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1.
Med Microbiol Immunol ; 213(1): 15, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-39008129

RESUMO

Chlamydiae are a large group of obligate endosymbionts of eukaryotes that includes the Chlamydiaceae family, comprising several animal pathogens. Among Chlamydiaceae, Chlamydia trachomatis causes widespread ocular and urogenital infections in humans. Like many bacterial pathogens, all Chlamydiae manipulate host cells by injecting them with type III secretion effector proteins. We previously characterized the C. trachomatis effector CteG, which localizes at the host cell Golgi and plasma membrane during distinct phases of the chlamydial infectious cycle. Here, we show that CteG is a Chlamydiaceae-specific effector with over 60 homologs phylogenetically categorized into two distinct clades (CteG I and CteG II) and exhibiting several inparalogs and outparalogs. Notably, cteG I homologs are syntenic to C. trachomatis cteG, whereas cteG II homologs are syntenic among themselves but not with C. trachomatis cteG. This indicates a complex evolution of cteG homologs, which is unique among C. trachomatis effectors, marked by numerous events of gene duplication and loss. Despite relatively modest sequence conservation, nearly all tested CteG I and CteG II proteins were identified as type III secretion substrates using Yersinia as a heterologous bacterial host. Moreover, most of the type III secreted CteG I and CteG II homologs were delivered by C. trachomatis into host cells, where they localized at the Golgi region and cell periphery. Overall, this provided insights into the evolution of bacterial effectors and revealed a Chlamydiaceae family of type III secreted proteins that underwent substantial divergence during evolution while conserving the capacity to localize at specific host cell compartments.


Assuntos
Proteínas de Bactérias , Chlamydia trachomatis , Filogenia , Sistemas de Secreção Tipo III , Humanos , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo III/genética , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Células HeLa , Yersinia/genética , Yersinia/metabolismo , Transporte Proteico , Interações Hospedeiro-Patógeno , Evolução Molecular , Chlamydiaceae/genética , Chlamydiaceae/metabolismo , Chlamydiaceae/classificação
2.
Infect Immun ; 91(4): e0040522, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-36877064

RESUMO

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.


Assuntos
Infecções por Chlamydia , Citocinese , Humanos , Citocinese/fisiologia , Chlamydia trachomatis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células HeLa , Vacúolos/metabolismo , Centrossomo/metabolismo , Infecções por Chlamydia/microbiologia , Interações Hospedeiro-Patógeno
3.
Cell Microbiol ; 18(7): 949-69, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26676327

RESUMO

Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Salmonella typhimurium/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Células HeLa/microbiologia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Vacúolos/metabolismo , Fatores de Virulência/genética
4.
Antimicrob Agents Chemother ; 60(6): 3323-32, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26976875

RESUMO

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.


Assuntos
Anti-Infecciosos/farmacologia , Chlamydia trachomatis/efeitos dos fármacos , Gonorreia/tratamento farmacológico , Compostos de Amônio Quaternário/farmacologia , Streptococcus/efeitos dos fármacos , Tensoativos/farmacologia , Células HeLa , Humanos , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Neisseria gonorrhoeae/efeitos dos fármacos , Infecções Sexualmente Transmissíveis/microbiologia
5.
Infect Immun ; 82(7): 2923-34, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24778114

RESUMO

Salmonella enterica serovar Typhimurium is a bacterial pathogen causing gastroenteritis in humans and a typhoid-like systemic disease in mice. S. Typhimurium virulence is related to its capacity to multiply intracellularly within a membrane-bound compartment, the Salmonella-containing vacuole (SCV), and depends on type III secretion systems that deliver bacterial effector proteins into host cells. Here, we analyzed the cellular function of the Salmonella effector SteA. We show that, compared to cells infected by wild-type S. Typhimurium, cells infected by ΔsteA mutant bacteria displayed fewer Salmonella-induced filaments (SIFs), an increased clustering of SCVs, and morphologically abnormal vacuoles containing more than one bacterium. The increased clustering of SCVs and the appearance of vacuoles containing more than one bacterium were suppressed by inhibition of the activity of the microtubule motor dynein or kinesin-1. Clustering and positioning of SCVs are controlled by the effectors SseF and SseG, possibly by helping to maintain a balanced activity of microtubule motors on the bacterial vacuoles. Deletion of steA in S. Typhimurium ΔsseF or ΔsseG mutants revealed that SteA contributes to the characteristic scattered distribution of ΔsseF or ΔsseG mutant SCVs in infected cells. Overall, this shows that SteA participates in the control of SCV membrane dynamics. Moreover, it indicates that SteA is functionally linked to SseF and SseG and suggests that it might contribute directly or indirectly to the regulation of microtubule motors on the bacterial vacuoles.


