Eukaryotic protein recruitment into the Chlamydia inclusion: implications for survival and growth.
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ABSTRACT:
SUBMITTER: Soupene E
PROVIDER: S-EPMC3348897 | biostudies-literature | 2012
REPOSITORIES: biostudies-literature
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Project description:Chlamydia trachomatis causes genital and ocular infections in humans. This bacterial pathogen multiplies exclusively within host cells in a characteristic vacuole (inclusion) and delivers proteins such as inclusion membrane proteins (Incs) into the host cell. Here, we identified CT006 as a novel C. trachomatis protein that when expressed ectopically eukaryotic cells can associate with lipid droplets (LDs). A screen using Saccharomyces cerevisiae identified two Incs causing vacuolar protein sorting defects and seven Incs showing tropism for eukaryotic organelles. Ectopic expression in yeast and mammalian cells of genes encoding different fragments of CT006 revealed tropism for the endoplasmic reticulum and LDs. We identified a LD-targeting region within the first 88 amino acid residues of CT006, and positively charged residues important for this targeting. Comparing with the parental wild-type strain, cells infected by a newly generated C. trachomatis strain overproducing CT006 with a double hemagglutinin tag showed a slight increase in the area occupied by LDs within the inclusion region. However, we could not correlate this effect with the LD-targeting regions within CT006. We further showed that both the amino and carboxy-terminal regions of CT006, flanking the Inc-characteristic bilobed hydrophobic domain, are exposed to the host cell cytosol during C. trachomatis infection, supporting their availability to interact with host cell targets. Altogether, our data suggest that CT006 might participate in the interaction of LDs with C. trachomatis inclusions.
| S-EPMC8863265 | biostudies-literature
Project description:Bacterial pathogens that reside in membrane bound compartment manipulate the host cell machinery to establish and maintain their intracellular niche. The hijacking of inter-organelle vesicular trafficking through the targeting of small GTPases or SNARE proteins has been well established. Here, we show that intracellular pathogens also establish direct membrane contact sites with organelles and exploit non-vesicular transport machinery. We identified the ER-to-Golgi ceramide transfer protein CERT as a host cell factor specifically recruited to the inclusion, a membrane-bound compartment harboring the obligate intracellular pathogen Chlamydia trachomatis. We further showed that CERT recruitment to the inclusion correlated with the recruitment of VAPA/B-positive tubules in close proximity of the inclusion membrane, suggesting that ER-Inclusion membrane contact sites are formed upon C. trachomatis infection. Moreover, we identified the C. trachomatis effector protein IncD as a specific binding partner for CERT. Finally we showed that depletion of either CERT or the VAP proteins impaired bacterial development. We propose that the presence of IncD, CERT, VAPA/B, and potentially additional host and/or bacterial factors, at points of contact between the ER and the inclusion membrane provides a specialized metabolic and/or signaling microenvironment favorable to bacterial development.
| S-EPMC3121800 | biostudies-literature
Project description:Chlamydia trachomatis is an obligate intracellular pathogen with global health and economic impact. Upon infection, C. trachomatis resides within a protective niche, the inclusion, wherein it replicates and usurps host cell machinery and resources. The inclusion membrane is the key host-pathogen interface that governs specific protein-protein interactions to manipulate host signaling pathways. At the conclusion of the infection cycle, C. trachomatis exits the host cell via lysis or extrusion. Extrusion depends on the phosphorylation state of myosin light chain 2 (MLC2); the extent of phosphorylation is determined by the ongoing opposing activities of myosin phosphatase (MYPT1) and myosin kinase (MLCK). Previously, it was shown that MYPT1 is recruited to the inclusion and interacts with CT228 for regulation of host cell egress. In this study, we generated a targeted chromosomal mutation of CT228 (L2-ΔCT228) using the TargeTron system and demonstrate a loss of MYPT1 recruitment and increase in extrusion production in vitro. Mutation of CT228 did not affect chlamydial growth in cell culture or recruitment of MLC2. Moreover, we document a delay in clearance of L2-ΔCT228 during murine intravaginal infection as well as a reduction in systemic humoral response, relative to L2-wild type. Taken together, the data suggest that loss of MYPT1 recruitment (as a result of CT228 disruption) regulates the degree of host cell exit via extrusion and affects the longevity of infection in vivo.
