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Chlamydia outer membrane protein discovery using genomics
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Chlamydia outer membrane protein discovery using genomics Richard S Stephens and Claudia J Lammel Outer membrane proteins of microbial pathogens serve essential roles in engaging the host environment and can be important immunotherapeutic targets. Because of the difficulty of growing large quantities of chlamydiae suitable for biochemical fractionation, little was known about their outer membrane protein composition prior to the recent sequencing of the C. trachomatis and C. pneumoniae genomes. Using bioinformatic approaches to characterize chlamydial open reading frames, novel outer membrane proteins were predicted. Several of the predicted outer membrane proteins recently have been shown to be translated and localized to the surface of the chlamydial outer membrane. Addresses Division of Infectious Diseases, School of Public Health, University of California, Berkeley, California 94720, USA, and The Francis I Proctor Foundation, University of California, San Francisco, CA 94143, USA Correspondence: Richard S Stephens Current Opinion in Microbiology 2001, 4:16–20 1369-5274/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations EB elementary body Inc inclusion membrane protein LPS lipopolysaccharide MOMP major outer membrane protein OMP outer membrane protein ORF open reading frame Pmp polymorphic membrane protein RB reticulate body
Introduction Among all the infectious agents that are reported to the United States Centers for Disease Control and Prevention, Chlamydia trachomatis is, by far, the most common. Sexually transmitted diseases caused by C. trachomatis infections may result in epididymitis, salpingitis, tubal factor infertility, chronic pelvic pain and ectopic pregnancy. C. trachomatis is also a leading cause of blindness in many regions of the developing world; of the approximately 150 million people with trachoma, six million have been irreversibly blinded by the disease. A recently-identified chlamydial organism, C. pneumoniae, causes acute respiratory infection, including pneumonia, pharyngitis and bronchitis, and may contribute to coronary artery disease [1], which is the leading cause of death in humans. The medical and public health importance of infection by chlamydiae provides the imperative for understanding the biological basis for infection and disease. For a comprehensive review of chlamydial epidemiology, immunology and biology, see [2••]. Although chlamydiae are bacterial pathogens, investigating their biology is often complicated by their requirement for growth within mammalian cells, and by the lack of
genetic methods that are suitable for these unique organisms and that may be productively applied to experimental designs [3]. Phylogenetically, chlamydiae are deeply separated from other bacteria and form the basis for a separate bacterial division [4]. In addition to their obligate intracellular growth, chlamydiae have a developmental cycle in which the infectious developmental form — the elementary body (EB) — is metabolically inactive and is highly resistant to lysis because of a complex of outer envelope proteins highly crosslinked by disulfide bonds. Following entry into eukaryotic host cells, chlamydiae reside within a membrane-bound vacuole called the chlamydial inclusion. The EB differentiates into the metabolically active developmental form — the reticulate body (RB) — that grows and replicates. The chlamydial envelope is similar to the outer envelope of Gram-negative organisms, in that it consists of an inner membrane, a periplasmic space and an outer membrane containing proteins and lipopolysaccharide (LPS). Unlike in Gram-negative organisms, however, chlamydial LPS is truncated, terminating in 3-deoxy-Dmanno-octulosonic acid (KDO) [5], and there is little detectable peptidoglycan. The completed genome sequencing of C. trachomatis [6] and C. pneumoniae [7] immediately and comprehensively clarified the biology of these human pathogens well beyond the scope of pre-genomic understanding, and challenged several existing paradigms in the field. Many discoveries involving metabolic pathways, genetic regulation, signal transduction and protein secretion have been gleaned from the chlamydial genome. One pertinent question is this: how does the genome sequence of Chlamydia allow researchers to elucidate the protein composition of the chlamydial outer membrane? This question is important because outer membrane proteins (OMPs) of microbial pathogens function at the environmental interface between the pathogen and its host. Given the marked differences in outer membrane organization between the EB and RB developmental forms, knowledge of the composition of chlamydial outer membranes is also important for understanding the differences in EB and RB function. Typically, OMPs are engaged in: firstly, specific nutrient and metabolite acquisition; secondly, adhesion to, or invasion of, host cells and tissues; thirdly, type III secretion [8] delivery of pathogen effector kinases or phosphatases that modulate host responses to infection; and fourthly, host immune evasion by antigenic or phase variation. However, unlike many of the genes whose functions are conserved in the microbial world and can be inferred by sequence similarity, OMPs, whose functions are often tailored to the virulence of particular pathogens, are not expected to be identified by similar genes in other organisms. We shall review the knowledge of chlamydial OMPs discovered using genomics.
Chlamydia outer membrane protein discovery using genomics Stephens and Lammel
Pre-genomic C. trachomatis and C. pneumoniae outer membrane proteins Caldwell et al. [9] provided the first extensive description of the major outer membrane protein (MOMP), OmpA, as a quantitatively predominant surface protein of chlamydiae. For the past 20 years, research has focused on this protein because it is a surface protein that is a target for antibody-mediated neutralization. While other putative surface proteins have been implicated as being present, only MOMP has been confirmed at the molecular level. Using chlamydial MOMP expressed in E. coli, it has been shown unequivocally to be a general diffusion porin [10]. Thus, until recently, the outer membranes of chlamydiae were known to consist only of MOMP and LPS.
The polymorphic outer membrane protein family Analysis of the genome sequences of C. trachomatis and C. pneumoniae for potential OMPs revealed a stunning surprise. The C. trachomatis genome encodes a family of nine polymorphic membrane proteins (Pmps) and the C. pneumoniae genome encodes 21 Pmp paralogs. Four nearly-identical Pmps were originally detected in C. psittaci strains that cause ovine abortion, although homologs were not detected in C. trachomatis or C. pneumoniae at that time [11]. Unlike the C. psittaci Pmp, Pmps encoded by C. trachomatis and C. pneumoniae differ extensively in their primary amino acid sequence, and are recognizable largely by shared repeated sequence motifs [12]. In addition, each protein contains a leader signal sequence. All of the pmp genes for C. trachomatis and C. pneumoniae are transcribed (J Grimwood, L Olinger, RS Stephens, unpublished data) [13], but only a few have been shown to be stably translated and present in the chlamydial outer membrane (J Grimwood, L Olinger, RS Stephens, unpublished data) [14]. Moreover, it has been shown that expression of several Pmps differs between C. pneumoniae strains CWL029, AR39 and TW183. For at least PmpG10, expression varies within a strain. Based upon the genome sequence for strain CWL029, pmpG10 contains a string of 13 guanine residues and is out of reading frame. A PmpG10-specific monoclonal antibody does not detect an expressed protein product in strain CWL029. A CWL029 strain with a different passage history contains 14 guanine residues, which are in frame, and does produce a protein [15]. Likewise, strain AR39 pmpG10 has 14 guanine residues and PmpG10 is detected (J Grimwood, L Olinger, RS Stephens, unpublished data). Strain TW183 also produces a protein detectable by the G10-specific antibody, and its gene contains 11 guanine residues and is in frame (J Grimwood, L Olinger, RS Stephens, unpublished data). Thus, interstrain and intrastrain variation in the number of guanine residues varies the production of PmpG10; however, other Pmp do not contain apparent runs of guanine residues. The function of Pmp is unknown, although, given their outer membrane, and often surface, exposure, and their extreme polymorphism,
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it is reasonable to expect an essential function for the virulence and pathogenesis of chlamydiae.
Porin-B One open reading frame (ORF) that was predicted to be in the outer membrane also has some very distant similarity to MOMP. The ORF PorB encodes a protein of 39 kDa and a predicted pI (isoelectric point) of 4.9. It has recently been shown that PorB is transcribed and expressed in chlamydiae and contains a leader signal sequence that produces a mature protein following expression in E. coli [16•]. Moreover, it was shown that PorB is present in the outer membrane complex of EB, and is surface accessible by PorB-specific antisera. Like MOMP, PorB is relatively cysteine-rich. However, unlike MOMP, PorB is expressed at nearly 1/50th of the amount of the quantitatively predominant MOMP. Also unlike MOMP, whose sequence varies significantly among C. trachomatis strains, PorB is highly conserved among chlamydial strains and even highly conserved for the C. pneumoniae (58% amino acid identity) ortholog. Coincidentally, MOMP is 40 kDa with a pI of 4.9, thus it is not surprising that PorB was not readily detected by biochemical methods. PorB has porin activity in liposomes reconstituted with purified PorB expressed in E. coli [16•]. The activity for solutes that diffuse readily through MOMP was substantially less for PorB, suggesting, along with its lower abundance, that the function of PorB for chlamydiae is as a substrate-specific porin responsible for the diffusion of some metabolite essential for chlamydial growth. Given the surface location of PorB, and given that its sequence is conserved among chlamydiae, PorB-specific antisera were tested in in vitro neutralization of infectivity assays, and it was shown to be a target for neutralization in vitro [16•]. MOMP is a target for antibody-mediated neutralization and is antigenically variable because it is thought to be under selection by the host immune response. If PorB is a target for neutralization, then why is it so highly conserved? One could hypothesize that the dominant immunogenicity of MOMP and its quantitative abundance may interfere with the development of potent natural immunity to PorB. Consistent with this hypothesis is that human antisera that are reactive to MOMP are rarely reactive to PorB, at least by immunoblot assays (A Kubo, RS Stephens, unpublished data).
