria was prepared. Vector align program was used to compare the amino acid sequences of these genes among Mycobacteria. Genes having substitutions, insertions or deletions in amino acid sequence were selected for further analysis. Prediction of Protein Stability Comparison of various physical and chemical parameters of proteins coded by the Esx genes in both strains was carried out via ProtParam tool from ExPASy portal (http:// web.expasy.org/protparam/). The computed parameters include the instability index, aliphatic index, and grand average of hydrophobicity (GRAVY). Protein Structure Prediction GlobPlot (http://globplot.embl.de/cgiDict.py) was used to predict globularity in the proteins with difference between M. tuberculosis and M. smegmatis.
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
561
Fig. 3 The esx family protein quantity among various Mycobacteria
Prediction of Phosphorylation Sites NetPhosBac (http://www.cbs.dtu.dk/services/NetPhosBac1.0/) was used to predict serine and threonine phosphorylation sites in the Esx proteins displaying difference between M. tuberculosis and M. smegmatis.
Result and Discusion The Distribution and Evolution of Esx Proteins Genome analyses suggest that a related secretion system is found in other Actinobacteria and in some Firmicutes [3]. The protein sequences of M. tuberculosis 23 Esx proteins were download from Tuberculist (http://tuberculist.epfl.ch/ index.html) except for esxM (there is no sequence information about this protein in M. tuberculosis H37Rv), and search the corresponding homologous genes of other Mycobacteria through blast in NCBI (http://www.ncbi.nlm. nih.gov/). Comparative genomic analysis found that esx proteins were largely limited to pathogenic Mycobacteria, such as M. tuberculosis, M. bovis, M. ulcerans, and M.marinum (Fig. 3). Evolutionary studies found that
various esx orthologues are deleted in avirulent mycobacterial strains, like M. tuberculosis H37Ra, M. bovis BCG, M. tuberculosis F11 Mycobacterium microti, another avirulent strain such as M. smegmatis and one that is attenuated like M. bovis BCG carries deletions of almost all these genes except esxE(Rv3904c), esxU(Rv3445c) and esxV(Rv3619c), and M. tuberculosis F11 carries only esxC (Supplementary Table 1). The distribution of esx proteins suggests their unique roles in the virulence, pathogenesis, and persistence of Mycobacteria. Analysis of the genome sequence of M. smegmatis revealed three ESAT-6 (esx) gene cluster regions (regions 4, 1, and 3), with absence of regions 2 and 5 [6]. Regions 2 and 5 might be deleted from the genome of this organism, which might be occurred after the divergence of M. smegmatis, as evidenced by the last two duplicates of the ESAT-6 (esx) gene cluster evolution [6]. This point is supported by comparative genomics analyses of the genomes of closely-related fast-growing Mycobacteria such as M. flavescens, M. vanbaalenii, M. sp. MCS, and M. sp. JLS in which ESAT-6 (esx) gene cluster. Regions 2 and 5 were also found to be absent, as well as M. sp KMS in which ESAT-6 (esx) gene cluster region 2 was present, but region 5 was absent (Supplementary Table 2). The fact that the genome of
123
562
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
M. smegmatis is *1.7 times larger than that of M. tuberculosis [50], and thus does not display the same reductive properties to that observed in the genome of, for example, M. leprae was confirmed to have lost ESAT-6 (esx) gene cluster region 2 and 4 by deletion [51]. Strikingly, of the 11 pairs of CFP-10/ESAT-6 family proteins found in M. tuberculosis, only orthologues of CFP-10 and ESAT-6 are conserved in M. leprae (ML0050c and ML0049c, respectively), which further emphasizes their importance in the lifecycle of Mycobacterial pathogens [30]. Comparative Genomics Yield Insights into the Extent of Sequence Variation To further examine the relationships between, and evolutionary history of, the members of the esx family, to determine the extent of esx sequence similarity and variation, orthologues in the fully sequenced and annotated genomes of Mycobacteria were analyzed by comparative genomics. During this analysis, a complete investigation of the presence and absence of genes, gene sizes, deletions, and conservative substitutions was performed. We found that the difference of some of esx family members among Mycobacteria originate from the deletion of C