H-NST induces LEE expression and the formation of attaching and effacing lesions in enterohemorrhagic Escherichia coli.
BACKGROUND
Enteropathogenic 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.


RESULTS
We 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.


CONCLUSIONS
H-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.
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America ; Graduate Program in Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland, United States of America.
Jonathan A
JA
Levine
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
Anne-Marie
AM
Hansen
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
Jane M
JM
Michalski
Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
Tracy H
TH
Hazen
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America ; Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
David A
DA
Rasko
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
James B
JB
Kaper
D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics; D020895:Enterocytes / Q000382:microbiology*; D054324:Enterohemorrhagic Escherichia coli / Q000235:genetics*; D054308:Enteropathogenic Escherichia coli / Q000235:genetics*; D004847:Epithelial Cells / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D044404:Genomic Islands / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000382:microbiology; D009154:Mutation / Q000235:genetics
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins