< > Home : FAQ : Professional : Chlamydiales : Immunobiology : Infections : Diagnosis & Treatment : Links : Contact Us

Interactions with the host cell:

Inc proteins of the inclusion membrane.

From within the inclusion, chlamydiae have to interact with the host cell across the inclusion membrane to ensure their survival. It has long been evident that the inclusion membrane must be modified by chlamydiae in some way, but, for a long time, technical problems got in the way of progress. A new approach was required, which was provided by Rockey and colleagues in 1995 [Rockey et al., 1995]. They first created an "expression library" of chlamydial proteins from assorted genes of C. caviae, the former C. psittaci GPIC agent. Antisera [mixed antibodies] were then prepared against purified chlamydiae themselves [to identify chlamydial elementary body-associated proteins] or were collected from animals convalescing from chlamydial infection [which additionally would react with any chlamydial proteins uniquely produced during intracellular infection]. This resulted in the identification of a gene encoding a protein of molecular weight 39 kDa  only recognised by convalescent sera. Subsequently the protein encoded by this gene was expressed and antibody was prepared against the recombinant [genetically engineered] protein. Using the antibody as a specific probe, it was found that the protein was present in infected cell lysates and reticulate bodies only [reticulate bodies are the intracellular replicating form of chlamydiae]. Virtually none of the protein was detected in elementary bodies [the extracellular infectious stage]. Furthermore, when the antibody tagged with fluorescent dye was used in conjunction with fluorescence microscopy on  chlamydial infected cells, it became clear that the protein was associated with the inclusion membrane. Accordingly the protein was named the IncA protein [Rockey et al., 1995], encoded by the incA gene. Unexpectedly the protein was also present in dramatic fibrillar structures which extended from the inclusion over the nucleus or into the cytoplasm of infected cells.

Subsequently, it was found that C. caviae IncA faces the cytoplasmic (i.e. host cell) side of the inclusion membrane where it is phosphorylated by host cell kinases at its serine and threonine residues [Rockey et al., 1997]. As host cell kinases are involved in intracellular signalling pathways, it seemed likely that IncA might subvert host cell signalling to serve the chlamydial invader. Moreover antibodies to IncA are also found in human subjects recovering from C. trachomatis infection [Bannantine et al., 1998]. A function for IncA is suggested by the finding that certain C. trachomatis strains that do not express an IncA homologue produced uncharacteristic multiple inclusions in cells [Suchland et al., 2000]. Furthermore, when an IncA-specific monoclonal antibody was micro-injected into C. trachomatis infected cells, it provoked the development of aberrant, multi-lobed inclusions [Hackstadt et al., 1999]. A key feature of the C. trachomatis developmental cycle is the fusion of small homotypic inclusions containing chlamydial reticulate bodies, into one large inclusion. This appears to be one of the functions of the Inc proteins. Critically for chlamydial survival, Inc proteins may also be involved in preventing fusion between the chlamydial endosome and inclusion and  lysosomes, and in getting nutrients from the cytosol of the host cell across the inclusion membrane to the chlamydiae [Hackstadt et al., 1997; Wyrick, 2001]. 

At least 11 different Inc proteins are known in C. trachomatis. In addition to IncA there are: IncB to IncG and IncS [Bannantine et al., 1998;  Stephens et al., 1998; Scidmore-Carlson et al., 1999; Kalman et al., 1999]. The functions of these proteins are still being identified [Hackstadt et al., 1997; Hackstadt 1999; Rockey & Matsumoto, 1999]. The various Inc proteins have very low  primary sequence homology, though a typical bilobed hydrophobic domain of 50 - 8- amino acids is present in all of them [Bannantine et al., 2000; Rockey et al., 2002]. Genes incB and incC are clustered together in one operon as are incD - G. Furthermore, incD - G, but not incA, are transcribed within the first two hours after the endocytosis of chlamydial elementary bodies, perhaps indicating involvement in modification of the nascent chlamydial inclusion. Whole genomic sequencing indicates that the inc genes are conserved among the family Chlamydiaceae. In another study, computer prediction identified 90 proteins in the C. pneumoniae J138 genome sequence and 36 in the C. trachomatis serovar D sequence that had hydropathy profiles similar to the inc proteins. Only a few Inc-like open reading frames were found in other organisms, suggesting that these proteins are unique to the chlamydiae. Comparative genome analysis suggests that the Inc-like open reading frames have multiplied and diverged as paralogues and orthologues in the chlamydial genomes and that some of them lacked the N-terminal portion or encoded a split form. The Inc-like gene products constitute a large protein family likely to play an important and unique role in chlamydial infection, growth and survival in the host cell [Toh et al., 2003].

