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Type three secretion in chlamydiae.

The identification of genes in chlamydiae encoding a type three secretion (tts) system provides a newly understood mechanism by which chlamydiae might interact with the host cell. The tts system functions as a kind of 'molecular syringe', enabling gram-negative bacteria to inject virulence-related proteins into the cytoplasm of host cells [Hueck, 1998]. The tts system differs from all other bacterial secretion mechanisms in that it requires and is triggered by the intimate contact of the bacterium with host cell membrane. The tts system is found in a wide range of Gram negative bacteria including  Yersinia, Salmonella, Shigella, Escherichia, Pseudomonas, Bordetella, Burkholderia, a number of plant pathogens or symbionts and of course Chlamydia [Winstanley & Hart, 2001]. Many but by no means all of these bacteria are facultative or obligate intracellular bacteria. Bacterial proteins injected via the tts system function to subvert the host cell for the benefit of the bacterium. In the case of chlamydiae, tts activity may be triggered extracellularly when the EB comes into contact with the host cell membrane, and intracellularly when it comes into contact with the inclusion membrane into which effector proteins are secreted and may become either embedded or alternatively translocated into the host cell cytoplasm [Hsia et al., 1997], possibly through the surface rosettes and fibres [Rockey & Matsumoto, 1999]. These stuctures locate to the area of contact between the bacterium and the host-derived inclusion membrane (see figures below) and are, superficially at least, similar to the tts organelles of Salmonella [Kubori et al., 1998].

It has been suggested that the oft-seen juxtaposition of reticulate bodies with the inclusion membrane might indicate that activation of the tts is a requisite trigger for the binary fission of reticulate bodies. Following active division, overcrowding might lead to detachment from the inclusion membrane, with tts inactivation a possible trigger for late differentiation, [Hackstadt et al., 1997; Rockey & Matsumoto, 1999]. Currently there is little supporting evidence for this hypothesis other than preliminary studies which suggest that some components of the tts system are either at middle or late in the developmental cycle [Kubo & Stephens, 1998]. The apparent juxtaposition of chlamydial reticulate bodies with the inclusion membrane might alternatively be fortuitous; an inevitable consequence of active reticulate body division within a finite, membrane-bound space. The contact activation hypothesis conflicts with the alternative notion of a temporally regulated developmental cycle, a developmental clock, implicit in circular representations of the cycle. However it would explain why chlamydial development, initially almost synchronous, becomes asynchronous in the mature inclusion as elementary bodies differentiate from reticulate bodies [Bavoil et al., 2000]. If a chlamydial tts system is triggered by membrane contact, then perhaps, as for the Ipa proteins of Shigella flexneri, the "pay-load" protein(s) are pre-synthesised 'ready to go' [Hueck, 1998]. Compared with reticulate bodies, elementary bodies too, although metabolically inert, are studded with a [lesser] number of tts-like surface projections, [see: elementary body structure]. Speculatively one can think of elementary bodies as being like primed mines, with the tts projections the detonator that releases pre-formed proteins.   It is necessary to postulate some such rapid mechanism to explain why phagolysosomal fusion is blocked by viable but not dead chlamydiae before early chlamydial differentiation gets under way [Bavoil et al., 2000]. However, in Yersinia species the crucial Yop proteins are only synthesised following activation of the tts system [Hueck, 1998]. Obvious candidates for tts secretion are the inclusion proteins, which lack the signal peptides otherwise required to target them to the inclusion membrane [Rockey & Matsumoto, 1999]. Tts-secreted proteins,  as in the Salmonella pathogenicity island 2, are likely to be important for chlamydial survival in macrophages [Bavoil et al., 2000]. Other likely tts-mediated functions include: the transportation of essential nutrients into the inclusion; blocking acidification of the nascent chlamydial endosome and its fusion with lysosomes; peturbation of host cell signalling events and membrane recycling and the modulation of apoptosis

Genome sequencing has shown that chlamydiae have most of the genes for a tts system [Stephens et al., 1998]. However, the genetic organisation of the tts system in chlamydiae is unlike that of some other gram negative pathogens, e.g. Salmonella or Yersinia [Winstanley & Hart, 2001] in that they are scattered across at least four distinct locations [Stephens et al., 1998] rather than clustered in obvious "pathogenicity islands". The presence of the necessary genes makes it likely that they are functional, but does not prove it. In the absence of systems for the genetic manipulation of chlamydiae, the necessary proofs are somewhat tedious, involving the determination of whether cloned paralogous proteins of the chlamydial tts system are able to compensate the tts system of other more amenable bacteria that has been functionally compromised in the corresponding gene. Fields & Hackstadt, 2000 focused on a locus containing genes encoding potential tts-secreted proteins including chaperones (Scc1), secretion pore components (Cds1 and Cds2) and CopN, the chlamydial homologue of the YopN tts-secreted protein of Yersinia.  Gene expression (specific RNA production) was tested by RT-PCR on RNA extracted from infected HeLa cell monolayers at 2, 6, 12 and 20 h after infection and normalized for the number of C. trachomatis genomes present. Message for Scc1 was detected at all times, whereas message for all other tested genes was detected in significant amounts at 12 and 20 hours post-infection. Expressed CopN and Scc1 proteins were demonstrated in elementary bodies, reticulate bodies and whole-culture extracts harvested 20 hours after infection. CopN but not Scc1 was demonstrated 20 hours after infection in the inclusion membrane. The putative chlamydial tts protein CopN, but not but not control cytoplasmic protein NrdB, was secreted by Y. enterocolitica in a Ca2+- and Yersinia virulence plasmid-dependent fashion. These data indicate that components of the putative type III apparatus of C. trachomatis are expressed and that at least one of these products is secreted by chlamydiae to the inclusion membrane. The observation that CopN is also secreted by the Yersinia type III apparatus provides elegant support for the hypothesis that chlamydiae secrete proteins via a tts mechanism [Fields & Hackstadt, 2000]. More recently, Subtil et al., 2001 have shown that chimeras of the N terminal portion of the chlamydial inclusion proteins IncA, IncB and IncC  with the reporter Cya protein of Bordetella pertussis [whooping cough] are secreted by the type III secretion system of Shigella flexneri. Again, this is elegant work strongly supporting the hypothesis that chlamydiae have a functional type III secretion pathway with which to deliver effector proteins into the eukaryotic host cell.

It has been suggested that the type III system may have evolved from the flagellar export system [Kim, 2001], perhaps in the last common ancestor ; [see: Moulder: evolution of chlamydial type III secretion]. 

When  chlamydiae are stressed by effectors of the cell mediated immune system, tts systems may become important for survival. Indeed, using gamma interferon to induce persistent C. pneumoniae infection, the type III secretion (SctN) protein was one of a group of proteins [others included MOMP, heat shock protein 60 (Hsp-60/GroEL), DNA gyrase (GyrA), and proteins concerned with transcription (RpoA, PnP), translation (Rrf) and glycolysis (PgK, GlgP)]. Thus SctN among these other proteins may help chlamydiae to resist stress from effectors of the T helper 1 cellular immune system [Molestina et al., 2002]. 

In another proteomics study of purified elementary bodies of C. pneumoniae VR 1390 , 167 genes representing 15% of the coding capacity of the genome were identified, including 31 hypothetical proteins. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk. [VanDahl et al., 2001].  Some of these proteins are shown in the table below, which is derived from the Aarhus University proteome database [VanDahl et al., 2001 & Shaw et al., 2002]. Most relevantly,  a number of  proteins of the putative C. pneumoniae type III secretion apparatus have been shown to be expressed [Table 1]. 

Table 1: Known expressed type III secretion mechanism proteins, C. pneumoniae VR 1390. Source Aarhus and STD genome databases. Click on Aarhus accession hyperlink and then on map for location of protein on 2D gels. Go to the STD database or other genomic databases and search using accession number or name of gene for detailed information on each protein.

At least 13 C. pneumoniae genes are thought to be homologous with other known type III secretion systems. The expression of these genes during C. pneumoniae infection of HEp 2 cells has been followed by rtPCR with and without the presence of gamma interferon, a cytokine thought to induce persistent chlamydial infection. Expression of groEL-1, ompA, and omcB genes were used as markers for the early, middle, and late stages of the developmental cycle, respectively. Inhibition of expression of the fstK gene was used as a marker for the effect of gamma interferon on C. pneumoniae. In the absence of gamma interferon, the putative type III secretion genes were expressed as follows:

• early cycle: (1.5 to 8 h), yscC, yscS, yscL, yscJ and lcrH-2;
• mid cycle (12 to 18 h post infection), lcrD, yscN, and yscR;
• late cycle (24 h), lcrE, sycE, lcrH-1, and yscT.

