RESUMEN
Zn(2+) uptake systems are required for many enteric pathogens to survive and form biofilm in zinc-deficient conditions. ykgM and zinT (formerly yodA), regulated by Zur (zinc uptake regulator), have been reported as being highly induced during zinc shortage. This work reports that ykgM and zinT in enterohemorrhagic Escherichia coli (EHEC) O157:H7 biofilms under fluidic conditions were highly expressed compared to those in stationary-phase planktonic cells and a mutation of either ykgM or zinT genes led to the inhibition of curli biosynthesis. Inductively coupled plasma mass spectroscopy showed that the ykgM and zinT mutants contained lower concentrations of Zn(2+) than the wild type. Both mutants were less attached to both the glass surface of a microchannel and epithelial cells than the wild type. Quantitative reverse-transcription PCR data indicated that the expression of csgA, which encodes the major curli subunit, was inhibited in both mutants with a zinc deficiency. Scanning electron microscopy showed that the mutants grown under zinc-deficient condition were covered with a lower amount of curli compared to the wild type and often became filamentous. Zn(2+) supplementation restored curli production and prevented filamentation in the mutants. Overall, under zinc-deficient conditions, YkgM and ZinT proteins are required for maintaining optimal zinc concentration in EHEC and intracellular zinc deficiency inhibits curli production.
Asunto(s)
Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Zinc/metabolismo , Proteínas Bacterianas/metabolismo , Biopelículas , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Expresión Génica/efectos de los fármacos , Plancton/metabolismoRESUMEN
Broccoli extract (BE) has numerous beneficial effects on human health including anticancer activity. Quorum sensing (QS), mediated by self-produced autoinducer (AI) molecules, is a key process for the production of virulence determinants in pathogenic bacteria. BE suppressed AI-2 synthesis and AI-2-mediated bacterial motility in a dose-dependent manner in Escherichia coli O157:H7. In addition, expression of the ler gene that regulates AI-3 QS system was also diminished in response to treatment with BE. Furthermore, in an in vivo efficacy test using Caenorhabditis elegans as a host organism, C. elegans fed on E. coli O157:H7 in the presence of BE survived longer than those fed solely on the pathogenic bacteria. Quantitative real-time PCR analysis indicated that quercetin was the most active among the tested broccoli-derived compounds in downregulating virulence gene expression, while treatment with myricetin significantly suppressed the expression of the eae gene involved in type III secretion system. These data suggest that BE and its flavonoid constituents can inhibit expression of QS-associated genes, thereby downregulating the virulence attributes of E. coli O157:H7 both in vitro and in vivo. This study clearly elucidates BE's QS-inhibitory activity and suggests that BE has the potential to be developed as an anti-infective agent.
Asunto(s)
Antibacterianos/farmacología , Brassica/química , Caenorhabditis elegans/microbiología , Escherichia coli O157/efectos de los fármacos , Escherichia coli O157/patogenicidad , Extractos Vegetales/farmacología , Percepción de Quorum/efectos de los fármacos , Agonistas alfa-Adrenérgicos/farmacología , Animales , Regulación hacia Abajo/efectos de los fármacos , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flavonoides/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Homoserina/análogos & derivados , Homoserina/genética , Homoserina/metabolismo , Lactonas/metabolismo , Norepinefrina/farmacología , Regiones Promotoras Genéticas/genética , Percepción de Quorum/genética , Transactivadores/genética , Transactivadores/metabolismo , Virulencia/efectos de los fármacosRESUMEN
The inhibitory effects of green tea polyphenol epigallocatechin gallate (EGCG) on virulence phenotypes and gene expression regulated by quorum sensing (QS) in Escherichia coli O157:H7 were demonstrated at concentrations of 1 to 100 microg/ml, which are lower than the MIC (539 +/- 22 microg/ml). At 25 microg/ml, the growth rate was not affected, but autoinducer 2 concentration, biofilm formation, and swarm motility decreased to 13.2, 11.8, and 50%, respectively. Survival at 5 days of nematodes (Caenorhabditis elegans) that were fed the pathogen without and with EGCG were 47.1 and 76%, respectively. Real-time PCR data indicated decreased transcriptional level in many quorum sensing-regulated virulence genes at 25 microg/ml. Our results suggest that EGCG at concentrations below itsMIC has significant antipathogenic effects against E. coli O157:H7.