Rare C. trachomatis strains with unusual non fusogenic inclusions have been identified [Suchland et al., 2000]. These strains lack IncA in the inclusion membrane. Sequence analysis of two strains with the non fusogenic characteristic showed the presence of two non synonymous mutations in IncA. One of these mutations, I47T leads to the replacement of isoleucine at codon 47 with threonine, modifying the characteristic hydrophobic domain of the protein. The second mutation E116K leads to acidic glutamate being exchanged for basic lysine at codon 116 outside the hydrophobic domain. C. trachomatis IncA is not transported to the inclusion membrane at 32C, at which temperature inclusion fusion does not occur [Fields & Hackstadt, 2002]. 

Pannekoek et al., 2001 sequenced the incA gene of 25 isolates covering the different serovars to test the strength between observed I47T mutation in the translated protein and non fusogenic phenotype .  Four major different IncA sequence types were found among the isolates [These are readily accessed using the nucleotide search section of the Multi-find facility on the home page following registration and log in]. Seven of the 25 isolates had the I47T substitution but they were not associated with a deficiency in IncA expression, a failure of IncA translocation to the inclusion membrane, or a failure of inclusion fusion. Thus I47T substitution does not necessarily cause non fusogenicity; more sophisticated hypotheses are necessary [Pannekoek et al., 2001].  Rockey et al., 2002 mention briefly that they have  observed a variety of IncA mutations which are associated with non fusogenic phenotype.  Geisler et al., 2001 reported that those non fusogenic strains which were unable to make IncA had a greater tendency to cause subclinical infection in natural human genital tract infection. [This observation needs to be confirmed by other studies in different geographical locations]. The IncG protein, in contrast, is a target for the cell signalling protein 14-3-3 and may be a means by which chlamydiae modulate the cell cycle, apoptosis and mitogenic signal transduction [See: MAPK signalling].

It is puzzling that, although the Inc proteins are membrane-associated proteins, they generally lack the characteristic signal peptides that target nascent proteins into bio-membranes. One speculation is that chlamydiae inject the Inc proteins into inclusion membrane using a type three secretion system [a kind of syringe for injecting bacterial proteins into host cells]. Chlamydiae have many of the genes for encoding a type three secretion / delivery system. Subtil et al., 2001 showed that the N-terminus, including the hydrophobicity motif, of IncA, IncB, IncC and three candidate Inc proteins are sufficient for targeting a fusion protein through the Shigella flexneri type three secretory apparatus. Moreover it has been suggested [but not yet proven] that the chlamydial surface projections observed penetrating the adjacent inclusion membrane might be part of such a system [Rockey & Matsumoto, 1999]. These are attractive hypotheses ultimately testable by experiment.

Two other proteins are found in the inclusion membrane that do not have the typical bi-lobed hydrophobic domain of the Inc proteins. These are: CopN, which, analagous to YopN of Yersinia species, is likely associated with a chlamydial type three secretion system [Fields & Hackstadt, 2000] and Cap1, a protein of unknown function, which generated a protective CD8+ T cell response in mice [Fling et al., 2001; See: Cap1].

[Comment: The discovery by Daniel Rockey, Ted Hackstadt and colleagues of the Inc proteins back in 1994, before the results of genomic sequencing, was a scientific tour de force. Their approach at the time was risky, but it paid off and  indicated what had long been suspected; that chlamydiae must exert control via the inclusion membrane on the cytoplasm of the host cell. Subsequently, genomic sequencing has revealed that chlamydiae have a number of environmental sensor mechanisms].

[MEW] August 2003

NEXT: Inc proteins and MAPK signalling

INDEX: Biology index.

