CT295 Is Chlamydia trachomatis' Phosphoglucomutase and a Type 3 Secretion Substrate.
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ABSTRACT:
SUBMITTER: Triboulet S
PROVIDER: S-EPMC9251005 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Chlamydia trachomatis is an obligate intracellular bacterial pathogen of humans that uses a type III secretion (T3S) system to manipulate host cells through the delivery of effector proteins into their cytosol and membranes. The function of T3S systems depends on small bacterial cytosolic chaperone-like proteins, which bind T3S substrates and ensure their appropriate secretion. To find novel T3S chaperone-substrate complexes of C. trachomatis we first searched its genome for genes encoding proteins with features of T3S chaperones. We then systematically tested for interactions between candidate chaperones and chlamydial T3S substrates by bacterial two-hybrid. This revealed interactions between Slc1 (a known T3S chaperone) or CT584 and several T3S substrates. Co-immunoprecipitation after protein expression in Yersinia enterocolitica and protein overlay binding assays indicated that Slc1 interacted with the N-terminal region of the known T3S substrates Tarp (a previously described substrate of Slc1), CT694, and CT695, and that CT584 interacted with a central region of CT082, which we identified as a C. trachomatis T3S substrate using Y. enterocolitica as a heterologous system. Further T3S assays in Yersinia indicated that Slc1 or CT584 increased the amount of secreted Tarp, CT694, and CT695, or CT082, respectively. Expression of CT584 increased the intra-bacterial stability of CT082, while Slc1 did not affect the stability of its substrates. Overall, this indicated that in C. trachomatis Slc1 is a chaperone of multiple T3S substrates and that CT584 is a chaperone of the newly identified T3S substrate CT082.
| S-EPMC3576375 | biostudies-literature
Project description:UnlabelledThe Scc4 protein (CT663) of the pathogenic bacterium Chlamydia has been described as a type III secretion (T3S) chaperone as well as an inhibitor of RNA polymerase. To examine if these roles are connected, we first investigated physical interactions between Chlamydia trachomatis Scc4 and the T3S chaperone Scc1 and a T3S substrate, CopN. In a yeast 3-hybrid assay, Scc4, Scc1, and CopN were all required to detect an interaction, which suggests that these proteins form a trimolecular complex. We also detected interactions between any two of these three T3S proteins in a pulldown assay using only recombinant proteins. We next determined whether these interactions affected the function of Scc4 as an inhibitor of RNA transcription. Using Escherichia coli as a heterologous in vivo system, we demonstrated that expression of C. trachomatis Scc4 led to a drastic decrease in transcript levels for multiple genes. However, coexpression of Scc4 with Scc1, CopN, or both alleviated Scc4-mediated inhibition of transcription. Scc4 expression also severely impaired E. coli growth, but this growth defect was reversed by coexpression of Scc4 with Scc1, CopN, or both, suggesting that the inhibitory effect of Scc4 on transcription and growth can be antagonized by interactions between Scc4, Scc1, and CopN. These findings suggest that the dual functions of Scc4 may serve as a bridge to link T3S and the regulation of gene expression in Chlamydia.ImportanceThis study investigates a novel mechanism for regulating gene expression in the pathogenic bacterium Chlamydia. The Chlamydia type III secretion (T3S) chaperone Scc4 has been shown to inhibit transcription by RNA polymerase. This study describes physical interactions between Scc4 and the T3S proteins Scc1 and CopN. Furthermore, Chlamydia Scc1 and CopN antagonized the inhibitory effects of Scc4 on transcription and growth in a heterologous Escherichia coli system. These results provide evidence that transcription in Chlamydia can be regulated by the T3S system through interactions between T3S proteins.
| S-EPMC4573728 | biostudies-literature
Project description:Bacterial type III secretion system (T3SS) chaperones pilot substrates to the export apparatus in a secretion-competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well-characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two-hybrid, co-precipitation, cross-linking and size exclusion chromatography we show that Slc1 (SycE-like chaperone 1; CT043) specifically interacts with a 200-amino-acid residue N-terminal region of TARP (TARP¹?²??). Slc1 formed homodimers in vitro, as shown in cross-linking and gel filtration experiments. Biochemical analysis of an isolated Slc1-TARP¹?²?? complex was consistent with a characteristic 2:1 chaperone-effector stoichiometry. Furthermore, Slc1 was co-immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, coexpression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP.
