Characterization and intracellular localization of putative Chlamydia pneumoniae effector proteins

We here describe four proteins of Chlamydia pneumoniae, which might play a role in host-pathogen interaction. The hypothetical bacterial proteins CPn0708 and CPn0712 were detected in Chlamydia pneumoniae-infected host cells by indirect immunofluorescence tests with polyclonal antisera raised against the respective proteins. While CPn0708 was localized within the inclusion body, CPn0712 was identified in the inclusion membrane and in the surrounding host cell cytosol. CPn0712 colocalizes with actin, indicating its possible interaction with components of the cytoskeleton. Investigations on CPn0809 and CPn1020, two Chlamydia pneumoniae proteins previously described to be secreted into the host cell cytosol, revealed colocalization with calnexin, a marker for the ER. Neither CPn0712, CPn0809 nor CPn1020 were able to inhibit host cell apoptosis. Furthermore, transient expression of CPn0712, CPn0809 and CPn1020 by the host cell itself had no effect on subsequent infection with Chlamydia pneumoniae. However, microarray analysis of CPn0712-expressing host cells revealed six host cell genes which were regulated as in host cells infected with Chlamydia pneumoniae, indicating the principal usefulness of heterologous expression to study the effect of Chlamydia pneumoniae proteins on host cell modulation

Hydrodynamic Regulation of Monocyte Inflammatory Response to an Intracellular Pathogen

Systemic bacterial infections elicit inflammatory response that promotes acute or chronic complications such as sepsis, arthritis or atherosclerosis. Of interest, cells in circulation experience hydrodynamic shear forces, which have been shown to be a potent regulator of cellular function in the vasculature and play an important role in maintaining tissue homeostasis. In this study, we have examined the effect of shear forces due to blood flow in modulating the inflammatory response of cells to infection. Using an in vitro model, we analyzed the effects of physiological levels of shear stress on the inflammatory response of monocytes infected with chlamydia, an intracellular pathogen which causes bronchitis and is implicated in the development of atherosclerosis. We found that chlamydial infection alters the morphology of monocytes and trigger the release of pro-inflammatory cytokines TNF-α, IL-8, IL-1β and IL-6. We also found that the exposure of chlamydia-infected monocytes to short durations of arterial shear stress significantly enhances the secretion of cytokines in a time-dependent manner and the expression of surface adhesion molecule ICAM-1. As a functional consequence, infection and shear stress increased monocyte adhesion to endothelial cells under flow and in the activation and aggregation of platelets. Overall, our study demonstrates that shear stress enhances the inflammatory response of monocytes to infection, suggesting that mechanical forces may contribute to disease pathophysiology. These results provide a novel perspective on our understanding of systemic infection and inflammation

Chlamydial Infection Induces Pathobiotype-Specific Protein Tyrosine Phosphorylation in Epithelial Cells

Members of the genus Chlamydia are strict obligate intracellular pathogens that exhibit marked differences in host range and tissue tropism despite sharing a remarkable level of genomic synteny. These pathobiotype differences among chlamydiae are also mirrored in their early interactions with cultured mammalian host cells. Chlamydial attachment and entry is known to trigger protein tyrosine phosphorylation. In this study, we examined the kinetics and pattern of protein tyrosine phosphorylation induced by infection with a comprehensive collection of chlamydial strains exhibiting diversity in host, tissue, and disease tropisms. We report new findings showing that protein tyrosine phosphorylation patterns induced by infection directly correlate with the pathobiotype of the infecting organism. Patterns of protein tyrosine phosphorylation were induced following early infection that unambiguously categorized chlamydial pathobiotypes into four distinct groups: (i) Chlamydia trachomatis trachoma biovars (serovars A to H), (ii) C. trachomatis lymphogranuloma venereum biovars (serovars L1 to L3), (iii) C. muridarum, and (iv) C. pneumoniae and C. caviae. Notably, chlamydia-infected murine and human epithelial cells exhibited the same protein tyrosine phosphorylation patterns; this is indirect evidence suggesting that the phosphorylated protein(s) is of chlamydial origin. If our hypothesis is correct, these heretofore-uncharacterized proteins may represent a novel class of bacterial molecules that influence pathogen-host range or tissue tropism