Expression of the yscU gene was not detected at any time point. Using gamma interferon as a model for persistent chlamydial infection, the cluster of type III secretion genes that were normally not expressed until mid to late cycle, i.e. lcrD, lcrE, and sycE, as well as lcrH-1, were down-regulated. Expression of other type III secretion  genes was unaffected by gamma interferon.  The lcrH-1 and lcrH-2 genes differed from one another in both their temporal expression and response to gamma interferon. In other bacteria, these genes code for proteins that regulate effector protein synthesis as well as serving as chaperones for proteins that provide for the translocation of bacterial effector proteins into the host cell. Thus, the expression pattern of the type III secretion genes of C. pneumoniae is temporally regulated throughout the developmental cycle and those genes expressed in the later stages of development are down-regulated when the organism is grown in the presence of gamma interferon [Slepenkin et al., 2003].

By proteomics, the core type III apparatus secretion component, CdsJ was identified in both elementary bodies and reticulate bodies of C. trachomatis and it has been shown that the chlamydial IncC inclusion protein can be secreted by the classic Yersinia pseudotuberculosis type III secretion apparatus. It was suggested that type III secretion pores present on the EB surface mediate secretion of early Inc proteins [which help modify the fusogenic properties of the early chlamydial vacuole] and possible other effectors. Mid cycle expression of type III genes would replenish the secretion apparatus on reticulate bodies and thereby serve as a source of secretion pores for the EBs derived from them [Fields et al., 2003].

[Comment: There is little doubt that chlamydiae do have a functioning type III secretion apparatus, although it is difficult to prove. The high degree of conservation in both C. trachomatis and C. pneumoniae of components of this system suggests that it might have been present early in chlamydial evolution, being retained because it confers, as yet unknown, survival advantages. The Parachlamydia UWE25 genome sequencing project at Munich [see: Parachlamydia] should provide information on whether this system was conserved before the divergence of the Chlamydiaceae].

[MEW] Updated June 2003

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References.

Bavoil, P. M. & Hsia, R-C. (1998). Type III secretion in Chlamydia. A case of déjà vu? Molecular Microbiology 28, 860 - 862.

Bavoil, P., Hsia, R-C. & Ojcius, D. M. (2000). Closing in on Chlamydia and its intracellular bag of tricks. Microbiology 146, 2723 - 2731 Full article   [Interesting, well written and illustrated review]

Fields, K. A. & Hackstadt, T. (2000). Evidence for the secretion of Chlamydia trachomatis CopN by a type III secretion mechanism. Molecular Microbiology 38, 1048 -1060.

Fields, K. A., Mead, D. J., Dooley, C. A. & Hackstadt. T. (2003). Chlamydia trachomatis type III secretion: evidence for a functional apparatus during early-cycle development. Molecular Microbiology 48, 671 - 683.

Hackstadt, T., Fischer, E. R., Scidmore, M. A. et al., (1997). Origins and functions of the chlamydial inclusion. Trends in Microbiology 5, 288 - 293.

Hsia, R-C., Pannekoek, Y., Ingerowski, E. & Bavoil, P. M. (1997). Type III secretion genes identify a putative virulence locus of Chlamydia. Molecular Microbiology 25, 351-359. [The paper that started interest in the type iii system of chlamydiae, in this case C. caviae].

Hueck, C. J. (1998). Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiology and Molecular Biology Reviews 62, 379 - 433. Full article [Excellent review, extraordinary scope]

Kim, J. F. (2001). Revisiting the chlamydial type III protein secretion system: clues to the origin of type III protein secretion. Trends in Genetics 17, 65 - 69.

Kubo, A. & Stephens, R. S. (1998). Temporal differences in transcription of type III secretion genes during the Chlamydia trachomatis developmental cycle. Page 539-542. In: Chlamydial infections. Proceedings of the ninth international symposium on human chlamydial infection. (Stephens, R. S. et al., eds). International Chlamydia Symposium, San Francisco, ISBN 0-9664383-02.

Kubori, T., Matsushima, Y., Nakamura, D. et al., (1998). Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280, 602-605.

Molestina, R. E., Klein, J. B., Miller, R. D., Pierce, W. H., Ramirez, J. A. & Summersgill, J. T. (2002). Proteomic analysis of differentially expressed Chlamydia pneumoniae genes during persistent infection of HEp-2 cells. Infection and Immunity 70, 2976 - 2981.

Subtil, A., Parsot, C. & Dautry-Varsat, A. (2001). Secretion of predicted Inc proteins of Chlamydia pneumoniae by a heterologous type III machinery. Molecular Microbiology 39, 792 - 800.

van Dahl, B. B., Birkelund, S. , Demol, H., Hoorelbeke, B., Christiansen, G., Vandekerckhove, J. & Gevaert K. (2001). Proteome analysis of the Chlamydia pneumoniae elementary body. Electrophoresis 22, 1204 - 1223.

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