terminal sequences. The deletion of about 45 amino in its C terminal of M. bovis esxF when compare with M. tuberculosis esxF. Moreover, we observed that the extension of M. bovis esxC in its C-terminal sequence whereas approximately deletion of 41 amino in C-terminal of M. tuberculosis esxC. The amino sequence of some members of M. tuberculosis esx family proteins are highly homologous sequence. There are several amino mutations between esxH and esxR or esxG and esxS (Supplementary Table 3). Besides, there is only one or two mutation between some members of esx family protein, such as esxJ and esxK, esxN and esxO, esxI and esxL, and esxP and esxW (Supplementary Table 3). The redundancy between esx family proteins might imply their functional overlapping. Analyses of Aliphatic Index and GRAVY The aliphatic index of a protein signifies the relative volume occupied by aliphatic side chains. Aliphatic hydrophobicity increases with increase in temperature and hence it is a positive factor for increase in thermal stability of globular proteins [52]. GRAVY is an indication of protein solubility where a positive value correlates with hydrophobicity and negative with hydrophilicity. More hydrophilic the protein greater will be the extent of hydrogen boding with water molecules and higher will be the solubility. The analysis of GRAVY by ProtParam (Fig. 4) shows that the esx protein esxG shared by M. tuberculsosis and M. smagmatis is hydrophobicity, while some esx
123
proteins such as esxH, esxR,esxB,esxT, and esxU are hydrophilicity. Among these five members, the GRAVY value of esxB in M. tuberculosis H37Rv (-0.703) is much lower than its homolog MSMEG_0065 in M. smegmatis (-0.063). Interestingly, some members of esx proteins are hydrophobic in M. tuberculosis H37Rv (esxE and esxA), while their homologous proteins in M. smegmatis (MSMEG_1871 and MSMEG_0066) are hydrophilic. The GRAVY value of M. tuberculosis H37Rv esxE is 0.004, while the homolog MSMEG_1871 is -0.823. On the contrary, the GRAVY value of well-documented virulence factor esxA in M. tuberculosis H37Rv is negative with hydrophilicity, but MSMEG_0066 in M. smagmatis is positive value correlates with hydrophobicity. Together, these data suggest that the variations in protein sequence of esx proteins between M. tuberculosis H37Rv and M. smagmatis manifest in changes in GRAVY values, resulting in the thermal stability of proteins, which might underlie the differential virulence characteristic of the shared esxA(ESAT6) and esxB(CFP10) between M. tuberculosis and non-pathogenic pathogen M. smagmatis, while as virulence factor in the former. Analyses of Instability Index The instability index is indicative of the stability of the protein under in vitro conditions. Instability index [40 is a sign of unstable protein and \40 is an indication of stable protein. The in vivo instability of proteins is possibly determined by the order of certain amino acids in its sequence, some dipeptides occurring differently in unstable and stable proteins. Presence of such dipeptides facilitates the analysis of protein stability [53]. Examples of instability index analyses of esx family proteins in M. tuberculosis H37Rv and their homologs in M. smegmatis are shown (Fig. 5). We found that some members of esx proteins are stable (esxE/Rv3904c/MSMEG_1871) or unstable (esxA/ Rv3875, MSMEG_0066, esxT/Rv3444c, MSMEG_1539 and esxU/Rv1538, MSMEG_1538) in both M. tuberculosis H37Rv and M. smegmatis. A perusal of the same clearly reveals that a few of the esx proteins are stable in H37Rv (esxG/Rv0287, esxH/Rv0288, esxR/Rv3019c, and esxB/ Rv3874), but the corresponding protein in M. smegmatis (MSMEG_0629, MSMEG-0621, and MSMEG_0065) are unstable. These differences between pathogenic M. tuberculosis H37Rv and non-pathogenic M. smegmatis might partly be responsible for the cytotoxic effect of esx proteins in M. tuberculosis H37Rv. Analyses of Globular Domains of Esx Proteins Globular domains in protein molecules confer special functions to a protein. Thus, addition or deletion of globular
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
563
Fig. 4 Protein sequence variations between M. tuberculosis and M. smegmatis manifest in changes in GRAVY values
Fig. 5 Variations in protein sequence variations between M. tuberculosis H37Rv and M. smegmatis affect the stability of esx proteins