Chlamydophila pneumoniae and Human Cytomegalovirus in Atherosclerotic Carotid Plaques - Combined Presence and Possible Interactions

The aim of our study was to investigate the combination of Chlamydophila pneumoniae and human cytomegalovirus (HCMV) as a pathogenic factor in atherosclerosis. Accordingly, we tested by means of PCR and immunohistochemistry the presence of these pathogens in the same atherosclerotic carotid specimen. The histology of the samples and the patients' antibodies against these pathogens were evaluated. Further, we examined the impact of C. pneumoniae and HCMV infection on the gene expression of the human monocytic cell line U937. Six of the 22 samples contained only C. pneumoniae, 4 contained only HCMV, 7 contained both C. pneumoniae DNA and/or antigens of both pathogens, and 5 samples were negative. No correlation was found between the presence of these microbes and either the cellular structure of the plaques, or the serostatus of the patients. The infection of U937 cells with HCMV and especially C. pneumoniae induced inflammation and atherosclerosis-related genes. Furthermore, the doubly-infected cells produced higher levels of the mRNA of pro-platelet basic protein and fatty acid binding protein 4. In conclusion, C. pneumoniae is often present in combination with HCMV in atherosclerotic carotid lesions. The in vitro coinfection model reveals that the doubly-infected monocytes are potent expressors of proatherosclerotic genes, suggesting that this coinfected population may accelerate the process of atherosclerosis

Generation of targeted Chlamydia trachomatis null mutants

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects hundreds of millions of individuals globally, causing blinding trachoma and sexually transmitted disease. More effective chlamydial control measures are needed, but progress toward this end has been severely hampered by the lack of a tenable chlamydial genetic system. Here, we describe a reverse-genetic approach to create isogenic C. trachomatis mutants. C. trachomatis was subjected to low-level ethyl methanesulfonate mutagenesis to generate chlamydiae that contained less then one mutation per genome. Mutagenized organisms were expanded in small subpopulations that were screened for mutations by digesting denatured and reannealed PCR amplicons of the target gene with the mismatch specific endonuclease CEL I. Subpopulations with mutations were then sequenced for the target region and plaque-cloned if the desired mutation was detected. We demonstrate the utility of this approach by isolating a tryptophan synthase gene (trpB) null mutant that was otherwise isogenic to its parental clone as shown by de novo genome sequencing. The mutant was incapable of avoiding the anti-microbial effect of IFN-γ–induced tryptophan starvation. The ability to genetically manipulate chlamydiae is a major advancement that will enhance our understanding of chlamydial pathogenesis and accelerate the development of new anti-chlamydial therapeutic control measures. Additionally, this strategy could be applied to other medically important bacterial pathogens with no or difficult genetic systems

Chlamydial IFN-γ immune evasion is linked to host infection tropism

Chlamydiae are obligate intracellular pathogens that can exhibit a broad host range in infection tropism despite maintaining near genomic identity. Here, we have investigated the molecular basis for this unique host-pathogen relationship. We show that human and murine chlamydial infection tropism is linked to unique host and pathogen genes that have coevolved in response to host immunity. This intimate host-pathogen niche revolves around a restricted repertoire of host species-specific IFN-γ-mediated effector responses and chlamydial virulence factors capable of inhibiting these effector mechanisms. In human epithelial cells, IFN-γ induces indoleamine 2,3-dioxygenase expression that inhibits chlamydial growth by depleting host tryptophan pools. Human chlamydial strains, but not the mouse strain, avoid this response by the production of tryptophan synthase that rescues them from tryptophan starvation. Conversely, in murine epithelial cells IFN-γ induces expression of p47 GTPases, but not indoleamine 2,3-dioxygenase. One of these p47 GTPases (Iigp1) was shown by small interfering RNA silencing experiments to specifically inhibit human strains, but not the mouse strain. Like human strains and their host cells, the murine strain has coevolved with its murine host by producing a large toxin possessing YopT homology, possibly to circumvent host GTPases. Collectively, our findings show chlamydial host infection tropism is determined by IFN-γ-mediated immunity