Omp85 Omp85 is a highly-conserved outer membrane protein present in a wide variety of Gram-negative pathogens, including Neisseria gonorrhoeae, Haemophilus influenzae, Treponema pallidum and other pathogens. The function of Omp85 is unknown, although it has been proposed to represent a common surface antigen and a vaccine candidate [17,18]. An ortholog of Omp85 is present in the chlamydial
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Host–microbe interactions: bacteria
Table 1 Predicted* outer membrane proteins for C. trachomatis and C. pneumoniae kDa
pI
Cys
C-term AA CT CPn
CT#
CPn#
Gene
CT
CPn
CT
CPn
CT
CPn
681 713 241 548 623 351 242 476 858 021 007
0695 0854 0300 0669 0729 0020 0301 0595 1016 0111 0441 0795 0796 0797 0798 0278
Pmp Family OmpA PorB Omp85 hypothetical protein hypothetical protein hypothetical protein OmpH-like hypothetical protein hypothetical protein hypothetical protein Hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein conserved outer membrane lipoprotein hypothetical protein
~100 40 35 86 20 46 78 17 34 64 25 36
~100 39 34 86 20 46 78 17 34 67 25 35 37 65 36 33 28
5-9 4.7 4.8 8.9 5.9 6.4 9.4 4.5 7.5 5.4 9.6 5.9
5-9 6.1 4.9 7.9 6.1 9.2 9.1 4.6 6.1 5.2 9.2 8.9 6.5 5.5 5.0 4.4 6.5
9-26 9 9 3 3 1 12 1 4 7 0 4
9-26 9 6 3 5 2 12 1 1 4 1 5 1 4 0 4 7
F F F F F F G N L F F F
F F F F F F F N N S Q F F F K D G
9
G
G
0498
33
5.1
*The encoded proteins for the C. trachomatis (CT) and C. pneumoniae (CPn) genome sequences were evaluated for leader peptide signal sequences using SPSCAN (GCG Wisconsin Package), and those with high scores for signal peptides were subjected to analysis by PSORT (http://psort.nibb.ac.jp), which provides a
probability estimate of outer membrane protein localization [26,27]. Additional evidence for outer membrane localization is the presence of a carboxyl-terminal phenylalanine [28]. C-term AA, carboxy-terminal amino acid single letter code; Cys, cysteine; pI, isoelectric point; #, assigned open reading frame reference number.
genomes. Omp85 is expressed and present in the chlamydial outer membrane complex, although when incorporated into liposomes, porin activity is not detectable (using solutes that diffuse through MOMP and PorB) [18]. Because Omp85 is surface-accessible to anti-Omp85 antibodies, and Omp85specific antibodies neutralize chlamydial infectivity in vitro (A Kubo, RS Stephens, unpublished data), it should be investigated for its vaccine potential.
apparatus is composed of a complex of many proteins that traverse the inner and outer membranes, as well as a needle structure that protrudes from the surface of the bacterium and penetrates the target eukaryotic cell. Although direct sequence-similarity comparisons to the S. typhimurium genome/type III secretion system apparatus provide little additional information about the composition of the chlamydial surface projections, the identifiable type III components in chlamydia are clustered in three operons and testing each protein associated with these operons is likely to resolve this question.
Type III secretion Salmonella typhimurium has small surface structures, which have been isolated and purified. The proteins that compose these structures were identified as components of the type III secretion system [19]. This system secretes proteins into target host cells and is essential for the virulence of a number of bacterial pathogens. Structures of similar size and morphology were described for Chlamydia over 20 years ago by Matsumoto [20], and later by Nichols [21]. The C. trachomatis and C. pneumoniae genomes contain identical genes for components of the machinery necessary for type III secretion, and it is presumed that the structures characterized by Matusmoto and Nichols are components of the chlamydial type III secretion system apparatus. Chlamydiae have orthologs of the YscC/GspD family, which is an outer membrane component of the type III secretion apparatus [7]. However, orthologous chlamydial proteins to PrgK and PrgH, which make up the S. typhimurium type III secretion system apparatus, were not identified. Likewise, a chlamydial ortholog was not found for the needle portion of the type III secretion apparatus, which is composed of a small protein, MxiH, in Shigella [22]. The type III secretion
Other predicted OMPs In addition to the genes encoding MOMP, PorB and Omp85 (which have been shown to be localized to the chlamydial outer membrane), there are several additional ORFs that encode proteins that can be predicted to be outer-membrane-localized (Table 1). Five proteins with strong predictions for outer membrane localization have orthologs in both C. trachomatis and C. pneumoniae (CT548/CPn0669, CT623/CPn0729, CT351/CPn0020, CT242/CPn0301 and CT476/CPn0595). CT351/CPn0020 share 61% amino acid sequence identity and each contains 12 cysteine residues. The other four orthologous protein pairs are not notable for their cysteine content but one, CT242/CPn0301, has weak similarity to Yersinia enterolitica OmpH. Two additional orthologous pairs, CT858/CPn1016 and CT021/CPn0111, are less confidently predicted for outer membrane localization and the orthologous pairs share only 40–48% sequence identity. However, they remain of interest for further investigation.