domains in a protein might leads to the loss or gain of function. In M. tuberculosis H37Rv, esxF(Rv3905c) has two globular domains, the deletion of a stretch of amino acids in C terminal of M. bovis esxF(Mb3935c) results in deletion of the globular domain, probably leading to a loss of function in M. bovis (Fig. 6a). Similarly, the extension and variation of C terminal of M. bovis esxC, leading to the deficiency of one globular domains (Fig. 6b). In addition, the substitutions of amino between M. tuberculosis esxB(Rv3874) and M. smegmatis esxB(MSMEG_0065) are responsible for the lose of globular domains in N and C terminal of M. smegmatis esxB(MSMEG_0065). Finally, we found that the lose of globular domains M. tuberculosis esxA(Rv3875) and its homolog M. smegmatis esxA(MSMEG_0066) in C and N terminal might be originated in mutation of some special amino in its N and C terminal, respectively (Supplementary Table 4). These suggest that the deletion of globular domains
of esx proteins in M. bovis and M. smegmatis might result in a loss of their function. Analyses of Protein Phosphorylation Sites in Esx Proteins The role of phosphorylation of serine/threonine residues in regulating cell signaling and host–pathogen interaction is well documented. Thus, differences in these sites, as a consequence of variations in nucleotide sequences between M. tuberculosis H37Rv and M. smegmatis, will highlight the likely differences in protein–protein interactions and binding of different domains consequently leading to altered downstream effects. Analysis of potential serine/threonine phosphorylation sites in two typical esx family protein ESAT6 and CFP10, which all exit in M. tuberculosis and M. smagmatis with high homology, revealed a number of
123
564
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
Fig. 6 Gain or loss of globular domain is a function of variation
123
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
565
Fig. 7 Nucleotide variations among Mycobacteria result in gain or loss of serine/threonine phosphorylation sites
differences between these two strains. Gain or loss in serine/ threonine phosphorylation sites mostly due to base substitution was observed in esxA and esxB (Fig. 7). Interestingly, gain of serine/threonine phosphorylation sites was noted in M. tuberculosis esxF(Rv3905c) and esxC(Rv3890c) due to C-terminal extension and variation in amino sequence, respectively. The results presented earlier clearly highlight the important consequence of amino sequence changes between Mycobacteria in esx genes. Such changes manifest in major structural and physicochemical alterations in the corresponding proteomes thereby likely impacting their ability to bring about protein–protein interactions that are very important for host pathogen cross talks and consequent virulence and pathogenesis.
Conclusion In summary, through a comparative genomic analysis, we identified several key changes in the amino sequence between esx homologies of Mycobacteria. These changes mainly included duplications and deletions, which altered the ORFs of the gene. These alterations in the genes quite often resulted in major physicochemical changes in the
encoded proteins. For example, the instability values of M. tuberculosis esxB is lower than its homolog in M. smegmatis as some substitution of few amino sequence of this protein. In addition, the gain or loss global domains may affect the function of the protein. The duplication, deletion, and variation of C terminal amino sequences are responsible for the loss of global domains of the proteins. As shown in our data, M. bovis esxF(Mb3935c) loss the globular domain as the deletion of a stretch of amino acids in its C terminal when compared with M. tuberculosis H37Rv esxF(Rv3905c). In addition, the extension and variation of C terminal of M. bovis esxC also lead to the deficiency of globular domains. Therefore, search of the literature established correlation of many such changes to in vivo function including virulence and pathogenesis. Further biochemical and functional studies are required to establish the role of such changes in amino acid sequence in the attenuation of M. tubeculosis H37Ra, M. bovis BCG, and non-pathogenic M. smegmatis. Such studies along with the combination of clinical data will improve our understanding of the mechanisms of virulence, pathogenesis, and latency of tuberculosis caused by M. tuberculosis and inform the design of better new interventions.
