RESUMO
Afa/Dr diffusely adhering Escherichia coli (DAEC) strains are responsible for urinary tract and intestinal infections. Both in intestine and kidney, the epithelial cells forming epithelium are sealed by junctional domains. We provide evidence that the Secreted autotransporter toxin, Sat, belonging to the subfamily of serine protease autotransporters of Enterobacteriaceae (SPATEs), acts as a virulence factor in Afa/Dr DAEC by promoting lesions in the tight junctions (TJs) of polarized epithelial Caco-2/TC7 cells. Southern blot analysis reveals that the prototype strains of the subclass-1 and subclass-2 typical Afa/Dr DAEC strains, hybridize with a sat probe. Using the wild-type IH11128 strain, the recombinant E. coli AAEC185 strain that expresses Sat, the recombinant E. coli that expresses both Dr adhesin and Sat, we report that Sat in monolayers of cultured enterocyte-like Caco-2/TC7 cells, induces rearrangements of the TJs-associated proteins ZO-1, ZO-3 and occludin, and increases the formation of domes as the result of an increase in the paracellular permeability without affecting the transepithelial electrical resistance of the cell monolayers. Moreover, we observe that Sat-induced disassembly of TJs-associated proteins is dependent on the serine protease motif. Finally, an analysis of the prevalence of the sat gene in three collections of Afa/Dr DAEC strains collected from the stools of children with and without diarrhoea, and from the urine of patients with urinary tract infection (UTI) shows that: (i) the sat gene is highly prevalent in UTI-associated Afa/Dr DAEC strains (88% positive), (ii) the sat gene is generally absent from Afa/Dr DAEC strains collected from the stools of children without diarrhoea (16% positive); whereas (iii) it is present in about half of the strains collected from the stools of children with diarrhoea (46% positive).
Assuntos
Adesinas de Escherichia coli/metabolismo , Toxinas Bacterianas/metabolismo , Células Epiteliais/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Junções Íntimas/ultraestrutura , Actinas/metabolismo , Motivos de Aminoácidos , Aderência Bacteriana , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Células CACO-2 , Criança , Diarreia/microbiologia , Células Epiteliais/citologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Humanos , Mutagênese Sítio-Dirigida , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Junções Íntimas/metabolismo , Infecções Urinárias/microbiologia , Fatores de Virulência/metabolismoRESUMO
In vivo Listeria monocytogenes infection results in the massive release of mucus by goblet cells into the lumen of the intestine. We have previously reported that apical infection by L. monocytogenes is followed by listeriolysin O (LLO)-dependent stimulation of mucus exocytosis, and the upregulation of the MUC genes. Here, we report that L. monocytogenes EGD wild-type bacteria enter cultured human polarized, mucin-secreting, HT29-MTX cells apically by an InlA-dependent mechanism. The LLO-induced increase in mucin secretion together with an increase in transcription of the MCU4 and MUC12 genes encoding for membrane-bound mucins, results in the inhibition of the cell-entry of L. monocytogenes into mucin-secreting, HT29-MTX cells. Moreover, we report that sialic acid residues in mucins are crucial for the inhibition of L. monocytogenes internalization. Based on these findings, we suggest that the LLO-induced mucin exocytosis and upregulation of the MUC genes encoding for membrane-bound mucins constitute a host cell defence response that inhibits the cell-entry of L. monocytogenes.
Assuntos
Proteínas de Choque Térmico/fisiologia , Listeria monocytogenes/patogenicidade , Mucinas/genética , Mucinas/metabolismo , Regulação para Cima , Toxinas Bacterianas , Linhagem Celular , Citoplasma/microbiologia , Células Epiteliais/microbiologia , Exocitose , Proteínas Hemolisinas , Humanos , Listeria monocytogenes/fisiologia , Mucina-4 , Ácido N-Acetilneuramínico/fisiologiaRESUMO
The human gastrointestinal microbiota produces antagonistic activities against gastrointestinal bacterial pathogens. We undertook a study to investigate the mechanism(s) by which a Lactobacillus acidophilus strain of human microbiota origin antagonizes the gram-negative enteroinvasive pathogen Salmonella enterica serovar Typhimurium. We showed that the cell-free culture supernatant of L. acidophilus strain LB (LB-CFCS) induced the following effects in S. enterica SL1344: (i) a decrease in intracellular ATP that paralleled bacterial death, (ii) the release of lipopolysaccharide, (iii) permeabilization of the bacterial membrane, and (iv) an increase in the sensitivity of Salmonella to the lytic action of sodium dodecyl sulfate. Finally, we showed using two mutant strains of Salmonella, PhoP MS7953s and PmrA JKS1170, that the two-component regulatory systems PhoP-PhoQ and PmrA-PmrB that regulate the mechanisms of resistance to antibacterial agents in Salmonella did not influence the anti-Salmonella effect of LB-CFCS.
Assuntos
Antibiose , Membrana Celular/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Lactobacillus acidophilus/crescimento & desenvolvimento , Salmonella typhimurium/crescimento & desenvolvimento , Trifosfato de Adenosina/metabolismo , Bacteriólise , Membrana Celular/patologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Regulação Bacteriana da Expressão Gênica , Humanos , Lactobacillus acidophilus/metabolismo , Lipopolissacarídeos/metabolismo , Salmonella typhimurium/patogenicidade , Transdução de SinaisRESUMO
The mechanism(s) underlying the antibacterial activity of probiotic Lactobacillus strains appears to be multifactorial and includes lowering of the pH and the production of lactic acid and of antibacterial compounds, including bacteriocins and nonbacteriocin, non-lactic acid molecules. Addition of Dulbecco's modified Eagle's minimum essential medium to the incubating medium delays the killing activity of lactic acid. We found that the probiotic strains Lactobacillus johnsonii La1, Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029, L. casei DN-114 001, and L. rhamnosus GR1 induced a dramatic decrease in the viability of Salmonella enterica serovar Typhimurium SL1344 mainly attributable to non-lactic acid molecule(s) present in the cell-free culture supernatant (CFCS). These molecules were more active against serovar Typhimurium SL1344 in the exponential growth phase than in the stationary growth phase. We also showed that the production of the non-lactic acid substance(s) responsible for the killing activity was dependent on growth temperature and that both unstable and stable substances with killing activity were present in the CFCSs. We found that the complete inhibition of serovar Typhimurium SL1344 growth results from a pH-lowering effect.