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Conservation of extrusion as an exit mechanism for Chlamydia.

ABSTRACT: Chlamydiae exit via membrane-encased extrusion or through lysis of the host cell. Extrusions are novel, pathogen-containing structures that confer infectious advantages to Chlamydia, and are hypothesized to promote cell-to-cell spread, dissemination to distant tissues and facilitate immune evasion. The extrusion phenomenon has been characterized for several Chlamydia trachomatis serovars, but a thorough investigation of extrusion for additional clinically relevant C. trachomatis strains and Chlamydia species has yet to be performed. The key parameters investigated in this study were: (i) the conservation of extrusion across the Chlamydia genus, (ii) the functional requirement for candidate Chlamydia genes in extrusion formation i.e. IncA and CT228 and (iii) extrusion-mediated uptake, and consequent survival of Chlamydia inside macrophages. Inclusion morphology was characterized by live fluorescence microscopy, using an inverted GFP strategy, at early and mid-stages of infection. Enriched extrusions were used to infect bone marrow-derived macrophages, and bacterial viability was measured following macrophage engulfment. Our results demonstrate that extrusion is highly conserved across chlamydiae, including ocular, STD and LGV biovars and divergent Chlamydia species. Consequently, this exit mechanism for Chlamydia may fulfill common advantages important for pathogenesis.

SUBMITTER: Zuck M

PROVIDER: S-EPMC5985487 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Conservation of extrusion as an exit mechanism for Chlamydia.

Zuck Meghan M Sherrid Ashley A Suchland Robert R Ellis Tisha T Hybiske Kevin K

Pathogens and disease 20160911 7

Chlamydiae exit via membrane-encased extrusion or through lysis of the host cell. Extrusions are novel, pathogen-containing structures that confer infectious advantages to Chlamydia, and are hypothesized to promote cell-to-cell spread, dissemination to distant tissues and facilitate immune evasion. The extrusion phenomenon has been characterized for several Chlamydia trachomatis serovars, but a thorough investigation of extrusion for additional clinically relevant C. trachomatis strains and Chla ...[more]

PMID: 27620201

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Project description:Neural crest cells (NCC) comprise a heterogeneous population of cells with variable potency, that contribute to nearly every tissue and organ system throughout the body. Considered unique to vertebrates, NCC are transiently generated within the dorsolateral region of the neural plate or neural tube, during neurulation. Their delamination and migration are crucial events in embryo development as the differentiation of NCC is heavily influenced by their final resting locations. Previous work in avian and aquatic species has shown that NCC delaminate via an epithelial-mesenchymal transition (EMT), which transforms these stem and progenitor cells from static polarized epithelial cells into migratory mesenchymal cells with fluid front and back polarity. However, the cellular and molecular drivers facilitating NCC delamination in mammals are poorly understood. We performed live timelapse imaging of NCC delamination in mouse embryos and discovered a group of cells that exit the neuroepithelium as isolated round cells, which then halt for a short period prior to acquiring the mesenchymal migratory morphology classically associated with most delaminating NCC. High magnification imaging and protein localization analyses of the cytoskeleton, together with measurements of pressure and tension of delaminating NCC and neighboring neuroepithelial cells, revealed these round NCC are extruded from the neuroepithelium prior to completion of EMT. Furthermore, we demonstrate that cranial NCC are extruded through activation of the mechanosensitive ion channel, PIEZO1, a key regulator of the live cell extrusion pathway, revealing a new role for PIEZO1 in neural crest cell development. Our results elucidating the cellular and molecular dynamics orchestrating NCC delamination support a model in which high pressure and tension in the neuroepithelium results in activation of the live cell extrusion pathway and delamination of a subpopulation of NCC in parallel with EMT. This model has broad implications for our understanding of cell delamination in development and disease.

Project description:Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia-host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX2 system of proximity-dependent biotinylation. APEX2 is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C. trachomatis to express the enzyme APEX2 fused to known inclusion membrane proteins, allowing biotinylation and purification of inclusion-associated proteins. Using quantitative mass spectrometry against APEX2 labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. Mass spectrometry analysis revealed a novel, early association between C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological and genetic disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. APEX2 is therefore a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth.

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Conservation of extrusion as an exit mechanism for Chlamydia.

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Conservation of extrusion as an exit mechanism for Chlamydia.

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