Chlamydia outer membrane protein discovery using genomics Stephens and Lammel
Six predicted OMPs in C. pneumoniae do not have orthologs in C. trachomatis. Two of these are cysteine-rich proteins, CPn0278 and CPn0498, and one of these, CPn0278, is similar to outer membrane lipoproteins from Deinococcus spp., Helicobacter spp. and E. coli. The remaining four C. pneumoniae proteins are encoded sequentially in a likely operon, CPn0795-CPn0798. If verified as outer membrane proteins, their operon organization suggests that they may act together in some function essential for C. pneumoniae but not for C. trachomatis. This suggests that these could be involved in the differential virulence or tropism of C. pneumoniae.
Conclusions The prediction of genes that are likely to encode chlamydial OMPs has led directly to the demonstration that Pmp, PorB and Omp85 are outer membrane surface proteins. This has greatly expanded the understanding of chlamydial outer membranes that were previously known to consist only of MOMP and LPS. Protein predictions from chlamydial genomic information have also proven useful for greatly expanding the number of inclusion membrane proteins (Inc). These proteins are secreted by chlamydiae to the vacuolar membrane of the chlamydial inclusion [23,24]. It was noted that for the three Inc proteins that had been characterized biochemically, each has a motif signature with bi-lobed hydrophobic domains at the amino terminus. Using these criteria, five of six Inc proteins predicted from the genome sequence were shown to be localized to the inclusion membrane [25••]. The surge in characterization of chlamydial proteins that are secreted or localized to the chlamydial outer membrane has been an outcome of the sequencing of the genomes of two species of Chlamydia. The expectation is that much more will result from the use of bioinformatics to approach functional characterization of the individual components of chlamydiae and, ultimately, their roles in the network of chlamydial biology both within chlamydiae and between chlamydiae and their host cells.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Grayston JT: Does Chlamydia pneumoniae cause atherosclerosis? Arch Surg 1999, 134:930-934.
2. ••
Stephens RS (Ed): Chlamydia: intracellular biology, pathogenesis, and immunity. Washington, DC: American Society for Microbiology; 1999. This book provides a comprehensive review of chlamydial developmental biology, metabolism, genetics, cellular microbiology, host immune response, and epidemiology.
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6.
Stephens RS, Kalman S, Lammel C, Fan J, Marathe R, Aravind L, Mitchell W, Olinger L, Tatusov RL, Zhao Q et al.: Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 1998, 282:754-759.
7.
Kalman S, Mitchell W, Marathe R, Lammel C, Fan J, Hyman RW, Olinger L, Grimwood J, Davis RW, Stephens RS: Comparative genomes of Chlamydia pneumoniae and C. trachomatis. Nat Genet 1999, 21:385-389.
8.
Hueck CJ: Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 1998, 62:379-433.
9.
Caldwell HD, Kromhout J, Schachter J: Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect Immun 1981, 31:1161-1176.
10. Jones HM, Kubo A, Stephens RS: Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein. Gene 2000, 258:173-181. 11. Longbottom D, Russell M, Dunbar SM, Jones GE, Herring AJ: Molecular cloning and characterization of the genes coding for the highly immunogenic cluster of 90-kilodalton envelope proteins from the Chlamydia psittaci subtype that causes abortion in sheep. Infect Immun 1998, 66:1317-1324. 12. Grimwood J, Stephens RS: Computational analysis of the polymorphic membrane protein superfamily of Chlamydia trachomatis and Chlamydia pneumoniae. Microbial & Comp Genomics 1999, 4:187-201. 13. Lindquist EA, Stephens RS: Transcriptional activity of a sequence variable protein family in Chlamydia trachomatis. In Chlamydial Infections. Proceedings of the Ninth International Symposium on Human Chlamydial Infection. Edited by Stephens RS, Byrne GI, Christiansen G et al. San Francisco, International Chlamydia Symposium; 1998:259-262. 14. Mygind PH, Christiansen G, Roepstorff P, Birkelund S: Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex. FEMS Microbiol Lett 2000, 186:163-169. 15. Knudsen K, Madsen AS, Mygind P, Christiansen G, Birkelund S: Identification of two novel genes encoding 97- to 99-kilodalton outer membrane proteins of Chlamydia pneumoniae. Infect Immun 1999, 67:375-383. 16. Kubo A, Stephens RS: Characterization and functional analysis of • PorB, a Chlamydia porin and neutralizing target. Mol Microbiol 2000, 38:772-780. This paper describes the use of genomic information for the discovery of a novel chlamydial outer membrane protein, and its functional characterization. 17.