123
566
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
Acknowledgments This work was funded by National Natural Science Foundation (Grant Nos. 81071316, 81271882, 81371), National Megaprojects for Key Infectious Diseases (No. 2008ZX10003-006), New Century Excellent Talents in Universities (NCET-11-0703), Excellent PhD thesis fellowship of southwest university (Grant Nos. kb2009010 and ky2011003), the Fundamental Research Funds for the Central Universities (Grant Nos. XDJK2012D011, XDJK2013D003), Natural Science Foundation Project of CQ CSTC (Grant Nos. CSTC, 2010BB5002). The Chongqing Municipal Committee of Education for postgraduates excellence program (No. YJG123104, No.), The Undergraduates Teaching Reform Program (No. 2011JY052).
References 1. Oxlade O, Schwartzman K, Behr MA, Benedetti A, Pai M, Heymann J, Menzies D (2009) Global tuberculosis trends: a reflection of changes in tuberculosis control or in population health? Int J Tuberc Lung Dis 13:1238–1246 2. Vashishtha VM (2009) WHO global tuberculosis control report 2009: tuberculosis elimination is a distant dream. Indian Pediatr 46:401–402 3. Pallen MJ (2002) The ESAT-6/WXG100 superfamily—and a new Gram-positive secretion system? Trends Microbiol 10:209–212 4. Brodin P, Rosenkrands I, Andersen P, Cole ST, Brosch R (2004) ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol 12:500–508 5. Abdallah AM, Gey van Pittius NC, Champion PA, Cox J, Luirink J, Vandenbroucke-Grauls CM, Appelmelk BJ, Bitter W (2007) Type VII secretion—mycobacteria show the way. Nat Rev Microbiol 5:883–891 6. Gey Van Pittius NC, Gamieldien J, Hide W, Brown GD, Siezen RJ, Beyers AD (2001) The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G?C Gram-positive bacteria. Genome Biol 2:RESEARCH0044 7. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 8. Akpe San Roman S, Facey PD, Fernandez-Martinez L, Rodriguez C, Vallin C, Del Sol R, Dyson P (2010) A heterodimer of EsxA and EsxB is involved in sporulation and is secreted by a type VII secretion system in Streptomyces coelicolor. Microbiology 156:1719–1729 9. Stinear TP, Seemann T, Harrison PF, Jenkin GA, Davies JK, Johnson PD, Abdellah Z, Arrowsmith C, Chillingworth T, Churcher C, Clarke K, Cronin A, Davis P, Goodhead I, Holroyd N, Jagels K, Lord A, Moule S, Mungall K, Norbertczak H, Quail MA, Rabbinowitsch E, Walker D, White B, Whitehead S, Small PL, Brosch R, Ramakrishnan L, Fischbach MA, Parkhill J, Cole ST (2008) Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Res 18:729–741 10. Abdallah AM, Savage ND, van Zon M, Wilson L, Vandenbroucke-Grauls CM, van der Wel NN, Ottenhoff TH, Bitter W (2008) The ESX-5 secretion system of Mycobacterium marinum modulates the macrophage response. J Immunol 181:7166–7175 11. Davis JM, Ramakrishnan L (2009) The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell 136:37–49 12. van der Wel N, Hava D, Houben D, Fluitsma D, van Zon M, Pierson J, Brenner M, Peters PJ (2007) M. tuberculosis and M.
