Chlamydia trachomatis variant with nonfusing inclusions: growth dynamic and host-cell transcriptional response.
ABSTRACT We compared growth rate and host-cell transcriptional responses of a Chlamydia trachomatis variant strain and a prototype strain. Growth dynamics were estimated by 16S rRNA level and by inclusion-forming units (IFUs) at different times after infection in HeLa cells. When inoculated at the same multiplicity of infection and observed 24-48 h after infection, the variant 16S rRNA transcriptional level was 3%-4% that of the prototype, and the IFUs of the variant strain were 0.1%-1% those of the prototype. Specific host-cell transcriptional responses to the variant were identified in a global-expression microarray in which variant strain-infected cells were compared with mock-infected and prototype strain-infected cells. In variant strain-infected cells, 47% (16/34) of specifically induced host genes were related to immunity and 32% (8/25) of specifically suppressed genes were related to lipid metabolism. The variant strain grew significantly more slowly and induced a modified host-cell transcriptional response, compared with the prototype strain.
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ABSTRACT: Pathogens use diverse molecular machines to penetrate host cells and manipulate intracellular vesicular trafficking. Viruses employ glycoproteins, functionally and structurally similar to the SNARE proteins, to induce eukaryotic membrane fusion. Intracellular pathogens, on the other hand, need to block fusion of their infectious phagosomes with various endocytic compartments to escape from the degradative pathway. The molecular details concerning the mechanisms underlying this process are lacking. Using both an in vitro liposome fusion assay and a cellular assay, we showed that SNARE-like bacterial proteins block membrane fusion in eukaryotic cells by directly inhibiting SNARE-mediated membrane fusion. More specifically, we showed that IncA and IcmG/DotF, two SNARE-like proteins respectively expressed by Chlamydia and Legionella, inhibit the endocytic SNARE machinery. Furthermore, we identified that the SNARE-like motif present in these bacterial proteins encodes the inhibitory function. This finding suggests that SNARE-like motifs are capable of specifically manipulating membrane fusion in a wide variety of biological environments. Ultimately, this motif may have been selected during evolution because it is an efficient structural motif for modifying eukaryotic membrane fusion and thus contribute to pathogen survival.PLoS ONE 10/2009; 4(10):e7375. DOI:10.1371/journal.pone.0007375 · 3.53 Impact Factor
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ABSTRACT: With an increase in the number of putative inclusion membrane proteins (incs) in chlamydial genomes, there is a need for understanding their contribution in host-pathogen interactions. Thus in this study we determined the host mucosal and peripheral immune responses to incs (IncB and IncC) of Chlamydia trachomatis (CT). Female patients (n = 296) attending the gynaecology out patient department of Safdarjung hospital, New Delhi were enrolled for the study and were clinically characterized into two groups; CT-positive fertile women (n = 38) and CT-positive women with fertility disorders (n = 29). Uninfected healthy fertile women were enrolled as controls (n = 31). Gene specific PCRs were used for detection of incB and incC genes in endocervical samples of CT-positive patients. ELISA and Western blot assay were used for detection of IgA and IgG antibodies to IncB and IncC in cervical washes and sera. Effect of IncB and IncC stimulation of cervical cells and PBMCs on cellular proliferation and cytotoxity was determined using MTT assay and Lactate dehydrogenase (LDH)-cytotoxicity assay respectively. Modulation of cytokines (Interleukin (IL)-1 Beta, IL-4, IL-5, IL-6, IL-10, Interferon-gamma, IL-12, Tumor Necrosis Factor-alpha and Granulocyte macrophage colony-stimulating factor (GM-CSF)) in cervical cells and PBMCs upon stimulation with IncB and IncC was determined by real-time reverse-transcriptase (RT)-PCR and ELISA. Further, CD4 positive T cells were purified from cervical cells and peripheral blood mononuclear cells (PBMCs) and secreted cytokines (Interferon-gamma and IL-4) were evaluated by ELISPOT and real-time RT-PCR. Using MTT assay, significantly high proliferative responses (P < 0.05) were observed in inc-stimulated cervical cells and PBMCs from CT-positive fertile women compared to CT-positive women with fertility disorders and controls. Interferon-gamma, IL-12 and GM-CSF were found to be elevated in inc-stimulated cervical cells and PBMCs of CT-positive fertile women compared to CT-positive women with fertility disorders and controls (P < 0.05). In contrast, IL-1 Beta, IL-4, IL-5, IL-6 and IL-10 levels were found to be higher in CT-positive women with fertility disorders compared to CT-positive fertile women and controls (P < 0.05). Interferon-gamma secreting cells and mRNA expression in inc-stimulated cervical and peripheral CD4 positive T cells were significantly higher (P < 0.05) in CT positive fertile women compared to CT-positive women with fertility disorders. Our data overall suggests that CT incs, IncB and IncC modulate host immune responses and may have a role in protection/pathogenesis of genital chlamydial infection in women.Reproductive Biology and Endocrinology 04/2009; 7(1):38. DOI:10.1186/1477-7827-7-38 · 2.41 Impact Factor