Manning DS, Reschke DK, Judd RC: Omp85 proteins of Neisseria gonorrhoeae and Neisseria meningitidis are similar to Haemophilus influenzae D-15-Ag and Pasteurella multocida Omp87. Microb Pathog 1998, 25:11-21.
18. Cameron CE, Lukehart SA, Castro C, Moini B, Godornes C, Van Voorhis WC: Opsonic potential, protective capacity, and sequence conservation of the Treponema pallidum subspecies pallidum Tp92. J Infect Dis 2000, 181:1401-1413. 19. Kubori T, Matsushima Y, Nakamura D, Uralil J, Lara-Tejero M, Sukhan A, Galan JE, Aizawa S-I: Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 1998, 280:602-605. 20. Matsumoto A: Electron microscope observations of surface projections and related intracellular structures of Chlamydia organisms. J Electron Microsc 1981, 30:315-320. 21. Nichols BA, Setzer PY, Pang F, Dawson CR: New view of the surface projections of Chlamydia trachomatis. J Bacteriol 1985, 164:344-349.
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Stephens RS: Challenge of Chlamydia research. Infect Agents Dis 1992, 1:279-293.
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Pace NR: A molecular view of microbial diversity and the biosphere. Science 1997, 276:734-740.
22. Tamano K, Aizawa SI, Katayama E, Nonaka T, Imajoh-Ohmi S, Kuwae A, Nagai S, Sasakawa C: Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors. EMBO J 2000, 19:3876-3887.
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Rund S, Lindner B, Brade H, Holst O: Structural analysis of the lipopolysaccharide from Chlamydia trachomatis serotype L2. J Biol Chem 1999, 274:16819-16824.
23. Bannantine JP, Rockey DD, Hackstadt T: Tandem genes of Chlamydia psittaci that encode proteins localized to the inclusion membrane. Mol Microbiol 1998, 28:1017-1026.
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Host–microbe interactions: bacteria
24. Rockey DD, Heinzen RA, Hackstadt T: Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells. Mol Microbiol 1995, 15:617-626.
26. Nakai K, Kanehisa M: Expert system for predicting protein localization sites in Gram-negative bacteria. Proteins 1991, 11:95-110.
25. Bannantine JP, Griffiths RS, Viratyosin W, Brown WJ, Rockey DD: A •• secondary structure motif predictive of protein localization to the chlamydial inclusion membrane. Cell Microbiol 2000, 2:35-47. This paper demonstrates the effective use of the chlamydial genome sequence and its amono acid translation to predict the biological functions for chlamydial proteins secreted to the host cell vacuolar membrane.
27.
von Heijne G: A new method for predicting signal sequence cleavage sites. Nucleic Acids Research 1986, 14:4683-4690.
28. Struyve M, Moons M, Tommassen J: Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. J Mol Biol 1991, 218:141-148.
Chlamydia outer membrane protein discovery using genomics Richard S Stephens and Claudia J Lammel Outer membrane proteins of microbial pathogens serve essential roles in engaging the host environment and can be important immunotherapeutic targets. Because of the difficulty of growing large quantities of chlamydiae suitable for biochemical fractionation, little was known about their outer membrane protein composition prior to the recent sequencing of the C. trachomatis and C. pneumoniae genomes. Using bioinformatic approaches to characterize chlamydial open reading frames, novel outer membrane proteins were predicted. Several of the predicted outer membrane proteins recently have been shown to be translated and localized to the surface of the chlamydial outer membrane. Addresses Division of Infectious Diseases, School of Public Health, University of California, Berkeley, California 94720, USA, and The Francis I Proctor Foundation, University of California, San Francisco, CA 94143, USA Correspondence: Richard S Stephens Current Opinion in Microbiology 2001, 4:16–20 1369-5274/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations EB elementary body Inc inclusion membrane protein LPS lipopolysaccharide MOMP major outer membrane protein OMP outer membrane protein ORF open reading frame Pmp polymorphic membrane protein RB reticulate body
Introduction Among all the infectious agents that are reported to the United States Centers for Disease Control and Prevention, Chlamydia trachomatis is, by far, the most common. Sexually transmitted diseases caused by C. trachomatis infections may result in epididymitis, salpingitis, tubal factor infertility, chronic pelvic pain and ectopic pregnancy. C. trachomatis is also a leading cause of blindness in many regions of the developing world; of the approximately 150 million people with trachoma, six million have been irreversibly blinded by the disease. A recently-identified chlamydial organism, C. pneumoniae, causes acute respiratory infection, including pneumonia, pharyngitis and bronchitis, and may contribute to coronary artery disease [1], which is the leading cause of death in humans. The medical and public health importance of infection by chlamydiae provides the imperative for understanding the biological basis for infection and disease. For a comprehensive review of chlamydial epidemiology, immunology and biology, see [2••]. Although chlamydiae are bacterial pathogens, investigating their biology is often complicated by their requirement for growth within mammalian cells, and by the lack of
