Neutrophil-Associated Responses to Vibrio cholerae Infection in a Natural Host Model.

Vibrio cholerae, the cause of human cholera, is an aquatic bacterium found in association with a variety of animals in the environment, including many teleost fish species. V. cholerae infection induces a proinflammatory response followed by a noninflammatory convalescent phase. Neutrophils are integral to this early immune response. However, the relationship between the neutrophil-associated protein calprotectin and V. cholerae has not been investigated, nor have the effects of limiting transition metals on V. cholerae growth. Zebrafish are useful as a natural V. cholerae model as the entire infectious cycle can be recapitulated in the presence of an intact intestinal microbiome and mature immune responses. Here, we demonstrate that zebrafish produce a significant neutrophil, interleukin 8 (IL-8), and calprotectin response following V. cholerae infection. Bacterial growth was completely inhibited by purified calprotectin protein or the chemical chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), but growth was recovered by the addition of the transition metals zinc and manganese. The expression of downstream calprotectin targets was also significantly increased in the zebrafish. These findings illuminate the role of host calprotectin in combating V. cholerae infection. Inhibition of V. cholerae growth through metal limitation may provide new approaches in the development of anti-V. cholerae therapeutics. This study also establishes a major role for calprotectin in combating infectious diseases in zebrafish.

H-NS and ToxT Inversely Control Cholera Toxin Production by Binding to Overlapping DNA Sequences.

Vibrio cholerae infects human hosts following ingestion of contaminated food or water, resulting in the severe diarrheal disease cholera. The watery diarrhea that is characteristic of the disease is directly caused by the production of cholera toxin (CT). A complex regulatory cascade controls the production of CT and other virulence factors. However, ultimately, a single protein, ToxT, directly binds to virulence gene promoters and activates their transcription. Previously, we identified two ToxT binding sites, or toxboxes, within the cholera toxin promoter (PctxAB). The toxboxes overlap the two promoter-proximal GATTTTT heptad repeats found within PctxAB in classical biotype V. cholerae strain O395. These heptad repeats were previously found to be located within a large DNA region bound by H-NS, a global transcriptional repressor present in Gram-negative bacteria. The current model for the control of PctxAB transcription proposes complete H-NS displacement from the DNA by ToxT, followed by direct activation by ToxT-RNA polymerase (RNAP) contacts. The goal of this study was to determine more precisely where H-NS binds to PctxAB and test the hypothesis that ToxT completely displaces H-NS from the PctxAB promoter before activating transcription. The results suggest that H-NS binds only to the region of PctxAB encompassing the heptad repeats and that ToxT displaces H-NS only from its most promoter-proximal binding sites, calling for a revision of the current model involving H-NS and ToxT at PctxAB. IMPORTANCE H-NS is a global negative regulator of transcription in Gram-negative bacteria, particularly in horizontally acquired genetic islands. Previous work in Vibrio cholerae suggested that H-NS represses the transcription of cholera toxin genes by binding to a large region upstream of its promoter and that the virulence activator ToxT derepresses transcription by removing H-NS from the promoter. Here, new data support a revised model in which ToxT displaces only H-NS bound to the most promoter-proximal DNA sites that overlap the ToxT binding sites, leaving the upstream sites occupied by H-NS. This introduces a higher-resolution mechanism for the antirepression of H-NS in the control of cholera toxin production.

The Vibrio cholerae Type Six Secretion System Is Dispensable for Colonization but Affects Pathogenesis and the Structure of Zebrafish Intestinal Microbiome.

Zebrafish (Danio rerio) are an attractive model organism for a variety of scientific studies, including host-microbe interactions. The organism is particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae, an intestinal pathogen. V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. While numerous studies have explored various aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms, few, if any, have examined how a V. cholerae infection alters the resident intestinal microbiome and the role of the type six secretion system (T6SS) in that process. In this study, 16S rRNA gene sequencing was utilized to investigate how strains of V. cholerae both with and without the T6SS alter the aforementioned microbial profiles following an infection. V. cholerae infection induced significant changes in the zebrafish intestinal microbiome, and while not necessary for colonization, the T6SS was important for inducing mucin secretion, a marker for diarrhea. Additional salient differences to the microbiome were observed based on the presence or absence of the T6SS in the V. cholerae utilized for challenging the zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiome to enable colonization and that the T6SS is important for pathogenesis induced by the examined V. cholerae strains. Furthermore, the presence or absence of T6SS differentially and significantly affected the composition and structure of the intestinal microbiome, with an increased abundance of other Vibrio bacteria observed in the absence of V. cholerae T6SS.

Vibrio cholerae Infection Induces Strain-Specific Modulation of the Zebrafish Intestinal Microbiome.

Zebrafish (Danio rerio) is an attractive model organism to use for an array of scientific studies, including host-microbe interactions. Zebrafish contain a core (i.e., consistently detected) intestinal microbiome consisting primarily of Proteobacteria. Furthermore, this core intestinal microbiome is plastic and can be significantly altered due to external factors. Zebrafish are particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae. As an intestinal pathogen, V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. Members of the resident intestinal microbial community likely must be reduced or eliminated by V. cholerae for colonization, and subsequent disease, to occur. Many studies have explored a variety of aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms but few have researched how a V. cholerae infection changes the resident intestinal microbiome. In this study, 16S rRNA gene sequencing was used to examine how five genetically diverse V. cholerae strains alter the intestinal microbiome following an infection. We found that V. cholerae colonization induced significant changes in the zebrafish intestinal microbiome. Notably, changes in the microbial profile were significantly different from each other, based on the particular strain of V. cholerae used to infect zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiota to enable colonization and that specific microbes that are targeted depend on the V. cholerae genotype.

Zebrafish Models for Pathogenic Vibrios.

Vibrio is a large and diverse genus of bacteria, of which most are nonpathogenic species found in the aquatic environment. However, a subset of the Vibrio genus includes several species that are highly pathogenic, either to humans or to aquatic animals. In recent years, Danio rerio, commonly known as the zebrafish, has emerged as a major animal model used for studying nearly every aspect of biology, including infectious diseases. Zebrafish are especially useful because the embryos are transparent, larvae are small and facilitate imaging studies, and numerous transgenic fish strains have been constructed. Zebrafish models for several pathogenic Vibrio species have been described, and indeed a fish model is highly relevant for the study of aquatic bacterial pathogens. Here, we summarize the zebrafish models that have been used to study pathogenic Vibrio species to date.

Anti-virulence activity of polyphenolic fraction isolated from Kombucha against Vibrio cholerae.

The use of traditional foods and beverages or their bioactive compounds as anti-virulence agents is a new alternative method to overcome the increased global emergence of antimicrobial resistance in enteric pathogens. In the present study, we investigated the anti-virulence activity of a polyphenolic fraction previously isolated from Kombucha, a 14-day fermented beverage of sugared black tea, against Vibrio cholerae O1. The isolated fraction was mainly composed of the polyphenols catechin and isorhamnetin. The fraction, the individual polyphenols and the combination of the individual polyphenols significantly inhibited bacterial swarming motility and expression of flagellar regulatory genes motY and flaC, even at sub-inhibitory concentrations. The polyphenolic compounds also decreased bacterial protease secretion and mucin penetration in vitro. In vivo study revealed that the polyphenolic fraction significantly inhibited V. cholerae induced fluid accumulation in the rabbit ileal loop model and intestinal colonization in suckling mice model. Therefore, the anti-virulence activity of the Kombucha polyphenolic fraction involved inhibition of motility and protease secretion of V. cholerae, thus preventing bacterial penetration through the mucin layer as well as fluid accumulation and bacterial colonization in the intestinal epithelial cells. The overall results implied that Kombucha might be considered as a potential alternative source of anti-virulence polyphenols against V. cholerae. To the best of our knowledge, this is the first report on the anti-virulence activity of Kombucha, mostly attributed to its polyphenolic content.

Characterization of the Vibrio cholerae Phage Shock Protein Response.

The phage shock protein (Psp) system is a stress response pathway that senses and responds to inner membrane damage. The genetic components of the Psp system are present in several clinically relevant Gram-negative bacteria, including Vibrio cholerae However, most of the current knowledge about the Psp response stems from in vitro studies in Escherichia coli and Yersinia enterocolitica In fact, the Psp response in V. cholerae has remained completely uncharacterized. In this study, we demonstrate that V. cholerae does have a functional Psp response system. We found that overexpression of GspD (EpsD), the type II secretion system secretin, induces the Psp response, whereas other V. cholerae secretins do not. In addition, we have identified several environmental conditions that induce this stress response. Our studies on the genetic regulation and induction of the Psp system in V. cholerae suggest that the key regulatory elements are conserved with those of other Gram-negative bacteria. While a psp null strain is fully capable of colonizing the infant mouse intestine, it exhibits a colonization defect in a zebrafish model, indicating that this response may be important for disease transmission in the environment. Overall, these studies provide an initial understanding of a stress response pathway that has not been previously investigated in V. choleraeIMPORTANCEVibrio cholerae leads a dual life cycle, as it can exist in the aquatic environment and colonize the human small intestine. In both life cycles, V. cholerae encounters a variety of stressful conditions, including fluctuating pH and temperature and exposure to other agents that may negatively affect cell envelope homeostasis. The phage shock protein (Psp) response is required to sense and respond to such insults in other bacteria but has remained unstudied in V. cholerae Interestingly, the Psp system has protein homologs, principally, PspA, in a number of bacterial clades as well as in archaea and plants. Therefore, our findings not only fill a gap in knowledge about an unstudied extracytoplasmic stress response in V. cholerae, but also may have far-reaching implications.

Glucose Metabolism by Escherichia coli Inhibits Vibrio cholerae Intestinal Colonization of Zebrafish.

