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Broad recruitment of mGBP family members to Chlamydia trachomatis inclusions.

ABSTRACT: Chlamydia, the most common sexually transmitted pathogen, is an exquisitely adapted Gram-negative obligate intracellular bacterium. Intracellular Chlamydia trachomatis replicate in a specialized vacuole, termed inclusion, which shields the bacterium from antimicrobial immunity of the host cells and acts as a signalling interface. Previously it was shown that members of the interferon induced guanylate binding protein (mGBP) family, in particular murine GBP1 and mGBP2, were found to accumulate at the bacterial inclusions, similar to previously published recruitment of GBPs to the parasitophorous vacuole of Toxoplasma gondii. Here, we provide a wide comparison of mGBPs roles within the host cell in the context of Chlamydia and Toxoplasma infection. By confocal microscopy on fixed and living infected cells we show localization of mGBP3, mGBP6, mGBP7, mGBP9, and mGBP10, in addition to mGBP1 and mGBP2, at chlamydia inclusions. In time lapse videos using GFP expressing Chlamydia we show rapid and transient dynamics of mGBP9 accumulation onto chlamydia inclusions. Taken together this study reveals a broad activation of mGBP recruitment towards Chlamydia trachomatis inclusions after infection and provides evidence for time limited action of mGBP9 at the chlamydia inclusion.

SUBMITTER: Lindenberg V

PROVIDER: S-EPMC5612764 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Broad recruitment of mGBP family members to Chlamydia trachomatis inclusions.

Lindenberg Valesca V Mölleken Katja K Kravets Elisabeth E Stallmann Sonja S Hegemann Johannes H JH Degrandi Daniel D Pfeffer Klaus K

PloS one 20170925 9

Chlamydia, the most common sexually transmitted pathogen, is an exquisitely adapted Gram-negative obligate intracellular bacterium. Intracellular Chlamydia trachomatis replicate in a specialized vacuole, termed inclusion, which shields the bacterium from antimicrobial immunity of the host cells and acts as a signalling interface. Previously it was shown that members of the interferon induced guanylate binding protein (mGBP) family, in particular murine GBP1 and mGBP2, were found to accumulate at ...[more]

PMID: 28945814

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Project description:The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multiple Chlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D-, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. The incA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D((s)) strains. Three nucleotide changes were discovered in the D((s)) incA gene, leading to two amino acid changes within the predicted D((s)) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein.

Project description:Protein phosphorylation has become increasingly recognized for its role in regulating bacterial physiology and virulence. Chlamydia spp. encode two validated Hanks'-type Ser/Thr protein kinases, which typically function with cognate protein phosphatases and appear capable of global protein phosphorylation. Consequently, we sought to identify a Ser/Thr protein phosphatase partner for the chlamydial kinases. CTL0511 from Chlamydia trachomatis L2 434/Bu, which has homologs in all sequenced Chlamydia spp., is a predicted type 2C Ser/Thr protein phosphatase (PP2C). Recombinant maltose-binding protein (MBP)-tagged CTL0511 (rCTL0511) hydrolyzed p-nitrophenyl phosphate (pNPP), a generic phosphatase substrate, in a MnCl2-dependent manner at physiological pH. Assays using phosphopeptide substrates revealed that rCTL0511 can dephosphorylate phosphorylated serine (P-Ser), P-Thr, and P-Tyr residues using either MnCl2 or MgCl2, indicating that metal usage can alter substrate preference. Phosphatase activity was unaffected by PP1, PP2A, and PP3 phosphatase inhibitors, while mutation of conserved PP2C residues significantly inhibited activity. Finally, phosphatase activity was detected in elementary body (EB) and reticulate body (RB) lysates, supporting a role for protein dephosphorylation in chlamydial development. These findings support that CTL0511 is a metal-dependent protein phosphatase with broad substrate specificity, substantiating a reversible phosphorylation network in C. trachomatisChlamydia spp. are obligate intracellular bacterial pathogens responsible for a variety of diseases in humans and economically important animal species. Our work demonstrates that Chlamydia spp. produce a PP2C capable of dephosphorylating P-Thr, P-Ser, and P-Tyr and that Chlamydia trachomatis EBs and RBs possess phosphatase activity. In conjunction with the chlamydial Hanks'-type kinases Pkn1 and PknD, validation of CTL0511 fulfills the enzymatic requirements for a reversible phosphoprotein network. As protein phosphorylation regulates important cellular processes, including metabolism, differentiation, and virulence, in other bacterial pathogens, these results set the stage for elucidating the role of global protein phosphorylation in chlamydial physiology and virulence.

Project description:Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry. We identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form large molecular weight complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to EBs at entry sites where it remained associated with nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.

Dataset Information

Broad recruitment of mGBP family members to Chlamydia trachomatis inclusions.

Publications

Broad recruitment of mGBP family members to Chlamydia trachomatis inclusions.

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