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Protocols
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Articles and Issues
Current Issue
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AI-Generated Material
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Submit a Protocol
Overview
Authored
(1)
Reviewed
(2)
Former Editor-in-Chief (2018-2020)
Jeffrey M. Milush
Associate Professor, University of California San Francisco
Research fields
Immunology
Peer-reviewed
Preprint
Intracellular Macrophage Infections with
E. coli
under Nitrosative Stress
Authors:
Stacey L. Bateman
and
Patrick Seed
,
date:
10/20/2012,
view:
13508,
Q&A:
0
Escherichia coli
(
E. coli
) produces disseminated infections of the urinary tract, blood, and central nervous system where it encounters professional phagocytes such as macrophages, which utilize reactive nitrogen intermediates (RNI) to arrest bacteria.
In vitro
, extraintestinal pathogenic
E. coli
(ExPEC) can survive within bone marrow-derived macrophages for greater than 24 h post-infection within a LAMP1+ vesicular compartment, and ExPEC strains, in particular, are better adapted to intracellular macrophage survival than commensal strains (Bokil
et al.
, 2011). This protocol details an intracellular murine macrophage-like cell infection, including modulation of the host nitrosative stress response, to model this host-pathogen interaction
in vitro
. To accomplish this, RAW 264.7 murine macrophage-like cells are pre-incubated with either L-arginine, an NO precursor, or IFNγ to yield a high nitric oxide (NO) physiological state, or L-NAME, an inducible NO synthase (iNOS)-specific inhibitor, to yield a low NO physiological state. This protocol has been successfully utilized to assess the contribution of a novel ExPEC regulator to intracellular survival and the nitrosative stress response during macrophage infections (Bateman and Seed, 2012), but can be adapted for use with a variety of
E. coli
strains or isogenic deletions.
More >
Promoter Orientation of Prokaryotic Phase-variable Genes by PCR
Authors:
Stacey L. Bateman
and
Patrick Seed
,
date:
10/20/2012,
view:
9925,
Q&A:
0
One major mechanism of phase variable gene expression in prokaryotes is through inversion of the promoter element for a gene or operon. This protocol describes how to detect the promoter orientation of a phase-variable gene by PCR. This protocol, including primer design, is specific to detection of the promoter orientations of hyxR, a LuxR-like response regulator in Extraintestinal Pathogenic
Escherichia coli
(ExPEC) isolates (Bateman and Seed, 2012); however, this protocol can be generalized to other organisms and genes to discriminate prokaryotic promoter inversions by PCR through size discrimination of the amplification products. Expression of hyxR is regulated through bidirectional phase inversion of the upstream promoter region mediated by a member of the family of site-specific tyrosine recombinases called Fim-like recombinases. The recombinases recognize inverted DNA repeat sequences flanking the promoter and produce a genomic rearrangement, orientating the promoter in favor or disfavor of gene expression.
More >
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