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Volume 4, Issue 4(Suppl)
J Infect Dis Ther 2016
ISSN: 2332-0877, JIDT an open access journal
Page 58
Notes:
Infectious Diseases 2016
August 24-26, 2016
conferenceseries
.com
August 24-26, 2016 Philadelphia, USA
&
Infectious Diseases
Joint Event on
2
nd
World Congress on
Pediatric Care & Pediatric Infectious Diseases
International Conference on
Multiple regulatory small RNAs control virulence in enteropathogenic
Escherichia coli
Shantanu Bhatt, Marisa Egan, Valerie Jenkins, Thomas Buerkert, Jasmine Ramirez, Christian Xander, Jamie Palmer
and
Elizabeth Storm
Saint Joseph’s University, USA
E
nteropathogenic
Escherichia coli
(EPEC) is responsible for considerable disease and death amongst infants in developing countries.
EPEC belongs to the attaching and effacing (A/E) family of bacterial pathogens, which are aptly named because they adhere
intimately to intestinal cells and destroy cellular microvilli to form characteristic pathomorphological A/E lesions, which lead to
diarrhea and dehydration. The ability to form A/E lesions resides within the virulence module locus of enterocyte effacement (LEE),
which encodes a type III secretion system (T3SS). To date, over fifty non-LEE encoded regulators have been identified in EPEC. The
vast majority of these regulators affect the expression of the LEE-encoded transcription factors, Ler, GrlR and GrlA. Intriguingly,
every regulator of the LEE that has been identified to date in EPEC is a proteinaceous factor. Thus far, not a single regulatory small
RNA (sRNA) has been implicated in its virulence. We set out to identify and characterize sRNA regulators of the LEE in EPEC. Our
preliminary data suggest that Hfq globally represses gene expression from all the LEE-encoded genes including the
grlRA
operon.
Because Hfq and Hfq-dependent sRNAs typically target the 5’ region of the first gene in an operon, we constructed a reporter
E. coli
strain in which only the 5’ UTR and 45 nucleotides of the
grlR
ORF were fused to a chromosomal N-terminally truncated
lacZ
gene
driven by the heterologous
P
araBAD
promoter (
P
araBAD
-grlR-lacZ
). Inactivation of
hfq
resulted in elevated β-galactosidase activity from
the
grlR’-‘lacZ
fusion suggesting that the cloned 5’ region of
grlR
was sufficient to elicit Hfq-dependent repression. These results also
suggest that one or more Hfq-dependent sRNAs, conserved between EPEC and
E. coli
, regulate
grlRA
. To identify these sRNAs, each
of the 27 conserved Hfq-dependent sRNAs was individually overproduced in the
grlR-lacZ
reporter strain. Three sRNAs-MgrR, RyhB
and McaS reproducibly repressed the
grlR-lacZ
fusion. Using IntaRNA we aligned each of the 3 sRNAs to the cloned 5’ region of
grlR
.
Bioinformatic analysis revealed that MgrR exhibited the most extensive and contiguous region of complementarity (~10 bp) with
the
grlR
leader region. The predicted base-pairing region in MgrR was substituted with a scrambled oligonucleotide sequence that
lacks complementarity to
grlR
. Predictably, mutation of the base-pairing region abolished the ability of MgrR to pair to and repress
the
grlR-lacZ
fusion, thereby providing genetic evidence for direct base-pairing between
grlR
and MgrR. Subsequent experiments
revealed that MgrR binds to the same region on the
grlR
A transcript as CsrA and counteracts its stimulatory effect. Meanwhile,
RyhB appeared to form a relatively shorter (~6 bp) duplex with the ribosome-binding site of
grlR
. An oligonucleotide substitution
in the base-pairing region of RyhB also prevented the sRNA from repressing the
grlR-lacZ
fusion, suggesting that RyhB, like MgrR,
base pairs to the 5’ region of
grlR
. In contrast to MgrR and RyhB, McaS did not possess any regions of complementarity to
grlR
and
presumably exerts its effect by sequestering CsrA. In summary, our results provide the first piece of evidence to implicate multiple
Hfq-dependent sRNAs in controlling the LEE-encoded virulence of EPEC. Future studies are aimed at elucidating the molecular
mechanism by which MgrR, RyhB and McaS regulate the LEE and the ensuing A/E lesion formation.
Biography
Shantanu Bhatt is affiliated to Saint Joseph’s University, USA
sbhatt@sju.eduShantanu Bhatt et al., J Infect Dis Ther 2016, 4:4(Suppl)
http://dx.doi.org/10.4172/2332-0877.C1.008