Browsing by Author "Mota, Luís Jaime"
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- CteG is a Chlamydia trachomatis effector protein that associates with the Golgi complex of infected host cellsPublication . Pais, Sara V.; Key, Charlotte E.; Borges, Vítor; Pereira, Inês S.; Gomes, João Paulo; Fisher, Derek J.; Mota, Luís JaimeChlamydia trachomatis is a bacterial pathogen causing ocular and genital infections in humans. C. trachomatis multiplies exclusively inside host cells within a characteristic vacuole, from where it manipulates host cells by injecting them with type III secretion effector proteins. Here, we identified CteG as the first C. trachomatis effector associated with the Golgi. For this, C. trachomatis strains expressing candidate effectors fused to a double hemagglutinin (2HA) tag were constructed. Then, among these strains, immunofluorescence microscopy revealed that CteG-2HA was delivered into the cytoplasm of infected cells. Between 16-20 h post-infection, CteG-2HA mostly associated with the Golgi; however, CteG-2HA also appeared at the host cell plasma membrane, and at 30 or 40 h post-infection this was its predominant localization. This change in the main localization of CteG-2HA was independent of intact microfilaments or microtubules. Ectopic expression of different regions of CteG (656 amino acid residues) in uninfected cells revealed that its first 100 residues contain a Golgi targeting region. Although a C. trachomatis cteG mutant did not display a defect in intracellular multiplication, CteG induced a vacuolar protein sorting defect when expressed in Saccharomyces cerevisiae. This suggested that CteG might function by subverting host cell vesicular transport.
- In Vitro Activity of Quaternary Ammonium Surfactants against Streptococcal, Chlamydial, and Gonococcal Infective AgentsPublication . Inácio, Ângela S.; Nunes, Alexandra; Milho, Catarina; Mota, Luís Jaime; Borrego, Maria J.; Gomes, João P.; Vaz, Winchil L.C.; Vieira, Otília V.Quaternary ammonium compounds (QAC) are widely used, cheap, and chemically stable disinfectants and topical antiseptics with wide-spectrum antimicrobial activities. Within this group of compounds, we recently showed that there are significant differences between the pharmacodynamics of n-alkyl quaternary ammonium surfactants (QAS) with a short (C12) alkyl chain when in vitro toxicities toward bacterial and mammalian epithelial cells are compared. These differences result in an attractive therapeutic window that justifies studying short-chain QAS as prophylactics for sexually transmitted infections (STI) and perinatal vertically transmitted urogenital infections (UGI). We have evaluated the antimicrobial activities of short-chain (C12) n-alkyl QAS against several STI and UGI pathogens as well as against commensal Lactobacillus species. Inhibition of infection of HeLa cells by Neisseria gonorrhoeae and Chlamydia trachomatis was studied at concentrations that were not toxic to the HeLa cells. We show that the pathogenic bacteria are much more susceptible to QAS toxic effects than the commensal vaginal flora and that QAS significantly attenuate the infectivity of N. gonorrhoeae and C. trachomatis without affecting the viability of epithelial cells of the vaginal mucosa. N-Dodecylpyridinium bromide (C12PB) was found to be the most effective QAS. Our results strongly suggest that short-chain (C12) n-alkyl pyridinium bromides and structurally similar compounds are promising microbicide candidates for topical application in the prophylaxis of STI and perinatal vertical transmission of UGI.
- A Search for Novel Legionella pneumophila Effector Proteins Reveals a Strain Specific Nucleotropic EffectorPublication . Monteiro, Inês P.; Sousa, Sofia; Borges, Vítor; Gonçalves, Paulo; Gomes, João Paulo; Mota, Luís Jaime; Franco, Irina S.Legionella pneumophila is an accidental human pathogen that causes the potentially fatal Legionnaires' disease, a severe type of pneumonia. The main virulence mechanism of L. pneumophila is a Type 4B Secretion System (T4SS) named Icm/Dot that transports effector proteins into the host cell cytosol. The concerted action of effectors on several host cell processes leads to the formation of an intracellular Legionella-containing vacuole that is replication competent and avoids phagolysosomal degradation. To date over 300 Icm/Dot substrates have been identified. In this study, we searched the genome of a L. pneumophila strain (Pt/VFX2014) responsible for the second largest L. pneumophila outbreak worldwide (in Vila Franca de Xira, Portugal, in 2014) for genes encoding potential novel Icm/Dot substrates. This strain Pt/VFX2014 belongs to serogroup 1 but phylogenetically segregates from all other serogroup 1 strains previously sequenced, displaying a unique mosaic genetic backbone. The ability of the selected putative effectors to be delivered into host cells by the T4SS was confirmed using the TEM-1 β-lactamase reporter assay. Two previously unknown Icm/Dot effectors were identified, VFX05045 and VFX10045, whose homologs Lpp1450 and Lpp3070 in clinical strain L. pneumophila Paris were also confirmed as T4SS substrates. After delivery into the host cell cytosol, homologs VFX05045/Lpp1450 remained diffused in the cell, similarly to Lpp3070. In contrast, VFX10045 localized to the host cell nucleus. To understand how VFX10045 and Lpp3070 (94% of identity at amino acid level) are directed to distinct sites, we carried out a comprehensive site-directed mutagenesis followed by analyses of the subcellular localization of the mutant proteins. This led to the delineation of region in the C-terminal part (residues 380 to 534) of the 583 amino acid-long VFX10045 as necessary and sufficient for nuclear targeting and highlighted the fundamental function of the VFX10045-specific R440 and I441 residues in this process. These studies revealed a strain-specific nucleotropism for new effector VFX10045/Lpp3070, which anticipates distinct functions between these homologs.
- The Type III Secretion Effector CteG Mediates Host Cell Lytic Exit of Chlamydia trachomatisPublication . Pereira, Inês Serrano; Pais, Sara Vilela; Borges, Vítor; Borrego, Maria José; Gomes, João Paulo; Mota, Luís JaimeChlamydia trachomatis is an obligate intracellular bacterium causing ocular and urogenital infections in humans that are a significant burden worldwide. The completion of its characteristic infectious cycle relies on the manipulation of several host cell processes by numerous chlamydial type III secretion effector proteins. We previously identified the C. trachomatis CteG effector and showed it localizes at the host cell plasma membrane at late stages of infection. Here, we showed that, from 48 h post-infection, mammalian cells infected by wild-type C. trachomatis contained more infectious chlamydiae in the culture supernatant than cells infected by a CteG-deficient strain. This phenotype was CteG-dependent as it could be complemented in cells infected by the CteG-deficient strain carrying a plasmid encoding CteG. Furthermore, we detected a CteG-dependent defect on host cell cytotoxicity, indicating that CteG mediates chlamydial lytic exit. Previous studies showed that Pgp4, a global regulator of transcription encoded in the C. trachomatis virulence plasmid, also mediates chlamydial lytic exit. However, by using C. trachomatis strains encoding or lacking Pgp4, we showed that production and localization of CteG are not regulated by Pgp4. A C. trachomatis strain lacking both CteG and Pgp4 was as defective in promoting host cell cytotoxicity as mutant strains lacking only CteG or Pgp4. Furthermore, CteG overproduction in a plasmid suppressed the host cell cytotoxic defect of CteG- and Pgp4-deficient chlamydiae. Overall, we revealed the first chlamydial type III secretion effector involved in host cell lytic exit. Our data indicates that CteG and Pgp4 participate in a single cascade of events, but involving multiple layers of regulation, leading to lysis of host cells and release of the infectious chlamydiae.