| S-EPMC6284022 | biostudies-literature
Project description:Chlamydiae are Gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impacts. Like other chlamydial species, Chlamydia pneumoniae possesses a unique developmental cycle, the infectious elementary body gains access to the susceptible host cell, where it transforms into the replicative reticulate body. The cytoplasmic vacuole where Chlamydia pneumoniae replicates is called an inclusion, which is extensively modified by the insertion of chlamydial effectors known as inclusion membrane proteins (Incs). The C. pneumoniae-specific inclusion membrane protein (Inc) Cpn0147 contains domains that are predicted to be exposed to the host cytoplasm. To map host cell binding partners of Cpn0147, a yeast two-hybrid system was used to screen Cpn0147 against a HeLa cell cDNA library, which led to the finding that Cpn0147 interacted with the host cell protein cyclic adenosine monophosphate (cAMP)-responsive element (CRE)-binding protein (CREB3). The interaction was validated by co-immunoprecipitation of Cpn0147 with CREB3 from HeLa cells ectopically expressing both. Furthermore, Cpn0147 and CREB3 were co-localised in HeLa cells under confocal fluorescence microscopy. The above observations suggest that CREB3 may directly bind to the cytoplasmic domain of Cpn0147 to mediate the interactions of chlamydial inclusions with host cell endoplasmic reticulum.
| S-EPMC5619797 | biostudies-literature
Project description:Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs.
| S-EPMC5084276 | biostudies-literature
Project description:Chlamydia trachomatis is an obligate intracellular human pathogen responsible for ocular and genital infections. To establish its membrane-bound intracellular niche, the inclusion, C. trachomatis relies on a set of effector proteins that are injected into the host cells or inserted into the inclusion membrane. We previously proposed that insertion of the C. trachomatis effector protein IncD into the inclusion membrane contributes to the recruitment of the lipid transfer protein CERT to the inclusion. Due to the genetically intractable status of C. trachomatis at that time, this model of IncD-CERT interaction was inferred from ectopic expression of IncD and CERT in the host cell. In the present study, we investigated the impact of conditionally expressing a FLAG-tagged version of IncD in C. trachomatis. This genetic approach allowed us to establish that IncD-3×FLAG localized to the inclusion membrane and caused a massive recruitment of the lipid transfer protein CERT that relied on the PH domain of CERT. In addition, we showed that the massive IncD-dependent association of CERT with the inclusion led to an increased recruitment of the endoplasmic reticulum (ER)-resident protein VAPB, and we determined that, at the inclusion, CERT-VAPB interaction relied on the FFAT domain of CERT. Altogether, the data presented here show that expression of the C. trachomatis effector protein IncD mediates the recruitment of the lipid transfer protein CERT and the ER-resident protein VAPB to the inclusion.
| S-EPMC3993449 | biostudies-literature
Project description:The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not 'flash', but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism.
| S-EPMC3469565 | biostudies-literature
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.
| S-EPMC7567582 | biostudies-literature
Project description:Chlamydia trachomatis actively subverts the minus-end directed microtubule motor, dynein, to traffic along microtubule tracks to the Microtubule Organizing Center (MTOC) where it remains within a membrane bound replicative vacuole for the duration of its intracellular development. Unlike most substrates of the dynein motor, disruption of the dynactin cargo-linking complex by over-expression of the p50 dynamitin subunit does not inhibit C. trachomatis transport. A requirement for chlamydial protein synthesis to initiate this process suggests that a chlamydial product supersedes a requirement for p50 dynamitin. A yeast 2-hybrid system was used to screen the chlamydia inclusion membrane protein CT850 against a HeLa cell cDNA library and identified an interaction with the dynein light chain DYNLT1 (Tctex1). This interaction was at least partially dependent upon an (R/K-R/K-X-X-R/K) motif that is characteristic of DYNLT1 binding domains. CT850 expressed ectopically in HeLa cells localized at the MTOC and this localization is similarly dependent upon the predicted DYNLT1 binding domain. Furthermore, DYNLT1 is enriched at focal concentrations of CT850 on the chlamydial inclusion membrane that are known to interact with dynein and microtubules. Depletion of DYNLT1 disrupts the characteristic association of the inclusion membrane with centrosomes. Collectively, the results suggest that CT850 interacts with DYNLT1 to promote appropriate positioning of the inclusion at the MTOC.
| S-EPMC4449824 | biostudies-literature
Project description:Chlamydiae are prokaryotic obligate intracellular parasites that undergo a biphasic life cycle involving an infectious, extracellular form known as elementary bodies and an intracellular, replicating form termed reticulate bodies. We have purified from Chlamydia trachomatis a very basic elementary body-specific protein with an apparent molecular mass of 18 kDa, determined its N-terminal amino acid sequence, and cloned the encoding gene. Sequence analysis of the cloned gene revealed some remarkable properties for its expressed product, including a high lysine content (29%), a correspondingly high pI, and significant homology to the H1 class of eukaryotic histones. Furthermore, a monoclonal antibody to this chlamydial histone analog, termed Hc1, displayed immunoblot and antinuclear specificity suggestive of cross-reactivity to H1 histones. The gene was expressed only during the late stages of the chlamydial life cycle concomitant with the reorganization of chlamydial reticulate bodies into elementary bodies, suggesting that the Hc1 protein plays a role in the condensation of chlamydial chromatin during intracellular differentiation.
| S-EPMC51568 | biostudies-other