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The Chlamydia trachomatis type III secretion substrates CT142, CT143, and CT144 are secreted into the lumen of the inclusion.

ABSTRACT: Chlamydia trachomatis is a human bacterial pathogen causing ocular and genital infections. It multiplies exclusively within an intracellular membrane-bound vacuole, the inclusion, and uses a type III secretion system to manipulate host cells by injecting them with bacterially-encoded effector proteins. In this work, we characterized the expression and subcellular localization in infected host cells of the C. trachomatis CT142, CT143, and CT144 proteins, which we previously showed to be type III secretion substrates. Transcriptional analyses in C. trachomatis confirmed the prediction that ct142, ct143 and ct144 are organized in an operon and revealed that their expression is likely driven by the main ? factor, ?66. In host cells infected by C. trachomatis, production of CT142 and CT143 could be detected by immunoblotting from 20-26 h post-infection. Immunofluorescence microscopy of infected cells revealed that from 20 h post-infection CT143 appeared mostly as globular structures outside of the bacterial cells but within the lumen of the inclusion. Furthermore, immunofluorescence microscopy of cells infected by C. trachomatis strains carrying plasmids producing CT142, CT143, or CT144 under the control of the ct142 promoter and with a C-terminal double hemagglutinin (2HA) epitope tag revealed that CT142-2HA, CT143-2HA or CT144-2HA showed an identical localization to chromosomally-encoded CT143. Moreover, CT142-2HA or CT144-2HA and CT143 produced by the same bacteria co-localized in the lumen of the inclusion. Overall, these data suggest that the CT142, CT143, and CT144 type III secretion substrates are secreted into the lumen of the inclusion where they might form a protein complex.

SUBMITTER: da Cunha M

PROVIDER: S-EPMC5473537 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The Chlamydia trachomatis type III secretion substrates CT142, CT143, and CT144 are secreted into the lumen of the inclusion.

da Cunha Maria M Pais Sara V SV Bugalhão Joana N JN Mota Luís Jaime LJ

PloS one 20170616 6

Chlamydia trachomatis is a human bacterial pathogen causing ocular and genital infections. It multiplies exclusively within an intracellular membrane-bound vacuole, the inclusion, and uses a type III secretion system to manipulate host cells by injecting them with bacterially-encoded effector proteins. In this work, we characterized the expression and subcellular localization in infected host cells of the C. trachomatis CT142, CT143, and CT144 proteins, which we previously showed to be type III ...[more]

PMID: 28622339

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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.

Project description:The obligate intracellular bacterial pathogen Chlamydia trachomatis is reliant on a developmental cycle consisting of two cell forms, termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but it does not replicate. The RB replicates in the host cell but is noninfectious. This developmental cycle is central to chlamydial pathogenesis. In this study, we developed mathematical models of the developmental cycle that account for potential factors influencing RB-to-EB cell type switching during infection. Our models predicted that two categories of regulatory signals for RB-to-EB development could be differentiated experimentally, an "intrinsic" cell-autonomous program inherent to each RB and an "extrinsic" environmental signal to which RBs respond. To experimentally differentiate between mechanisms, we tracked the expression of C. trachomatis development-specific promoters in individual inclusions using fluorescent reporters and live-cell imaging. These experiments indicated that EB production was not influenced by increased multiplicity of infection or by superinfection, suggesting the cycle follows an intrinsic program that is not directly controlled by environmental factors. Additionally, live-cell imaging revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a progression with expression from the euo and ihtA promoters evident in RBs, while expression from the promoter for hctA was apparent in early EBs/IBs. Finally, expression from the promoters for the true late genes, hctB, scc2, and tarp, was evident in the maturing EB.IMPORTANCEChlamydia trachomatis is an obligate intracellular bacterium that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells, Chlamydia must complete a multiple-cell-type developmental cycle. The developmental cycle consists of specialized cells, the EB cell, which mediates infection of new host cells, and the RB cell, which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB-to-EB cell type switching, we demonstrate that RB-to-EB development follows a cell-autonomous program that does not respond to environmental cues. Additionally, we show that RB-to-EB development is a function of chlamydial growth and division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacterium's pathogenesis.

Dataset Information

The Chlamydia trachomatis type III secretion substrates CT142, CT143, and CT144 are secreted into the lumen of the inclusion.

Publications

The Chlamydia trachomatis type III secretion substrates CT142, CT143, and CT144 are secreted into the lumen of the inclusion.

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