Assuntos
Proteínas de Bactérias/metabolismo , Salmonella typhimurium/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Dineínas , Regulação da Expressão Gênica/imunologia , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Mutação , Fatores de Virulência/genética
6.
BMC Microbiol ; 14: 40, 2014 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-24533538

RESUMO

BACKGROUND: Chlamydia trachomatis is an obligate intracellular human pathogen causing ocular and urogenital infections that are a significant clinical and public health concern. This bacterium uses a type III secretion (T3S) system to manipulate host cells, through the delivery of effector proteins into their cytosol, membranes, and nucleus. In this work, we aimed to find previously unidentified C. trachomatis T3S substrates. RESULTS: We first analyzed the genome of C. trachomatis L2/434 strain for genes encoding mostly uncharacterized proteins that did not appear to possess a signal of the general secretory pathway and which had not been previously experimentally shown to be T3S substrates. We selected several genes with these characteristics and analyzed T3S of the encoding proteins using Yersinia enterocolitica as a heterologous system. We identified 23 C. trachomatis proteins whose first 20 amino acids were sufficient to drive T3S of the mature form of ß-lactamase TEM-1 by Y. enterocolitica. We found that 10 of these 23 proteins were also type III secreted in their full-length versions by Y. enterocolitica, providing additional support that they are T3S substrates. Seven of these 10 likely T3S substrates of C. trachomatis were delivered by Y. enterocolitica into host cells, further suggesting that they could be effectors. Finally, real-time quantitative PCR analysis of expression of genes encoding the 10 likely T3S substrates of C. trachomatis showed that 9 of them were clearly expressed during infection of host cells. CONCLUSIONS: Using Y. enterocolitica as a heterologous system, we identified 10 likely T3S substrates of C. trachomatis (CT053, CT105, CT142, CT143, CT144, CT161, CT338, CT429, CT656, and CT849) and could detect translocation into host cells of CT053, CT105, CT142, CT143, CT161, CT338, and CT429. Therefore, we revealed several C. trachomatis proteins that could be effectors subverting host cell processes.


Assuntos
Sistemas de Secreção Bacterianos , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Fatores de Virulência/metabolismo , Clonagem Molecular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Expressão Gênica , Células HeLa , Humanos , Transporte Proteico , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo
7.
Commun Biol ; 7(1): 839, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38987278

RESUMO

Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells.


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Esporos Bacterianos , Esporos Bacterianos/metabolismo , Esporos Bacterianos/genética , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Animais , Chlorocebus aethiops , Células Vero , Enterotoxinas/metabolismo , Enterotoxinas/genética
8.
J Bacteriol ; 194(23): 6574-85, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23042990

RESUMO

Chlamydia trachomatis is a human bacterial pathogen that multiplies only within an intracellular membrane-bound vacuole, the inclusion. C. trachomatis includes ocular and urogenital strains, usually causing infections restricted to epithelial cells of the conjunctiva and genital mucosa, respectively, and lymphogranuloma venereum (LGV) strains, which can infect macrophages and spread into lymph nodes. However, C. trachomatis genomes display >98% identity at the DNA level. In this work, we studied whether C. trachomatis Inc proteins, which have a bilobed hydrophobic domain that may mediate their insertion in the inclusion membrane, could be a factor determining these different types of infection and tropisms. Analyses of polymorphisms and phylogeny of 48 Inc proteins from 51 strains encompassing the three disease groups showed significant amino acid differences that were mainly due to variations between Inc proteins from LGV and ocular or urogenital isolates. Studies of the evolutionary dynamics of inc genes suggested that 10 of them are likely under positive selection and indicated that most nonsilent mutations are LGV specific. Additionally, real-time quantitative PCR analyses in prototype and clinical strains covering the three disease groups identified three inc genes with LGV-specific expression. We determined the transcriptional start sites of these genes and found LGV-specific nucleotides within their promoters. Thus, subtle variations in the amino acids of a subset of Inc proteins and in the expression of inc genes may contribute to the unique tropism and invasiveness of C. trachomatis LGV strains.