123
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129:1287–1298 Volkman HE, Clay H, Beery D, Chang JC, Sherman DR, Ramakrishnan L (2004) Tuberculous granuloma formation is enhanced by a Mycobacterium virulence determinant. PLoS Biol 2:e367 Raghavan S, Manzanillo P, Chan K, Dovey C, Cox JS (2008) Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature 454:717–721 Frigui W, Bottai D, Majlessi L, Monot M, Josselin E, Brodin P, Garnier T, Gicquel B, Martin C, Leclerc C, Cole ST, Brosch R (2008) Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP. PLoS Pathog 4:e33 Gonzalo-Asensio J, Mostowy S, Harders-Westerveen J, Huygen K, Hernandez-Pando R, Thole J, Behr M, Gicquel B, Martin C (2008) PhoP: a missing piece in the intricate puzzle of Mycobacterium tuberculosis virulence. PLoS ONE 3:e3496 Ohol YM, Goetz DH, Chan K, Shiloh MU, Craik CS, Cox JS (2010) Mycobacterium tuberculosis MycP1 protease plays a dual role in regulation of ESX-1 secretion and virulence. Cell Host Microbe 7:210–220 Serafini A, Boldrin F, Palu G, Manganelli R (2009) Characterization of a Mycobacterium tuberculosis ESX-3 conditional mutant: essentiality and rescue by iron and zinc. J Bacteriol 191:6340–6344 Siegrist MS, Unnikrishnan M, McConnell MJ, Borowsky M, Cheng TY, Siddiqi N, Fortune SM, Moody DB, Rubin EJ (2009) Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition. Proc Natl Acad Sci USA 106:18792–18797 Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84 Maciag A, Dainese E, Rodriguez GM, Milano A, Provvedi R, Pasca MR, Smith I, Palu G, Riccardi G, Manganelli R (2007) Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon. J Bacteriol 189:730–740 Rodriguez GM, Voskuil MI, Gold B, Schoolnik GK, Smith I (2002) ideR, An essential gene in Mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response. Infect Immun 70:3371–3381 Berthet FX, Rasmussen PB, Rosenkrands I, Andersen P, Gicquel B (1998) A Mycobacterium tuberculosis operon encoding ESAT6 and a novel low-molecular-mass culture filtrate protein (CFP10). Microbiology 144:3195–3203 Okkels LM, Andersen P (2004) Protein-protein interactions of proteins from the ESAT-6 family of Mycobacterium tuberculosis. J Bacteriol 186:2487–2491 Arbing MA, Kaufmann M, Phan T, Chan S, Cascio D, Eisenberg D (2010) The crystal structure of the Mycobacterium tuberculosis Rv3019c-Rv3020c ESX complex reveals a domain-swapped heterotetramer. Protein Sci 19:1692–1703 Lightbody KL, Ilghari D, Waters LC, Carey G, Bailey MA, Williamson RA, Renshaw PS, Carr MD (2008) Molecular features governing the stability and specificity of functional complex formation by Mycobacterium tuberculosis CFP-10/ESAT-6 family proteins. J Biol Chem 283:17681–17690 Lightbody KL, Renshaw PS, Collins ML, Wright RL, Hunt DM, Gordon SV, Hewinson RG, Buxton RS, Williamson RA, Carr MD (2004) Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility. FEMS Microbiol Lett 238:255–262 Meher AK, Bal NC, Chary KV, Arora A (2006) Mycobacterium tuberculosis H37Rv ESAT-6-CFP-10 complex formation confers thermodynamic and biochemical stability. FEBS J 273:1445–1462 Renshaw PS, Lightbody KL, Veverka V, Muskett FW, Kelly G, Frenkiel TA, Gordon SV, Hewinson RG, Burke B, Norman J,
W. Deng et al.: Genomic and Proteomic Anatomy of M. Ubiquitous Esx Family Proteins
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
Williamson RA, Carr MD (2005) Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6. EMBO J 24:2491–2498 Renshaw PS, Panagiotidou P, Whelan A, Gordon SV, Hewinson RG, Williamson RA, Carr MD (2002) Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6*CFP-10 complex. Implications for pathogenesis and virulence. J Biol Chem 277:21598–21603 Bitter W, Houben EN, Bottai D, Brodin P, Brown EJ, Cox JS, Derbyshire K, Fortune SM, Gao LY, Liu J, Gey van Pittius NC, Pym AS, Rubin EJ, Sherman DR, Cole ST, Brosch R (2009) Systematic genetic nomenclature for type VII secretion systems. PLoS Pathog 5:e1000507 Fortune SM, Jaeger A, Sarracino DA, Chase MR, Sassetti CM, Sherman DR, Bloom BR, Rubin EJ (2005) Mutually dependent secretion of proteins required for mycobacterial