genetic methods that are suitable for these unique organisms and that may be productively applied to experimental designs [3]. Phylogenetically, chlamydiae are deeply separated from other bacteria and form the basis for a separate bacterial division [4]. In addition to their obligate intracellular growth, chlamydiae have a developmental cycle in which the infectious developmental form — the elementary body (EB) — is metabolically inactive and is highly resistant to lysis because of a complex of outer envelope proteins highly crosslinked by disulfide bonds. Following entry into eukaryotic host cells, chlamydiae reside within a membrane-bound vacuole called the chlamydial inclusion. The EB differentiates into the metabolically active developmental form — the reticulate body (RB) — that grows and replicates. The chlamydial envelope is similar to the outer envelope of Gram-negative organisms, in that it consists of an inner membrane, a periplasmic space and an outer membrane containing proteins and lipopolysaccharide (LPS). Unlike in Gram-negative organisms, however, chlamydial LPS is truncated, terminating in 3-deoxy-Dmanno-octulosonic acid (KDO) [5], and there is little detectable peptidoglycan. The completed genome sequencing of C. trachomatis [6] and C. pneumoniae [7] immediately and comprehensively clarified the biology of these human pathogens well beyond the scope of pre-genomic understanding, and challenged several existing paradigms in the field. Many discoveries involving metabolic pathways, genetic regulation, signal transduction and protein secretion have been gleaned from the chlamydial genome. One pertinent question is this: how does the genome sequence of Chlamydia allow researchers to elucidate the protein composition of the chlamydial outer membrane? This question is important because outer membrane proteins (OMPs) of microbial pathogens function at the environmental interface between the pathogen and its host. Given the marked differences in outer membrane organization between the EB and RB developmental forms, knowledge of the composition of chlamydial outer membranes is also important for understanding the differences in EB and RB function. Typically, OMPs are engaged in: firstly, specific nutrient and metabolite acquisition; secondly, adhesion to, or invasion of, host cells and tissues; thirdly, type III secretion [8] delivery of pathogen effector kinases or phosphatases that modulate host responses to infection; and fourthly, host immune evasion by antigenic or phase variation. However, unlike many of the genes whose functions are conserved in the microbial world and can be inferred by sequence similarity, OMPs, whose functions are often tailored to the virulence of particular pathogens, are not expected to be identified by similar genes in other organisms. We shall review the knowledge of chlamydial OMPs discovered using genomics.
Chlamydia outer membrane protein discovery using genomics Stephens and Lammel
Pre-genomic C. trachomatis and C. pneumoniae outer membrane proteins Caldwell et al. [9] provided the first extensive description of the major outer membrane protein (MOMP), OmpA, as a quantitatively predominant surface protein of chlamydiae. For the past 20 years, research has focused on this protein because it is a surface protein that is a target for antibody-mediated neutralization. While other putative surface proteins have been implicated as being present, only MOMP has been confirmed at the molecular level. Using chlamydial MOMP expressed in E. coli, it has been shown unequivocally to be a general diffusion porin [10]. Thus, until recently, the outer membranes of chlamydiae were known to consist only of MOMP and LPS.
The polymorphic outer membrane protein family Analysis of the genome sequences of C. trachomatis and C. pneumoniae for potential OMPs revealed a stunning surprise. The C. trachomatis genome encodes a family of nine polymorphic membrane proteins (Pmps) and the C. pneumoniae genome encodes 21 Pmp paralogs. Four nearly-identical Pmps were originally detected in C. psittaci strains that cause ovine abortion, although homologs were not detected in C. trachomatis or C. pneumoniae at that time [11]. Unlike the C. psittaci Pmp, Pmps encoded by C. trachomatis and C. pneumoniae differ extensively in their primary amino acid sequence, and are recognizable largely by shared repeated sequence motifs [12]. In addition, each protein contains a leader signal sequence. All of the pmp genes for C. trachomatis and C. pneumoniae are transcribed (J Grimwood, L Olinger, RS Stephens, unpublished data) [13], but only a few have been shown to be stably translated and present in the chlamydial outer membrane (J Grimwood, L Olinger, RS Stephens, unpublished data) [14]. Moreover, it has been shown that expression of several Pmps differs between C. pneumoniae strains CWL029, AR39 and TW183. For at least PmpG10, expression varies within a strain. Based upon the genome sequence for strain CWL029, pmpG10 contains a string of 13 guanine residues and is out of reading frame. A PmpG10-specific monoclonal antibody does not detect an expressed protein product in strain CWL029. A CWL029 strain with a different passage history contains 14 guanine residues, which are in frame, and does produce a protein [15]. Likewise, strain AR39 pmpG10 has 14 guanine residues and PmpG10 is detected (J Grimwood, L Olinger, RS Stephens, unpublished data). Strain TW183 also produces a protein detectable by the G10-specific antibody, and its gene contains 11 guanine residues and is in frame (J Grimwood, L Olinger, RS Stephens, unpublished data). Thus, interstrain and intrastrain variation in the number of guanine residues varies the production of PmpG10; however, other Pmp do not contain apparent runs of guanine residues. The function of Pmp is unknown, although, given their outer membrane, and often surface, exposure, and their extreme polymorphism,
17
it is reasonable to expect an essential function for the virulence and pathogenesis of chlamydiae.