Assuntos
Chlamydia trachomatis/genética , Chlamydia trachomatis/patogenicidade , Linfogranuloma Venéreo/microbiologia , Polimorfismo Genético , Fatores de Virulência/genética , Chlamydia trachomatis/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/genética , Evolução Molecular , Humanos , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Transcrição Gênica , Tropismo
9.
Front Cell Infect Microbiol ; 12: 864626, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35711665

RESUMO

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 ß-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.


Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Legionella/metabolismo , Legionella pneumophila/metabolismo
10.
Front Cell Infect Microbiol ; 12: 902210, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35903198

RESUMO

Chlamydia 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.


Assuntos
Infecções por Chlamydia , Chlamydia trachomatis , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/microbiologia , Células HeLa , Humanos , Mamíferos/genética , Plasmídeos/genética
11.
PLoS One ; 17(2): e0264292, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35192658

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/patogenicidade , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Chlorocebus aethiops , Células HeLa , Humanos , Gotículas Lipídicas/microbiologia , Células Vero
12.
Cell Microbiol ; 11(8): 1236-53, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19438519

RESUMO

Salmonella enterica are facultative intracellular bacterial pathogens that proliferate within host cells in a membrane-bounded compartment, the Salmonella-containing vacuole (SCV). Intracellular replication of Salmonella is mediated by bacterial effectors translocated on to the cytoplasmic face of the SCV membrane by a type III secretion system. Some of these effectors manipulate the host endocytic pathway, resulting in the formation in epithelial cells of tubules enriched in late endosomal markers, known as Salmonella-induced filaments (SIFs). However, much less is known about possible interference of Salmonella with the secretory pathway. Here, a small-interference RNA screen revealed that secretory carrier membrane proteins (SCAMPs) 2 and 3 contribute to the maintenance of SCVs in the Golgi region of HeLa cells. This is likely to reflect a function of SCAMPs in vacuolar membrane dynamics. Moreover, SCAMP3, which accumulates on the trans-Golgi network in uninfected cells, marked tubules induced by Salmonella effectors that overlapped with SIFs but which also comprised distinct tubules lacking late endosomal proteins. We propose that SCAMP3 tubules reflect a manipulation of specific post-Golgi trafficking that might allow Salmonella to acquire nutrients and membrane, or to control host immune responses.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Infecções por Salmonella/metabolismo , Salmonella enterica/fisiologia , Rede trans-Golgi/metabolismo , Biomarcadores/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Microscopia de Fluorescência , RNA Interferente Pequeno , Infecções por Salmonella/virologia , Salmonella enterica/citologia , Salmonella enterica/patogenicidade , Via Secretória , Virulência
13.
Nucleic Acids Res ; 35(14): 4755-66, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17617643

RESUMO

In the absence of arabinose, the AraR transcription factor represses the expression of genes involved in the utilization of arabinose, xylose and galactose in Bacillus subtilis. AraR exhibits a chimeric organization: the N-terminal DNA-binding region belongs to the GntR family and the C-terminal effector-binding domain is homologous to the GalR/LacI family. Here, the AraR-DNA-binding interactions were characterized in vivo and in vitro. The effect of residue substitutions in the AraR N-terminal domain and of base-pair exchanges into an AraR-DNA-binding operator site were examined by assaying for AraR-mediated regulatory activity in vivo and DNA-binding activity in vitro. The results showed that residues K4, R45 and Q61, located in or near the winged-helix DNA-binding motif, were the most critical amino acids required for AraR function. In addition, the analysis of the various mutations in an AraR palindromic operator sequence indicated that bases G9, A11 and T16 are crucial for AraR binding. Moreover, an AraR mutant M34T was isolated that partially suppressed the effect of mutations in the regulatory cis-elements. Together, these findings extend the knowledge on the nature of AraR nucleoprotein complexes and provide insight into the mechanism that underlies the mode of action of AraR and its orthologues.


