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Projeto de investigação
Identification and characterization of Chlamydia trachomatisvirulence proteins interfering with host cell vesicular trafficking
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Identification and characterization of IncL: a Chlamydia trachomatis protein associating with host cell lipid droplets and 14-3-3 proteins
Publication . Bugalhão, Joana Margarida Nunes; Mota, Luís
Chlamydia trachomatis causes genital and ocular infections in humans. This obligate intracellular bacterial pathogen multiplies within a characteristic vacuole, known as inclusion, and utilizes a type III secretion system to deliver chlamydial proteins, such as inclusion membrane proteins (Incs), into host cells. This work aimed to increase the knowledge on C. trachomatis Incs. A screen using Saccharomyces cerevisiae led to the identification of two Incs causing vacuolar protein sorting defects and seven Incs showing tropism for eukaryotic organelles. In particular, the transient production in yeast and mammalian cells of different fragments of CT006 (renamed IncL) revealed its tropism for the endoplasmic reticulum and lipid droplets (LDs), an organelle that regulates storage and hydrolysis of neutral lipids. We identified a LD-targeting region within the first 88 amino acid residues of IncL and positively charged residues important for this targeting. Comparing with the parental C. trachomatis strain, cells infected by a strain overproducing IncL 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 IncL. In addition, a previous proteomics screen suggested that IncL could bind mammalian 14-3-3 proteins, which regulate several signaling pathways in host cells. Here, co-immunoprecipitation assays validated the predicted interactions between IncL and 14-3-3β, η and γ isoforms and revealed an interaction with the 14-3-3σ isoform. The carboxy-terminal region of IncL was essential and sufficient for the IncL-14-3-3β interaction. We further showed that both the amino and carboxy-terminal regions of IncL, flanking the Inc-characteristic bilobed hydrophobic domain, are exposed to the host cell cytosol during C. trachomatis infection, and therefore available to interact with host cell targets. In conclusion, we characterized a chlamydial protein interacting with host cell LDs and 14-3-3 proteins via different protein regions, thus expanding the understanding of C. trachomatis-host cell interactions.
The Chlamydia trachomatis IncM Protein Interferes with Host Cell Cytokinesis, Centrosome Positioning, and Golgi Distribution and Contributes to the Stability of the Pathogen-Containing Vacuole
Publication . Luís, Maria Pequito; Pereira, Inês Serrano; Bugalhão, Joana N.; Simões, Catarina N.; Mota, Cristiano; Romão, Maria João; Mota, Luís Jaime; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; American Society for Microbiology
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes ocular and urogenital infections in humans. The ability of C. trachomatis to grow intracellularly in a pathogen-containing vacuole (known as an inclusion) depends on chlamydial effector proteins transported into the host cell by a type III secretion system. Among these effectors, several inclusion membrane proteins (Incs) insert in the vacuolar membrane. Here, we show that human cell lines infected by a C. trachomatis strain deficient for Inc CT288/CTL0540 (renamed IncM) displayed less multinucleation than when infected by IncM-producing strains (wild type or complemented). This indicated that IncM is involved in the ability of Chlamydia to inhibit host cell cytokinesis. The capacity of IncM to induce multinucleation in infected cells was shown to be conserved among its chlamydial homologues and appeared to require its two larger regions predicted to be exposed to the host cell cytosol. C. trachomatis-infected cells also displayed IncM-dependent defects in centrosome positioning, Golgi distribution around the inclusion, and morphology and stability of the inclusion. The altered morphology of inclusions containing IncM-deficient C. trachomatis was further affected by depolymerization of host cell microtubules. This was not observed after depolymerization of microfilaments, and inclusions containing wild-type C. trachomatis did not alter their morphology upon depolymerization of microtubules. Overall, these findings suggest that IncM may exert its effector function by acting directly or indirectly on host cell microtubules.
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Fundação para a Ciência e a Tecnologia
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OE
Número da atribuição
PD/BD/128214/2016