Porin-B One open reading frame (ORF) that was predicted to be in the outer membrane also has some very distant similarity to MOMP. The ORF PorB encodes a protein of 39 kDa and a predicted pI (isoelectric point) of 4.9. It has recently been shown that PorB is transcribed and expressed in chlamydiae and contains a leader signal sequence that produces a mature protein following expression in E. coli [16•]. Moreover, it was shown that PorB is present in the outer membrane complex of EB, and is surface accessible by PorB-specific antisera. Like MOMP, PorB is relatively cysteine-rich. However, unlike MOMP, PorB is expressed at nearly 1/50th of the amount of the quantitatively predominant MOMP. Also unlike MOMP, whose sequence varies significantly among C. trachomatis strains, PorB is highly conserved among chlamydial strains and even highly conserved for the C. pneumoniae (58% amino acid identity) ortholog. Coincidentally, MOMP is 40 kDa with a pI of 4.9, thus it is not surprising that PorB was not readily detected by biochemical methods. PorB has porin activity in liposomes reconstituted with purified PorB expressed in E. coli [16•]. The activity for solutes that diffuse readily through MOMP was substantially less for PorB, suggesting, along with its lower abundance, that the function of PorB for chlamydiae is as a substrate-specific porin responsible for the diffusion of some metabolite essential for chlamydial growth. Given the surface location of PorB, and given that its sequence is conserved among chlamydiae, PorB-specific antisera were tested in in vitro neutralization of infectivity assays, and it was shown to be a target for neutralization in vitro [16•]. MOMP is a target for antibody-mediated neutralization and is antigenically variable because it is thought to be under selection by the host immune response. If PorB is a target for neutralization, then why is it so highly conserved? One could hypothesize that the dominant immunogenicity of MOMP and its quantitative abundance may interfere with the development of potent natural immunity to PorB. Consistent with this hypothesis is that human antisera that are reactive to MOMP are rarely reactive to PorB, at least by immunoblot assays (A Kubo, RS Stephens, unpublished data).
Omp85 Omp85 is a highly-conserved outer membrane protein present in a wide variety of Gram-negative pathogens, including Neisseria gonorrhoeae, Haemophilus influenzae, Treponema pallidum and other pathogens. The function of Omp85 is unknown, although it has been proposed to represent a common surface antigen and a vaccine candidate [17,18]. An ortholog of Omp85 is present in the chlamydial
18
Host–microbe interactions: bacteria
Table 1 Predicted* outer membrane proteins for C. trachomatis and C. pneumoniae kDa
pI
Cys
C-term AA CT CPn
CT#
CPn#
Gene
CT
CPn
CT
CPn
CT
CPn
681 713 241 548 623 351 242 476 858 021 007
0695 0854 0300 0669 0729 0020 0301 0595 1016 0111 0441 0795 0796 0797 0798 0278
Pmp Family OmpA PorB Omp85 hypothetical protein hypothetical protein hypothetical protein OmpH-like hypothetical protein hypothetical protein hypothetical protein Hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein conserved outer membrane lipoprotein hypothetical protein
~100 40 35 86 20 46 78 17 34 64 25 36
~100 39 34 86 20 46 78 17 34 67 25 35 37 65 36 33 28
5-9 4.7 4.8 8.9 5.9 6.4 9.4 4.5 7.5 5.4 9.6 5.9
5-9 6.1 4.9 7.9 6.1 9.2 9.1 4.6 6.1 5.2 9.2 8.9 6.5 5.5 5.0 4.4 6.5
9-26 9 9 3 3 1 12 1 4 7 0 4
9-26 9 6 3 5 2 12 1 1 4 1 5 1 4 0 4 7
F F F F F F G N L F F F
F F F F F F F N N S Q F F F K D G
9
G
G
0498
33
5.1
*The encoded proteins for the C. trachomatis (CT) and C. pneumoniae (CPn) genome sequences were evaluated for leader peptide signal sequences using SPSCAN (GCG Wisconsin Package), and those with high scores for signal peptides were subjected to analysis by PSORT (http://psort.nibb.ac.jp), which provides a
probability estimate of outer membrane protein localization [26,27]. Additional evidence for outer membrane localization is the presence of a carboxyl-terminal phenylalanine [28]. C-term AA, carboxy-terminal amino acid single letter code; Cys, cysteine; pI, isoelectric point; #, assigned open reading frame reference number.
