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1.
Environ Microbiol ; 24(3): 1035-1051, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34431194

RESUMO

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new YehD fimbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.


Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Aderência Bacteriana/genética , Células CACO-2 , Citocinas/metabolismo , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Células HeLa , Humanos , Virulência/genética
2.
Environ Microbiol ; 20(9): 3363-3377, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30062827

RESUMO

Avian pathogenic Escherichia coli (APEC) causes localized and systemic avian infections and is responsible for considerable economic losses in the poultry industry. This organism adheres and invades human and avian cells, however, the regulatory networks that dictate its virulence are largely unknown. The CpxRA two-component system is responsible for sensing and controlling outer-membrane stress and detecting misfolded proteins in the bacterial periplasmic space. CpxA is a membrane sensor kinase and CpxR is a cytoplasmic transcriptional regulator. In this study, we found that the CpxRA system regulates the virulence properties of APEC. Adherence, invasiveness, motility, production of type 1 fimbriae and biofilm were negatively affected in the ΔcpxA mutant indicating that the CpxA is required for full manifestation of these phenotypes. We also found that CpxR-P directly bound to the fimA promoter, locking the fimS region of type 1 fimbriae in the phase-OFF orientation. In addition, the absence of CpxA also reduced flagella production strongly suggesting that CpxRA regulates these two important surface organelles in APEC. This study provides compelling evidence of the role of the CpxRA two-component system in the regulation of virulence factors of avian pathogenic E. coli.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Doenças das Aves Domésticas/microbiologia , Proteínas Quinases/metabolismo , Animais , Proteínas de Bactérias/genética , Galinhas , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Humanos , Ligação Proteica , Proteínas Quinases/genética , Virulência , Fatores de Virulência/genética
3.
mSphere ; 9(5): e0006024, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38647313

RESUMO

Enterobacter cloacae is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of E. cloacae harbors two T6SS gene clusters (T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in E. cloacae. Single deletions of the genes encoding the HU subunits (hupA and hupB) decreased mRNA levels of both T6SS. In contrast, the hupA hupB double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the hup genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were trans-complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria. IMPORTANCE: T6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche. Enterobacter cloacae expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in E. cloacae, affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on E. cloacae pathogenesis.


Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA , Enterobacter cloacae , Regulação Bacteriana da Expressão Gênica , Sistemas de Secreção Tipo VI , Enterobacter cloacae/genética , Enterobacter cloacae/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Família Multigênica
4.
Front Cell Infect Microbiol ; 12: 916247, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36204636

RESUMO

The genome of Mycobacterium tuberculosis (Mtb) harbors the genetic machinery for assembly of the Fimbrial low-molecular-weight protein (Flp) type IV pilus. Presumably, the Flp pilus is essential for pathogenesis. However, it remains unclear whether the pili genes are transcribed in culture or during infection of host cells. This study aimed to shed light on the expression of the Flp pili-assembly genes (tadZ, tadA, tadB, tadC, flp, tadE, and tadF) in Mtb growing under different growth conditions (exponential phase, stationary phase, and dormancy NRP1 and NRP2 phases induced by hypoxia), during biofilm formation, and in contact with macrophages and alveolar epithelial cells. We found that expression of tad/flp genes was significantly higher in the stationary phase than in exponential or NRP1 or NRP2 phases suggesting that the bacteria do not require type IV pili during dormancy. Elevated gene expression levels were recorded when the bacilli were in contact for 4 h with macrophages or epithelial cells, compared to mycobacteria propagated alone in the cultured medium. An antibody raised against a 12-mer peptide derived from the Flp pilin subunit detected the presence of Flp pili on intra- and extracellular bacteria infecting eukaryotic cells. Altogether, these are compelling data showing that the Flp pili genes are expressed during the interaction of Mtb with host cells and highlight a role for Flp pili in colonization and invasion of the host, subsequently promoting bacterial survival during dormancy.


Assuntos
Proteínas de Fímbrias , Mycobacterium tuberculosis , Células Epiteliais Alveolares/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Macrófagos/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Óperon
5.
Artigo em Inglês | MEDLINE | ID: mdl-31380298

RESUMO

Klebsiella pneumoniae successfully colonizes host tissues by recognizing and interacting with cholesterol present on membrane-associated lipid rafts. In this study, we evaluated the role of cholesterol in the expression of capsule polysaccharide genes of K. pneumoniae and its implication in resistance to phagocytosis. Our data revealed that exogenous cholesterol added to K. pneumoniae increases macrophage-mediated phagocytosis. To explain this event, the expression of capsular galF, wzi, and manC genes was determined in the presence of cholesterol. Down-regulation of these capsular genes occurred leading to increased susceptibility to phagocytosis by macrophages. In contrast, depletion of cholesterol from macrophage membranes led to enhanced expression of galF, wzi, and manC genes and to capsule production resulting in resistance to macrophage-mediated phagocytosis. Cholesterol-mediated repression of capsular genes was dependent on the RcsA and H-NS global regulators. Finally, cholesterol also down-regulated the expression of genes responsible for LPS core oligosaccharides production and OMPs. Our results suggest that cholesterol plays an important role for the host by reducing the anti-phagocytic properties of the K. pneumoniae capsule facilitating bacterial engulfment by macrophages during the bacteria-eukaryotic cell interaction mediated by lipid rafts.


Assuntos
Colesterol/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Polissacarídeos Bacterianos/antagonistas & inibidores , Células A549 , Cápsulas Bacterianas/efeitos dos fármacos , Cápsulas Bacterianas/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Colesterol/metabolismo , Genes Bacterianos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/metabolismo , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/microbiologia , Polissacarídeos Bacterianos/biossíntese , Células THP-1
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