| S-EPMC3214626 | biostudies-literature
Project description:Secretion of effector proteins into the eukaryotic host cell is required for Chlamydia trachomatis virulence. In the infection process, Scc1 and Scc4, two chaperones of the type III secretion (T3S) system, facilitate secretion of the important effector and plug protein, CopN, but little is known about the details of this event. Here we use biochemistry, mass spectrometry, nuclear magnetic resonance spectroscopy, and genetic analyses to characterize this trimolecular event. We find that Scc4 complexes with Scc1 and CopN in situ at the late developmental cycle of C. trachomatis. We show that Scc4 and Scc1 undergo dynamic interactions as part of the unique bacterial developmental cycle. Using alanine substitutions, we identify several amino acid residues in Scc4 that are critical for the Scc4-Scc1 interaction, which is required for forming the Scc4·Scc1·CopN ternary complex. These results, combined with our previous findings that Scc4 plays a role in transcription (Rao, X., Deighan, P., Hua, Z., Hu, X., Wang, J., Luo, M., Wang, J., Liang, Y., Zhong, G., Hochschild, A., and Shen, L. (2009) Genes Dev. 23, 1818-1829), reveal that the T3S process is linked to bacterial transcriptional events, all of which are mediated by Scc4 and its interacting proteins. A model describing how the T3S process may affect gene expression is proposed.
| S-EPMC4653673 | biostudies-literature
Project description:Chlamydia trachomatis is an obligate intracellular bacterium causing ocular and urogenital infections in humans that are a significant burden worldwide. The completion of its characteristic infectious cycle relies on the manipulation of several host cell processes by numerous chlamydial type III secretion effector proteins. We previously identified the C. trachomatis CteG effector and showed it localizes at the host cell plasma membrane at late stages of infection. Here, we showed that, from 48 h post-infection, mammalian cells infected by wild-type C. trachomatis contained more infectious chlamydiae in the culture supernatant than cells infected by a CteG-deficient strain. This phenotype was CteG-dependent as it could be complemented in cells infected by the CteG-deficient strain carrying a plasmid encoding CteG. Furthermore, we detected a CteG-dependent defect on host cell cytotoxicity, indicating that CteG mediates chlamydial lytic exit. Previous studies showed that Pgp4, a global regulator of transcription encoded in the C. trachomatis virulence plasmid, also mediates chlamydial lytic exit. However, by using C. trachomatis strains encoding or lacking Pgp4, we showed that production and localization of CteG are not regulated by Pgp4. A C. trachomatis strain lacking both CteG and Pgp4 was as defective in promoting host cell cytotoxicity as mutant strains lacking only CteG or Pgp4. Furthermore, CteG overproduction in a plasmid suppressed the host cell cytotoxic defect of CteG- and Pgp4-deficient chlamydiae. Overall, we revealed the first chlamydial type III secretion effector involved in host cell lytic exit. Our data indicates that CteG and Pgp4 participate in a single cascade of events, but involving multiple layers of regulation, leading to lysis of host cells and release of the infectious chlamydiae.
| S-EPMC9318579 | biostudies-literature
Project description:UnlabelledProtein 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. trachomatisImportanceChlamydia 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.