genomes. Omp85 is expressed and present in the chlamydial outer membrane complex, although when incorporated into liposomes, porin activity is not detectable (using solutes that diffuse through MOMP and PorB) [18]. Because Omp85 is surface-accessible to anti-Omp85 antibodies, and Omp85specific antibodies neutralize chlamydial infectivity in vitro (A Kubo, RS Stephens, unpublished data), it should be investigated for its vaccine potential.
apparatus is composed of a complex of many proteins that traverse the inner and outer membranes, as well as a needle structure that protrudes from the surface of the bacterium and penetrates the target eukaryotic cell. Although direct sequence-similarity comparisons to the S. typhimurium genome/type III secretion system apparatus provide little additional information about the composition of the chlamydial surface projections, the identifiable type III components in chlamydia are clustered in three operons and testing each protein associated with these operons is likely to resolve this question.
Type III secretion Salmonella typhimurium has small surface structures, which have been isolated and purified. The proteins that compose these structures were identified as components of the type III secretion system [19]. This system secretes proteins into target host cells and is essential for the virulence of a number of bacterial pathogens. Structures of similar size and morphology were described for Chlamydia over 20 years ago by Matsumoto [20], and later by Nichols [21]. The C. trachomatis and C. pneumoniae genomes contain identical genes for components of the machinery necessary for type III secretion, and it is presumed that the structures characterized by Matusmoto and Nichols are components of the chlamydial type III secretion system apparatus. Chlamydiae have orthologs of the YscC/GspD family, which is an outer membrane component of the type III secretion apparatus [7]. However, orthologous chlamydial proteins to PrgK and PrgH, which make up the S. typhimurium type III secretion system apparatus, were not identified. Likewise, a chlamydial ortholog was not found for the needle portion of the type III secretion apparatus, which is composed of a small protein, MxiH, in Shigella [22]. The type III secretion
Other predicted OMPs In addition to the genes encoding MOMP, PorB and Omp85 (which have been shown to be localized to the chlamydial outer membrane), there are several additional ORFs that encode proteins that can be predicted to be outer-membrane-localized (Table 1). Five proteins with strong predictions for outer membrane localization have orthologs in both C. trachomatis and C. pneumoniae (CT548/CPn0669, CT623/CPn0729, CT351/CPn0020, CT242/CPn0301 and CT476/CPn0595). CT351/CPn0020 share 61% amino acid sequence identity and each contains 12 cysteine residues. The other four orthologous protein pairs are not notable for their cysteine content but one, CT242/CPn0301, has weak similarity to Yersinia enterolitica OmpH. Two additional orthologous pairs, CT858/CPn1016 and CT021/CPn0111, are less confidently predicted for outer membrane localization and the orthologous pairs share only 40–48% sequence identity. However, they remain of interest for further investigation.
Chlamydia outer membrane protein discovery using genomics Stephens and Lammel
Six predicted OMPs in C. pneumoniae do not have orthologs in C. trachomatis. Two of these are cysteine-rich proteins, CPn0278 and CPn0498, and one of these, CPn0278, is similar to outer membrane lipoproteins from Deinococcus spp., Helicobacter spp. and E. coli. The remaining four C. pneumoniae proteins are encoded sequentially in a likely operon, CPn0795-CPn0798. If verified as outer membrane proteins, their operon organization suggests that they may act together in some function essential for C. pneumoniae but not for C. trachomatis. This suggests that these could be involved in the differential virulence or tropism of C. pneumoniae.
Conclusions The prediction of genes that are likely to encode chlamydial OMPs has led directly to the demonstration that Pmp, PorB and Omp85 are outer membrane surface proteins. This has greatly expanded the understanding of chlamydial outer membranes that were previously known to consist only of MOMP and LPS. Protein predictions from chlamydial genomic information have also proven useful for greatly expanding the number of inclusion membrane proteins (Inc). These proteins are secreted by chlamydiae to the vacuolar membrane of the chlamydial inclusion [23,24]. It was noted that for the three Inc proteins that had been characterized biochemically, each has a motif signature with bi-lobed hydrophobic domains at the amino terminus. Using these criteria, five of six Inc proteins predicted from the genome sequence were shown to be localized to the inclusion membrane [25••]. The surge in characterization of chlamydial proteins that are secreted or localized to the chlamydial outer membrane has been an outcome of the sequencing of the genomes of two species of Chlamydia. The expectation is that much more will result from the use of bioinformatics to approach functional characterization of the individual components of chlamydiae and, ultimately, their roles in the network of chlamydial biology both within chlamydiae and between chlamydiae and their host cells.
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