References

Bannantine, J. P., Stamm, W. E., Suchland, P. & Rockey, D. D. (1998). C. trachomatis IncA is localized to the inclusion membrane and is recognized by antisera from infected humans and primates. Infection and Immunity 66, 6017 - 6021. Full article

Bannantine, J. P., Griffiths, R. S., Viratyosin, W., Brown, W. J. & Rockey, D. D. (2000). A secondary structure motif predictive of protein localization to the chlamydial inclusion membrane. Cellular Microbiology 2, 35 - 47.  

Fields, K. A., Fischer, E. & Hackstadt, T. (2002). Inhibition of fusion of Chlamydia trachomatis inclusions at 32 degrees C correlates with restricted export of IncA. Infection and Immunity 70, 3816 - 3823.

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.

Geisler, W. M., Suchland, R. J., Rockey, D. D. & Stamm, W. E. (2001). Epidemiology and clinical manifestations of unique Chlamydia trachomatis isolates that occupy nonfusogenic inclusions. Journal of Infectious Diseases 184, 879 - 884.

Hackstadt, T., Fischer, E. R., Scidmore, M. A., Rockey, D. D. & Heinzen, R. A. (1997). Origins and functions of the chlamydial inclusion. Trends in Microbiology 5, 288 293. [Review].

Hackstadt, T. (1999). Cell biology. In Chlamydia: Intracellular Biology, Pathogenesis, and Immunity, pp. 101-138. Edited by R. S. Stephens. Washington, D.C.: ASM Press.

Hackstadt, T., Scidmore-Carlson, M. A., Shaw, E. I. & Fischer, E. R. (1999). The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion. Cellular Microbiology 1, 119 - 130.  

Kalman, S., Mitchell, W., Marathe, R., et al., (1999). Comparative genomes of Chlamydia pneumoniae and C. trachomatis. Nature Genetics 21, 385-389. Full article Chromosome map C. pneumoniae Functional assignments table of genes in C. pneumoniae and C. trachomatis 

Pannekoek, Y., van der Ende, A., Eijk, P. P., van Marle, J., de Witte, M. A., Ossewaarde, J. M., van den Brule, A. J., Morre, S. A. & Dankert, J. (2001). Normal IncA expression and fusogenicity of inclusions in Chlamydia trachomatis isolates with the incA I47T mutation. Infection and Immunity 69, 4654 - 4656. Full article    

Rockey, D. D., Heinzen, R. A. & Hackstadt, T. (1995). Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells. Molecular Microbiology 15, 617 - 626.

Rockey, D. D., Grosenbach, D., Hruby, D. E. et al., (1997). Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion. Molecular Microbiology 24, 217 - 228.  

Rockey, D. D. & Matsumoto, A. (1999). The chlamydial developmental cycle. In Prokaryotic Development, pp. 403 - 425. Edited by Y. V. Brun & L. J. Shimkets. Washington, D.C.: ASM Press.

Rockey, D. D., Lenart, J. & Stephens, R. S. (2000). Genome sequencing and our understanding of chlamydiae. Infection and Immunity 68, [Useful review].

Rockey, D. D., Scidmore, M. A., Bannantine, J. P., Brown, W. J. (2002). Proteins in the chlamydial inclusion membrane. Microbes and Infection 4, 333 - 340. [Key Review superseding the previous review]. Full article

Scidmore-Carlson, M. A., Shaw, E. I., Dooley, C. A. et al., (1999). Identification and characterization of a Chlamydia trachomatis early operon encoding four novel inclusion membrane proteins. Molecular Microbiology 33, 753 - 765.

Stephens, R. S., Kalman, S., Lammel, C. et al., (1998). Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282, 754 - 759. + [The first genomic sequence, and high quality]

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.

Suchland, R. J., Rockey, D. D., Bannantine, J. P. & Stamm, W. E. (2000). Isolates of Chlamydia trachomatis that occupy non-fusogenic inclusions lack IncA, a protein localized to the inclusion membrane. Infection and Immunity 68, 360 - 367. Full article

Toh, H., Miura, K., Shirai, M. & Hattori, M. (2003). In silico inference of inclusion membrane protein family in obligate intracellular parasites chlamydiae. DNA Research 10, 9 - 17.

Wyrick, P. B. (2000). Intracellular survival by Chlamydia. Cellular Microbiology 2, 275 - 282. [Useful review].

NEXT: Inc proteins and MAPK signalling