| S-EPMC4907117 | biostudies-literature
Project description: Not available
| EMPIAR-10047 | biostudies-other
Project description:BackgroundChlamydia trachomatis is an obligate intracellular human pathogen causing ocular and urogenital infections that are a significant clinical and public health concern. This bacterium uses a type III secretion (T3S) system to manipulate host cells, through the delivery of effector proteins into their cytosol, membranes, and nucleus. In this work, we aimed to find previously unidentified C. trachomatis T3S substrates.ResultsWe first analyzed the genome of C. trachomatis L2/434 strain for genes encoding mostly uncharacterized proteins that did not appear to possess a signal of the general secretory pathway and which had not been previously experimentally shown to be T3S substrates. We selected several genes with these characteristics and analyzed T3S of the encoding proteins using Yersinia enterocolitica as a heterologous system. We identified 23 C. trachomatis proteins whose first 20 amino acids were sufficient to drive T3S of the mature form of ?-lactamase TEM-1 by Y. enterocolitica. We found that 10 of these 23 proteins were also type III secreted in their full-length versions by Y. enterocolitica, providing additional support that they are T3S substrates. Seven of these 10 likely T3S substrates of C. trachomatis were delivered by Y. enterocolitica into host cells, further suggesting that they could be effectors. Finally, real-time quantitative PCR analysis of expression of genes encoding the 10 likely T3S substrates of C. trachomatis showed that 9 of them were clearly expressed during infection of host cells.ConclusionsUsing Y. enterocolitica as a heterologous system, we identified 10 likely T3S substrates of C. trachomatis (CT053, CT105, CT142, CT143, CT144, CT161, CT338, CT429, CT656, and CT849) and could detect translocation into host cells of CT053, CT105, CT142, CT143, CT161, CT338, and CT429. Therefore, we revealed several C. trachomatis proteins that could be effectors subverting host cell processes.
| S-EPMC3931295 | biostudies-literature
Project description:CPAF (chlamydial protease-like activity factor), a Chlamydia serine protease, is activated via proximity-induced intermolecular dimerization that triggers processing and removal of an inhibitory peptide occupying the CPAF substrate-binding groove. An active CPAF is a homodimer of two identical intramolecular heterodimers, each consisting of 29-kDa N-terminal and 35-kDa C-terminal fragments. However, critical residues for CPAF intermolecular dimerization, catalytic activity, and processing were defined in cell-free systems. Complementation of a CPAF-deficient chlamydial organism with a plasmid-encoded CPAF has enabled us to characterize CPAF during infection. The transformants expressing CPAF mutated at intermolecular dimerization, catalytic, or cleavage residues still produced active CPAF, although at a lower efficiency, indicating that CPAF can tolerate more mutations inside Chlamydia-infected cells than in cell-free systems. Only by simultaneously mutating both intermolecular dimerization and catalytic residues was CPAF activation completely blocked during infection, both indicating the importance of the critical residues identified in the cell-free systems and exploring the limit of CPAF's tolerance for mutations in the intracellular environment. We further found that active CPAF was always detected in the host cell cytoplasm while nonactive CPAF was restricted to within the chlamydial inclusions, regardless of how the infected cell samples were treated. Thus, CPAF translocation into the host cell cytoplasm correlates with CPAF enzymatic activity and is not altered by sample treatment conditions. These observations have provided new evidence for CPAF activation and translocation, which should encourage continued investigation of CPAF in chlamydial pathogenesis.
| S-EPMC4432759 | biostudies-literature
Project description:Chlamydia are obligate intracellular bacterial pathogens that cause a variety of diseases. Like many Gram-negative bacteria, they employ type III secretion systems (T3SS) for invasion, establishing and maintaining their unique intracellular niche, and possibly cellular exit. Computational structure prediction indicated that ORF CT584 is homologous to other T3SS needle tip proteins. Tip proteins have been shown to be localized to the extracellular end of the T3SS needle and play a key role in controlling secretion of effector proteins. We have previously demonstrated that T3SS needle tip proteins from different bacteria share many biophysical characteristics. To support the hypothesis that CT584 is a T3SS needle tip protein, biophysical properties of CT584 were explored as a function of pH and temperature, using spectroscopic techniques. Far-UV circular dichroism, Fourier transform infrared spectroscopy, UV absorbance spectroscopy, ANS extrinsic fluorescence, turbidity, right angle static light scattering, and analytical ultracentrifugation were all employed to monitor the secondary, tertiary, quaternary, and aggregation behavior of this protein. An empirical phase diagram approach is also employed to facilitate such comparisons. These analyses demonstrate that CT584 shares many biophysical characteristics with other T3SS needle tip proteins. These data support the hypothesis that CT584 is a member of the same functional family, although future biologic analyses are required.
| S-EPMC4285778 | biostudies-literature