Abstract
BackgroundEnteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli are important causes of morbidity and mortality worldwide. These enteric pathogens contain a type III secretion system (T3SS) responsible for the attaching and effacing (A/E) lesion phenotype. The T3SS is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. The H-NS-mediated repression of LEE expression is counteracted by Ler, the major activator of virulence gene expression in A/E pathogens. A regulator present in EPEC, H-NST, positively affects expression of H-NS regulon members in E. coli K-12, although the effect of H-NST on LEE expression and virulence of A/E pathogens has yet-to-be determined.ResultsWe examine the effect of H-NST on LEE expression and A/E lesion formation on intestinal epithelial cells. We find that H-NST positively affects the levels of LEE-encoded proteins independently of ler and induces A/E lesion formation. We demonstrate H-NST binding to regulatory regions of LEE1 and LEE3, the first report of DNA-binding by H-NST. We characterize H-NST mutants substituted at conserved residues including Ala16 and residues Arg60 and Arg63, which are part of a potential DNA-binding domain. The single mutants A16V, A16L, R60Q and the double mutant R60Q/R63Q exhibit a decreased effect on LEE expression and A/E lesion formation. DNA mobility shift assays reveal that these residues are important for H-NST to bind regulatory LEE DNA targets. H-NST positively affects Ler binding to LEE DNA in the presence of H-NS, and thereby potentially helps Ler displace H-NS bound to DNA.ConclusionsH-NST induces LEE expression and A/E lesion formation likely by counteracting H-NS-mediated repression. We demonstrate that H-NST binds to DNA and identify arginine residues that are functionally important for DNA-binding. Our study suggests that H-NST provides an additional means for A/E pathogens to alleviate repression of virulence gene expression by H-NS to promote virulence capabilities.
Highlights
The histone-like nucleoid structuring protein (H-NS) of Escherichia coli is the prototype of an important family of regulatory proteins that repress transcription of numerous genes in Gramnegative bacteria [1,2]
Production of H-NS truncated protein (H-NST) from pQEH-NST in Enteropathogenic E. coli (EPEC) grown to the exponential phase in DMEM did not affect levels of locus of enterocyte effacement (LEE)-encoded proteins, which might be due to the possibility that an effect of H-NST is masked by a relatively high basal level of LEE-encoded proteins present in EPEC
Since the abundance of LEE-encoded proteins in EHEC is minimal in the exponential phase [48], we assessed the regulatory effect of H-NST on the LEE using the EHEC O157:H7 strain TUV93-0, a stx-deleted derivative of EDL933 that exhibits
Summary
The histone-like nucleoid structuring protein (H-NS) of Escherichia coli is the prototype of an important family of regulatory proteins that repress transcription of numerous genes in Gramnegative bacteria [1,2]. H-NS-mediated modulation of gene expression can involve multiple mechanisms including binding of H-NS to regulatory regions of H-NS regulon genes to block association of RNA polymerase or by preventing open-complex formation after RNAP has already associated with the promoter [1,4,5,6]. These mechanisms can be augmented or countered by other nucleoid-associated proteins such as Hha, YmoA, Fis, HU, and IHF [1,6]. A regulator present in EPEC, H-NST, positively affects expression of H-NS regulon members in E. coli K-12, the effect of H-NST on LEE expression and virulence of A/E pathogens has yet-to-be determined
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