The bacterium Vibrio cholerae is native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across the V. cholerae genome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of many V. cholerae genes in response to lifestyle changes.
Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen which can cause diarrhea, vomiting, and, in some cases, severe complications such as kidney failure in humans. Up to 30% of cattle are colonized with EHEC, which can enter the food chain through contaminated meat, dairy, and vegetables. In order to control infections and stop transmission, it is important to understand what factors allow EHEC to colonize its hosts, cause virulence, and aid transmission. Since this cannot be systematically studied in humans, it is important to develop animal models of infection and transmission. We developed a model which allows us to study foodborne infection in zebrafish, a vertebrate host that is transparent and genetically tractable. Our results show that foodborne infection is more efficient than waterborne infection and that the locus of enterocyte effacement is a key virulence determinant in the zebrafish model. It is induced early during infection, and loss of tight LEE regulation leads to a decreased bacterial burden and decreased host mortality. Overall, the zebrafish model allows us to study foodborne infection, including pathogen release from the food vehicle and gene regulation and its context of host-microbe interactions, as well as environmental shedding and transmission to naive hosts.
Vibrio cholerae is a Gram-negative bacterium found
in aquatic environments and a human pathogen of global significance.
Its transition between host-associated and environmental lifestyles
involves the tight regulation of niche-specific phenotypes such as
motility, biofilm formation, and virulence. V. cholerae’s transition from the host to environmental dispersal usually
involves suppression of virulence and dispersion of biofilm communities.
In contrast to this naturally occurring transition, bacterial aggregation
by cationic polymers triggers a unique response, which is to suppress
virulence gene expression while also triggering biofilm formation
by V. cholerae, an artificial combination of traits
that is potentially very useful to bind and neutralize the pathogen
from contaminated water. Here, we set out to uncover the mechanistic
basis of this polymer-triggered bacterial behavior. We found that
bacteria–polymer aggregates undergo rapid autoinduction and
achieve quorum sensing at bacterial densities far below those required
for autoinduction in the absence of polymers. We demonstrate this
induction of quorum sensing is due both to a rapid formation of autoinducer
gradients and local enhancement of autoinducer concentrations within
bacterial clusters as well as the stimulation of CAI-1 and AI-2 production
by aggregated bacteria. We further found that polymers cause an induction
of the biofilm-specific regulator VpsR and the biofilm structural
protein RbmA, bypassing the usual suppression of biofilm during autoinduction.
Overall, this study highlights that synthetic materials can be used
to cross-wire natural bacterial responses to achieve a combination
of phenotypes with potentially useful applications.
Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting "bacteriomimetic" materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial -host interactions.
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