Analysis of O-island deletions in Escherichia coli O157:H7
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Date
30/11/2012Author
Flockhart, Allen Forrest
Metadata
Abstract
Escherichia coli (E. coli) are a diverse species of bacteria that reside, often
harmoniously and beneficially, in the gastrointestinal tracts of humans and
other mammals. However, some strains are associated with serious intestinal
and extra-intestinal disease and are considered pathogens. The main differences
between strains of these different E. coli pathotypes can be explained by the
acquisition of genetic information introduced by mobile genetic elements, in
particular bacteriophage. In enterohaemorrhagic E. coli (EHEC) O157:H7 strain
EDL933, a pathotype of E. coli containing prophage-encoded Shiga toxins
associated with severe gastrointestinal and systemic disease in humans, these
horizontally acquired elements have been termed O-islands (OIs) and include
both fully functional and cryptic prophages. The overall aim of this research
was to try and determine what these OIs are actually doing for the bacteria.
Systems pertinent in the life cycle and virulence of this pathogen were therefore
investigated by phenotypically screening a large library of OI deletions in EHEC
strain TUV93-0, a Shiga toxin-negative derivative strain of EDL933, and then
comparing these with the parent strain. These analyses highlighted a subset of
OIs with the potential to regulate motility and type III secretion (T3S), the latter
being an essential colonisation factor for EHEC that is encoded by the locus of
enterocyte effacement (LEE). Deletion of OI-51, a 14.93 Kb cryptic prophage
designated as CP-933C, significantly reduced persistence of faecal shedding in
sheep and levels of T3S expression in vitro. Cloning and complementation
together with targeted allelic replacements in OI-51 identified a novel positive
regulator of the LEE, encoded by ecs1581 in the sequenced E. coli O157:H7 strain
Sakai that is present but not annotated in the EDL933 sequence. Functionally
important residues of ECs1581 were identified by site-directed mutagenesis
based on phenotypic variants present in strains from different E. coli pathotypes,
including strains not harbouring a LEE-encoded T3S system. This regulator was
subsequently termed RgdR based on a motif demonstrated to be important for
stimulation of gene expression from LEE1. Purified RgdR protein was able to
form multiple complexes on a PCR generated LEE1 promoter fragment, and
activation of this operon appeared to require this DNA binding capacity as a
non-T3S inducing variant was unable to bind this same LEE1 promoter
fragment. RgdR did not directly activate LEE1 transcription in vitro, nor did it
activate transcription by relieving H-NS repression as proposed for the global
regulator Ler (LEE-encoded regulator). However, RgdR activation did require a
wild type LEE1 promoter and the Ler auto-induction cycle to induce LEE2-5
expression and T3S. RgdR was able to increase binding to Congo red and was
capable of repressing bacterial motility. Further analyses demonstrated that
RgdR did not regulate T3S and cell motility via GrlA (global regulator of LEE
activator) and QseC (quorum sensing E. coli regulator C), two established
regulators in E. coli that control LEE gene expression and motility in conjunction
with their partners, GrlR (global regulator of LEE repressor) and QseB (quorum
sensing E. coli regulator B) respectively. RgdR is therefore identified as a novel
regulator able to co-ordinate T3S and motility expression. This research has
identified OI-51 as being important for EHEC O157:H7 colonisation in sheep
and has identified a completely new family of small bacterial regulators that
control surface factor expression in E. coli.