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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-[gamma]-mediated effector responses and chlamydial virulence factors capable of inhibiting these effector mechanisms. In human epithelial cells, IFN-[gamma] 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-[gamma] induces expression of p47 GTPases, but not indoleamine 2,3-dioxygenase. One of these p47 GTPases (ligp1) 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-[gamma]-mediated immunity. immunity | virulence factors | pathogenesis | defense | coevolution

Background: Recombinant peptide chips could constitute a versatile complementation to state-of-the-art in situ (chemical on-chip) synthesis, particle-based printing or pre-manufactured peptide spotting. Bottlenecks still impeding a routine implementation - from restricted peptide lengths, low diversity and low array densities to high costs - could so be overcome. Methods: To assess overall performance, we assembled recombinant chips composed of 38,400 individual peptide spots on the area of a standard 96-well microtiter plate from comprehensive, highly diverse (>107 single clones) short random peptide libraries. Results: Screening of altogether 476,160 clones against Streptavidin uncovered 2 discrete new binders: a characteristic HPQ-motif containing VSHPQAPF and a cyclic CSGSYGSC peptide. Interactions were technically confirmed by fluorescence polarization as well as biolayer-interferometry and their potential suitability as novel detection tags evaluated by detection of a peptide-fused exemplary test protein. Conclusion: From our data we conclude that the presented technical pipeline can reliably identify novel hits, useful as first-generation binders or templates for subsequent ligand design plus engineering. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis is an obligate intracellular bacterium causing infections of the eyes, urogenital and respiratory tracts. Asymptomatic, repeat and chronic infections with C. trachomatis are common in the urogenital tract potentially causing severe reproductive pathology. Animal models of infection and epidemiological studies suggested the gastrointestinal tract as a reservoir of chlamydiae and as a source of repeat urogenital infections. Thus, we investigated the growth characteristics of C. trachomatis in human intestinal epithelial Caco-2 cells and the infection-induced defensin production. Immunofluorescence staining and transmission electron microscopy showed the presence of chlamydial inclusions in the cells. Chlamydial DNA and viable C. trachomatis were recovered from Caco-2 cells in similar quantity compared to that detected in the usual in vitro host cell of this bacterium. The kinetics of expression of selected C. trachomatis genes in Caco-2 cells indicated prolonged replication with persisting high expression level of late genes and of heat shock protein gene groEL. Replication of C. trachomatis induced moderate level of β-defensin-2 production by Caco-2 cells, which might contribute to avoidance of immune recognition in the intestine. According to our results, Caco-2 cells support C. trachomatis replication, suggesting that the gastrointestinal tract is a site of residence for these bacteria.

Chlamydiae are obligate intracellular bacteria that propagate in the inclusion, a specific niche inside the host cell. The standard method for counting chlamydiae is immunofluorescent staining and manual counting of chlamydial inclusions. High- or medium-throughput estimation of the reduction in chlamydial inclusions should be the basis of testing antichlamydial compounds and other drugs that positively or negatively influence chlamydial growth, yet low-throughput manual counting is the common approach. To overcome the time-consuming and subjective manual counting, we developed an automatic inclusion-counting system based on a commercially available DNA chip scanner. Fluorescently labeled inclusions are detected by the scanner, and the image is processed by ChlamyCount, a custom plug-in of the ImageJ software environment. ChlamyCount was able to measure the inclusion counts over a 1-log-unit dynamic range with a high correlation to the theoretical counts. ChlamyCount was capable of accurately determining the MICs of the novel antimicrobial compound PCC00213 and the already known antichlamydial antibiotics moxifloxacin and tetracycline. ChlamyCount was also able to measure the chlamydial growth-altering effect of drugs that influence host-bacterium interaction, such as gamma interferon, DEAE-dextran, and cycloheximide. ChlamyCount is an easily adaptable system for testing antichlamydial antimicrobials and other compounds that influence Chlamydia-host interactions.

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.