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      The DUF582 Proteins of Chlamydia trachomatis Bind to Components of the ESCRT Machinery, Which Is Dispensable for Bacterial Growth In vitro

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          Abstract

          Chlamydiae are Gram negative bacteria that develop exclusively inside eukaryotic host cells, within a membrane-bounded compartment. Members of the family Chlamydiaceae, such as Chlamydia trachomatis, are pathogenic species infecting vertebrates. They have a very reduced genome and exploit the capacities of their host for their own development, mainly through the secretion of proteins tailored to interfere with eukaryotic processes, called effector proteins. All Chlamydiaceae possess genes coding for four to five effectors that share a domain of unknown function (DUF582). Here we show that four of these effectors, which represent the conserved set in all Chlamydiaceae, accumulate in the infectious form of C. trachomatis, and are therefore likely involved in an early step of the developmental cycle. The fifth member of the family, CT621, is specific to C. trachomatis, and is secreted during the growth phase. Using a two-hybrid screen in yeast we identified an interaction between the host protein Hrs and the DUF582, which we confirmed by co-immunoprecipitations in co-transfected mammalian cells. Furthermore, we provide biochemical evidence that a second domain of one of the DUF582 proteins, CT619, binds the host protein Tsg101. Hrs and Tsg101 are both implicated in a well conserved machinery of the eukaryotic cell called the ESCRT machinery, which is involved in several cellular processes requiring membrane constriction. Using RNA interference targeting proteins implicated at different stages of ESCRT-driven processes, or inhibition by expression of a dominant negative mutant of VPS4, we demonstrated that this machinery was dispensable for bacterial entry, multiplication and differentiation into infectious progeny, and for uptake of glycogen into the parasitophorous vacuole. In light of these observations we discuss how the DUF582 proteins might target the ESCRT machinery during infection.

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          Most cited references26

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          Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis.

          Analysis of the 1,042,519-base pair Chlamydia trachomatis genome revealed unexpected features related to the complex biology of chlamydiae. Although chlamydiae lack many biosynthetic capabilities, they retain functions for performing key steps and interconversions of metabolites obtained from their mammalian host cells. Numerous potential virulence-associated proteins also were characterized. Several eukaryotic chromatin-associated domain proteins were identified, suggesting a eukaryotic-like mechanism for chlamydial nucleoid condensation and decondensation. The phylogenetic mosaic of chlamydial genes, including a large number of genes with phylogenetic origins from eukaryotes, implies a complex evolution for adaptation to obligate intracellular parasitism.
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            Mechanisms of host cell exit by the intracellular bacterium Chlamydia.

            The mechanisms that mediate the release of intracellular bacteria from cells are poorly understood, particularly for those that live within a cellular vacuole. The release pathway of the obligate intracellular bacterium Chlamydia from cells is unknown. Using a GFP-based approach to visualize chlamydial inclusions within cells by live fluorescence videomicroscopy, we identified that Chlamydia release occurred by two mutually exclusive pathways. The first, lysis, consisted of an ordered sequence of membrane permeabilizations: inclusion, nucleus and plasma membrane rupture. Treatment with protease inhibitors abolished inclusion lysis. Intracellular calcium signaling was shown to be important for plasma membrane breakdown. The second release pathway was a packaged release mechanism, called extrusion. This slow process resulted in a pinching of the inclusion, protrusion out of the cell within a cell membrane compartment, and ultimately detachment from the cell. Treatment of Chlamydia-infected cells with specific pharmacological inhibitors of cellular factors demonstrated that extrusion required actin polymerization, neuronal Wiskott-Aldrich syndrome protein, myosin II and Rho GTPase. The participation of Rho was unique in that it functioned late in extrusion. The dual nature of release characterized for Chlamydia has not been observed as a strategy for intracellular bacteria.
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              Trachoma.

              Trachoma is the most common infectious cause of blindness. Repeated episodes of infection with Chlamydia trachomatis in childhood lead to severe conjunctival inflammation, scarring, and potentially blinding inturned eyelashes (trichiasis or entropion) in later life. Trachoma occurs in resource-poor areas with inadequate hygiene, where children with unclean faces share infected ocular secretions. Much has been learnt about the epidemiology and pathophysiology of trachoma. Integrated control programmes are implementing the SAFE Strategy: surgery for trichiasis, mass distribution of antibiotics, promotion of facial cleanliness, and environmental improvement. This strategy has successfully eliminated trachoma in several countries and global efforts are underway to eliminate blinding trachoma worldwide by 2020.
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                Author and article information

                Contributors
                Journal
                Front Cell Infect Microbiol
                Front Cell Infect Microbiol
                Front. Cell. Infect. Microbiol.
                Frontiers in Cellular and Infection Microbiology
                Frontiers Media S.A.
                2235-2988
                07 October 2016
                2016
                : 6
                : 123
                Affiliations
                [1] 1Institut Pasteur, Unité de Biologie Cellulaire de l'Infection Microbienne Paris, France
                [2] 2CNRS UMR 3691 Paris, France
                [3] 3Université Pierre et Marie Curie, Cellule Pasteur UPMC Paris, France
                Author notes

                Edited by: Rey Carabeo, Washington State University, USA

                Reviewed by: Luís Jaime Mota, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Portugal; David Gondek, Ithaca College, USA

                *Correspondence: Agathe Subtil agathe.subtil@ 123456pasteur.fr
                Article
                10.3389/fcimb.2016.00123
                5053991
                27774439
                a94d9f3e-7865-42f5-8027-1b3bbab70c6b
                Copyright © 2016 Vromman, Perrinet, Gehre and Subtil.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 09 July 2016
                : 22 September 2016
                Page count
                Figures: 7, Tables: 0, Equations: 0, References: 36, Pages: 14, Words: 9241
                Funding
                Funded by: European Research Council 10.13039/501100000781
                Award ID: StG NUChLEAR 282046
                Funded by: Fondation pour la Recherche Médicale 10.13039/501100002915
                Award ID: FDT20130928198
                Funded by: Institut Pasteur 10.13039/501100003762
                Funded by: Centre National de la Recherche Scientifique 10.13039/501100004794
                Categories
                Microbiology
                Original Research

                Infectious disease & Microbiology
                host pathogen interactions,chlamydia trachomatis,type iii effectors,escrt,hrs,tsg101

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