virulence. Proc Natl Acad Sci USA 102:10676–10681 McLaughlin B, Chon JS, MacGurn JA, Carlsson F, Cheng TL, Cox JS, Brown EJ (2007) A mycobacterium ESX-1-secreted virulence factor with unique requirements for export. PLoS Pathog 3:e105 Xu J, Laine O, Masciocchi M, Manoranjan J, Smith J, Du SJ, Edwards N, Zhu X, Fenselau C, Gao LY (2007) A unique Mycobacterium ESX-1 protein co-secretes with CFP-10/ESAT-6 and is necessary for inhibiting phagosome maturation. Mol Microbiol 66:787–800 Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, Griffiths KE, Marchal G, Leclerc C, Cole ST (2003) Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat Med 9:533–539 Hsu T, Hingley-Wilson SM, Chen B, Chen M, Dai AZ, Morin PM, Marks CB, Padiyar J, Goulding C, Gingery M, Eisenberg D, Russell RG, Derrick SC, Collins FM, Morris SL, King CH, Jacobs WR Jr (2003) The primary mechanism of attenuation of bacillus Calmette–Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc Natl Acad Sci USA 100:12420–12425 Sassetti CM, Rubin EJ (2003) Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci USA 100:12989–12994 Stanley SA, Raghavan S, Hwang WW, Cox JS (2003) Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc Natl Acad Sci USA 100:13001–13006 Guinn KM, Hickey MJ, Mathur SK, Zakel KL, Grotzke JE, Lewinsohn DM, Smith S, Sherman DR (2004) Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol Microbiol 51:359–370 Alderson MR, Bement T, Day CH, Zhu L, Molesh D, Skeiky YA, Coler R, Lewinsohn DM, Reed SG, Dillon DC (2000) Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(?) T cells. J Exp Med 191:551–560
567
41. Rosenkrands I, Weldingh K, Jacobsen S, Hansen CV, Florio W, Gianetri I, Andersen P (2000) Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection. Electrophoresis 21:935–948 42. Skjot RL, Oettinger T, Rosenkrands I, Ravn P, Brock I, Jacobsen S, Andersen P (2000) Comparative evaluation of low-molecularmass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens. Infect Immun 68:214–220 43. Andersen P, Andersen AB, Sorensen AL, Nagai S (1995) Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J Immunol 154:3359–3372 44. Pollock JM, Andersen P (1997) Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle. Infect Immun 65:2587–2592 45. Ravn P, Demissie A, Eguale T, Wondwosson H, Lein D, Amoudy HA, Mustafa AS, Jensen AK, Holm A, Rosenkrands I, Oftung F, Olobo J, von Reyn F, Andersen P (1999) Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis. J Infect Dis 179:637–645 46. Elhay MJ, Oettinger T, Andersen P (1998) Delayed-type hypersensitivity responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the guinea pig. Infect Immun 66:3454–3456 47. van Pinxteren LA, Ravn P, Agger EM, Pollock J, Andersen P (2000) Diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10. Clin Diagn Lab Immunol 7:155–160 48. Majlessi L, Rojas MJ, Brodin P, Leclerc C (2003) CD8?-T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex class I-restricted epitope shared by proteins of the ESAT-6 family. Infect Immun 71:7173–7177 49. Skjot RL, Brock I, Arend SM, Munk ME, Theisen M, Ottenhoff TH, Andersen P (2002) Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat-6 gene family. Infect Immun 70:5446–5453 50. Reyrat JM, Kahn D (2001) Mycobacterium smegmatis: an absurd model for tuberculosis? Trends Microbiol 9:472–474 51. Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson NR, Wheeler PR, Honore N, Garnier T, Churcher C, Harris D, Mungall K, Basham D, Brown D, Chillingworth T, Connor R, Davies RM, Devlin K, Duthoy S, Feltwell T, Fraser A, Hamlin N, Holroyd S, Hornsby T, Jagels K, Lacroix C, Maclean J, Moule S, Murphy L, Oliver K, Quail MA, Rajandream MA, Rutherford KM, Rutter S, Seeger K, Simon S, Simmonds M, Skelton J, Squares R, Squares S, Stevens K, Taylor K, Whitehead S, Woodward JR, Barrell BG (2001) Massive gene decay in the leprosy bacillus. Nature 409:1007–1011 52. Ikai A (1980) Thermostability and aliphatic index of globular proteins. J Biochem 88:1895–1898 53. Guruprasad K, Reddy BV, Pandit MW (1990) Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng 4:155–161
123