Assuntos
Arabinose/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica , Regiões Operadoras Genéticas , Regulon , Proteínas Repressoras/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
14.
Microb Cell ; 6(9): 414-449, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31528632

RESUMO

Chlamydia trachomatis serovars are obligate intracellular bacterial pathogens mainly causing ocular and urogenital infections that affect millions of people worldwide and which can lead to blindness or sterility. They reside and multiply intracellularly within a membrane-bound vacuolar compartment, known as inclusion, and are characterized by a developmental cycle involving two morphologically and physiologically distinct chlamydial forms. Completion of the developmental cycle involves the secretion of > 70 C. trachomatis proteins that function in the host cell cytoplasm and nucleus, in the inclusion membrane and lumen, and in the extracellular milieu. These proteins can, for example, interfere with the host cell cytoskeleton, vesicular and non-vesicular transport, metabolism, and immune signalling. Generally, this promotes C. trachomatis invasion into, and escape from, host cells, the acquisition of nutrients by the chlamydiae, and evasion of cell-autonomous, humoral and cellular innate immunity. Here, we present an in-depth review on the current knowledge and outstanding questions about these C. trachomatis secreted proteins.

15.
Sci Rep ; 9(1): 6133, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992493

RESUMO

Chlamydia 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.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/patologia , Chlamydia trachomatis/patogenicidade , Complexo de Golgi/metabolismo , Animais , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/metabolismo , Chlorocebus aethiops , Células HeLa , Humanos , Mutação , Vesículas Transportadoras/metabolismo , Vacúolos/metabolismo , Células Vero
16.
Artigo em Inglês | MEDLINE | ID: mdl-30094225

RESUMO

Chlamydia trachomatis is an obligate intracellular human pathogen causing mainly ocular and genital infections of significant clinical and public health impact. C. trachomatis multiplies intracellularly in a membrane bound vacuole, known as inclusion. Both extracellularly and from within the inclusion, C. trachomatis uses a type III secretion system to deliver several effector proteins into the cytoplasm of host cells. A large proportion of these effectors, the inclusion membrane (Inc) proteins, are exposed to the host cell cytosol but possess a characteristic hydrophobic domain mediating their insertion in the inclusion membrane. By yeast two-hybrid, we found that C. trachomatis Inc CT288 interacts with the human centrosomal protein CCDC146 (coiled-coil domain-containing protein 146). The interaction was also detected by co-immunoprecipitation in mammalian cells either ectopically expressing CCDC146 and CT288 or ectopically expressing CCDC146 and infected by a C. trachomatis strain expressing epitope-tagged and inclusion membrane-localized CT288. In uninfected mammalian cells, ectopically expressed full-length CCDC146 (955 amino acid residues) localized at the centrosome; but in cells infected by wild-type C. trachomatis, its centrosomal localization was less evident and CCDC146 accumulated around the inclusion. Recruitment of CCDC146 to the inclusion periphery did not require intact host Golgi, microtubules or microfilaments, but was dependent on chlamydial protein synthesis. Full-length CCDC146 also accumulated at the periphery of the inclusion in cells infected by a C. trachomatis ct288 mutant; however, a C-terminal fragment of CCDC146 (residues 692-955), which interacts with CT288, showed differences in localization at the periphery of the inclusion in cells infected by wild-type or ct288 mutant C. trachomatis. This suggests a model in which chlamydial proteins other than CT288 recruit CCDC146 to the periphery of the inclusion, where the CT288-CCDC146 interaction might contribute to modulate the function of this host protein.


Assuntos
Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/fisiologia , Interações Hospedeiro-Patógeno , Corpos de Inclusão/microbiologia , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Vacúolos/microbiologia , Animais , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Corpos de Inclusão/química , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/química , Células Vero
17.
mBio ; 9(5)2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279280

