Dataset Information

Chlamydial development is blocked in host cells transfected with Chlamydophila caviae incA.

ABSTRACT:

Background

Chlamydiae produce a set of proteins, termed Inc proteins, that are localized to the inclusion membrane and exposed to the host cell cytosol. Little information exists regarding the interaction of Inc proteins with the eukaryotic cell. To examine these interactions, Vaccinia virus vectors and mammalian plasmid-based systems were used to express inc genes in mammalian cells.

Results

Cells transfected with plasmids expressing Chlamydophila caviae incA were not productively infected by C. caviae. Expression of C. caviae incA also reduced inclusion formation by Chlamydia trachomatis, but not to the degree seen for C. caviae. Chlamydia trachomatis incA did not block development of either C. trachomatis or C. caviae. Deletion mutagenesis was used to demonstrate that plasmids encoding either the amino or carboxy-terminal regions of the protein, as well as the changing of a single amino acid within IncA (serine 17) could not block C. caviae infection. Immunoblot analysis of truncated IncA in a Vaccinia virus system provided evidence that serine 17 of C. caviae IncA is a target for phosphorylation.

Conclusions

These experiments provide insight into the interaction of Inc proteins with the host cell and introduce a model system where these interactions can be explored further.

SUBMITTER: Alzhanov D

PROVIDER: S-EPMC459217 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Publications

Chlamydial development is blocked in host cells transfected with Chlamydophila caviae incA.

Alzhanov Damir D Barnes Jennifer J Hruby Dennis E DE Rockey Daniel D DD

BMC microbiology 20040701

<h4>Background</h4>Chlamydiae produce a set of proteins, termed Inc proteins, that are localized to the inclusion membrane and exposed to the host cell cytosol. Little information exists regarding the interaction of Inc proteins with the eukaryotic cell. To examine these interactions, Vaccinia virus vectors and mammalian plasmid-based systems were used to express inc genes in mammalian cells.<h4>Results</h4>Cells transfected with plasmids expressing Chlamydophila caviae incA were not productivel ...[more]

PMID: 15230981

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Project description:UnlabelledChlamydia trachomatis is an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (?pgp1 to ?pgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and ?pgp4 organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknown pgp4-regulated chromosomal T3S effector gene.ImportanceChlamydia's obligate intracellular life style requires both entry into and exit from host cells. Virulence factors that function in exiting are unknown. The chlamydial inclusion is stabilized late in the infection cycle by F-actin. A prerequisite of chlamydial exit is its ability to disassemble actin from the inclusion. We show that chlamydial plasmid-free organisms, and also a plasmid gene protein 4 (pgp4) null mutant, do not disassociate actin from the inclusion and fail to exit cells. We further provide evidence that Pgp4-regulated exit is dependent on the chlamydial type III secretion system. This study is the first to define a genetic mechanism that functions in chlamydial lytic exit from host cells. The findings also have practical implications for understanding why plasmid-free chlamydiae are highly attenuated and have the ability to elicit robust protective immune responses.

Project description:BackgroundThe chlamydial developmental cycle involves the alternation between the metabolically inert elementary body (EB) and the replicating reticulate body (RB). The triggers that mediate the interchange between these particle types are unknown and yet this is crucial for understanding basic Chlamydia biology.Presentation of the hypothesisWe have proposed a hypothesis to explain key chlamydial developmental events whereby RBs are replicating strictly whilst in contact with the host cell membrane-derived inclusion via type three secretion (T3S) injectisomes. As the inclusion expands, the contact between each RB and the inclusion membrane decreases, eventually reaching a threshold, beyond which T3S is inactivated upon detachment and this is the signal for RB-to-EB differentiation.Testing the hypothesisWe explore this hypothesis through the development of a detailed mathematical model. The model uses knowledge and data of the biological system wherever available and simulates the chlamydial developmental cycle under the assumptions of the hypothesis in order to predict various outcomes and implications under a number of scenarios.Implications of the hypothesisWe show that the concept of in vitro persistent infection is not only consistent with the hypothesis but in fact an implication of it. We show that increasing the RB radius, and/or the maximum length of T3S needles mediating contact between RBs and the inclusion membrane, and/or the number of inclusions per infected cell, will contribute to the development of persistent infection. The RB radius is the most important determinant of whether persistent infection would ensue, and subsequently, the magnitude of the EB yield. We determine relationships between the length of the T3S needle and the RB radius within an inclusion, and between the RB radius and the number of inclusions per host cell to predict whether persistent infection or normal development would occur within a host cell. These results are all testable experimentally and could lead to significantly greater understanding of one of the most crucial steps in chlamydial development.

Project description:The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Dataset Information

Chlamydial development is blocked in host cells transfected with Chlamydophila caviae incA.

Background

Results

Conclusions

Publications

Chlamydial development is blocked in host cells transfected with Chlamydophila caviae incA.

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