RESUMO

Nonflagellar type III secretion systems (nf T3SSs) form a cell surface needle-like structure and an associated translocon that deliver bacterial effector proteins into eukaryotic host cells. This involves a tightly regulated hierarchy of protein secretion. A switch involving SctP and SctU stops secretion of the needle protein. The gatekeeper protein SctW is required for secretion of translocon proteins and controls a second switch to start effector secretion. Salmonella enterica serovar Typhimurium encodes two T3SSs in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. The acidic vacuole containing intracellular bacteria stimulates assembly of the SPI-2 T3SS and its translocon. Sensing the nearly neutral host cytosolic pH is required for effector translocation. Here, we investigated the involvement of SPI-2-encoded proteins SsaP (SctP), SsaU (SctU), SsaV (SctV), and SsaL (SctW) in regulation of secretion. We found that SsaP and SsaU are involved in the first but not the second secretion switch. A random-mutagenesis screen identified amino acids of SsaV that regulate translocon and effector secretion. Single substitutions in subdomain 4 of SsaV or InvA (SPI-1-encoded SctV) phenocopied mutations of their corresponding gatekeepers with respect to translocon and effector protein secretion and host cell interactions. SsaL interacted with SsaV in bacteria exposed to low ambient pH but not after the pH was raised to 7.2. We propose that SsaP and SsaU enable the apparatus to become competent for a secretion switch and facilitate the SsaL-SsaV interaction. This mediates secretion of translocon proteins until neutral pH is sensed, which causes their dissociation, resulting in arrest of translocon secretion and derepression of effector translocation.IMPORTANCESalmonella Typhimurium is an intracellular pathogen that uses the SPI-2 type III secretion system to deliver virulence proteins across the vacuole membrane surrounding intracellular bacteria. This involves a tightly regulated hierarchy of protein secretion controlled by two molecular switches. We found that SPI-2-encoded proteins SsaP and SsaU are involved in the first but not the second secretion switch. We identify key amino acids of the inner membrane protein SsaV that are required to interact with the so-called gatekeeper protein SsaL and show that the dissociation of SsaV-SsaL causes the second switch, leading to delivery of effector proteins. Our results provide insights into the molecular events controlling virulence-associated type III secretion and suggest a broader model describing how the process is regulated.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Mapeamento de Interação de Proteínas , Sistemas de Secreção Tipo III/genética , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Análise Mutacional de DNA , Concentração de Íons de Hidrogênio , Ligação Proteica , Multimerização Proteica
18.
Trends Microbiol ; 14(5): 197-200, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16564172

RESUMO

Type III secretion is used by many Gram-negative pathogenic bacteria to inject effector proteins into eukaryotic host cells. Effector delivery requires a secretion apparatus, called an injectisome or needle complex, and the assembly of a translocation pore in a target-cell membrane. Recent work provides evidence that enlightens the view of how pore assembly might occur and of how the injectisome and the pore might be linked.


Assuntos
Antígenos de Bactérias/fisiologia , Yersinia/fisiologia , Antígenos de Bactérias/genética , Proteínas Citotóxicas Formadoras de Poros , Yersinia/genética , Yersinia/metabolismo , Yersinia/ultraestrutura
20.
PLoS One ; 12(6): e0178856, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28622339

RESUMO

Chlamydia trachomatis is a human bacterial pathogen causing ocular and genital infections. It multiplies exclusively within an intracellular membrane-bound vacuole, the inclusion, and uses a type III secretion system to manipulate host cells by injecting them with bacterially-encoded effector proteins. In this work, we characterized the expression and subcellular localization in infected host cells of the C. trachomatis CT142, CT143, and CT144 proteins, which we previously showed to be type III secretion substrates. Transcriptional analyses in C. trachomatis confirmed the prediction that ct142, ct143 and ct144 are organized in an operon and revealed that their expression is likely driven by the main σ factor, σ66. In host cells infected by C. trachomatis, production of CT142 and CT143 could be detected by immunoblotting from 20-26 h post-infection. Immunofluorescence microscopy of infected cells revealed that from 20 h post-infection CT143 appeared mostly as globular structures outside of the bacterial cells but within the lumen of the inclusion. Furthermore, immunofluorescence microscopy of cells infected by C. trachomatis strains carrying plasmids producing CT142, CT143, or CT144 under the control of the ct142 promoter and with a C-terminal double hemagglutinin (2HA) epitope tag revealed that CT142-2HA, CT143-2HA or CT144-2HA showed an identical localization to chromosomally-encoded CT143. Moreover, CT142-2HA or CT144-2HA and CT143 produced by the same bacteria co-localized in the lumen of the inclusion. Overall, these data suggest that the CT142, CT143, and CT144 type III secretion substrates are secreted into the lumen of the inclusion where they might form a protein complex.


Assuntos
Proteínas de Bactérias , Sistemas de Secreção Bacterianos , Chlamydia trachomatis , Plasmídeos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/metabolismo , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Chlorocebus aethiops , Células HeLa , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo , Células Vero
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