The Vibrio cholerae O1 serogroup is responsible for pandemic cholera and is divided into the classical and El Tor biotypes. Classical V. cholerae produces acid when using glucose as a carbon source, whereas El Tor V. cholerae produces the neutral product acetoin when using glucose as a carbon source. An earlier study demonstrated that Escherichia coli strains that metabolize glucose to acidic by-products drastically reduced the survival of V. cholerae strains in vitro In the present study, zebrafish were fed 1% glucose and either inoculated with single V. cholerae or E. coli strains or coinfected with both V. cholerae and E. coli A significant decrease in classical biotype colonization was observed after glucose feeding due to acid production in the zebrafish intestine. El Tor colonization was unaffected by glucose alone. However, the El Tor strain exhibited significantly lower colonization of the zebrafish when either of the acid-producing E. coli strains was coinoculated in the presence of glucose. An E. coli sugar transport mutant had no effect on V. cholerae colonization even in presence of glucose. Glucose and E. coli produced a prophylactic effect on El Tor colonization in zebrafish when E. coli was inoculated before V. cholerae infection. Thus, the probiotic feeding of E. coli inhibits V. cholerae colonization in a natural host. This suggests that a similar inhibitory effect could be seen in cholera patients, especially if a glucose-based oral rehydration solution (ORS) is administered in combination with probiotic E. coli during cholera treatment.

Quantifying Vibrio cholerae Enterotoxicity in a Zebrafish Infection Model.

Vibrio cholerae is the etiological agent of cholera, an acute intestinal infection in humans characterized by voluminous watery diarrhea. Cholera is spread through ingestion of contaminated food or water, primarily in developing countries that lack the proper infrastructure for proper water and sewage treatment. Vibrio cholerae is an aquatic bacterium that inhabits coastal and estuarine areas, and it is known to have several environmental reservoirs, including fish. Our laboratory has recently described the use of the zebrafish as a new animal model for the study of V. cholerae intestinal colonization, pathogenesis, and transmission. As early as 6 h after exposure to V. cholerae, zebrafish develop diarrhea. Prior work in our laboratory has shown that this is not due to the action of cholera toxin. We hypothesize that accessory toxins produced by V. cholerae are the cause of diarrhea in infected zebrafish. In order to assess the effects of accessory toxins in the zebrafish, it was necessary to develop a method of quantifying diarrheal volume as a measure of pathogenesis. Here, we have adapted cell density, protein, and mucin assays, along with enumeration of V. cholerae in the zebrafish intestinal tract and in the infection water, to achieve this goal. Combined, these assays should help us determine which toxins have the greatest diarrheagenic effect in fish and, consequently, which toxins may play a role in environmental transmission.IMPORTANCE Identification of the accessory toxins that cause diarrhea in zebrafish can help us understand more about the role of fish in the wild as aquatic reservoirs for V. cholerae It is plausible that accessory toxins can act to prolong colonization and subsequent shedding of V. cholerae back into the environment, thus perpetuating and facilitating transmission during an outbreak. It is also possible that accessory toxins help to maintain low levels of intestinal colonization in fish, giving V. cholerae an advantage when environmental conditions are not optimal for survival in the water. Studies such as this one are critical because fish could be an overlooked source of cholera transmission in the environment.

Mechanism for inhibition of Vibrio cholerae ToxT activity by the unsaturated fatty acid components of bile.

UNLABELLED: The Gram-negative curved bacillus Vibrio cholerae causes the severe diarrheal illness cholera. During host infection, a complex regulatory cascade results in production of ToxT, a DNA-binding protein that activates the transcription of major virulence genes that encode cholera toxin (CT) and toxin-coregulated pilus (TCP). Previous studies have shown that bile and its unsaturated fatty acid (UFA) components reduce virulence gene expression and therefore are likely important signals upon entering the host. However, the mechanism for the bile-mediated reduction of TCP and CT expression has not been clearly defined. There are two likely hypotheses to explain this reduction: (i) UFAs decrease DNA binding by ToxT, or (ii) UFAs decrease dimerization of ToxT. The work presented here elucidates that bile or UFAs directly affect DNA binding by ToxT. UFAs, specifically linoleic acid, can enter V. cholerae when added exogenously and are present in the cytoplasm, where they can then interact with ToxT. Electrophoretic mobility shift assays (EMSAs) with ToxT and various virulence promoters in the presence or absence of UFAs showed a direct reduction in ToxT binding to DNA, even in promoters with only one ToxT binding site. Virstatin, a synthetic ToxT inhibitor, was previously shown to reduce ToxT dimerization. Here we show that virstatin affects DNA binding only at ToxT promoters with two binding sites, unlike linoleic acid, which affects ToxT binding promoters having either one or two ToxT binding sites. This suggests a mechanism in which UFAs, unlike virstatin, do not affect dimerization but affect monomeric ToxT binding to DNA. IMPORTANCE: Vibrio cholerae must produce the major virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) to cause cholera. CT and TCP production depends on ToxT, the major virulence transcription activator. ToxT activity is negatively regulated by unsaturated fatty acids (UFAs) present in the lumen of the upper small intestine. This study investigated the mechanism for inhibition of ToxT activity by UFAs and found that UFAs directly reduce specific ToxT binding to DNA at virulence promoters and subsequently reduce virulence gene expression. UFAs inhibit ToxT monomers from binding DNA. This differs from the inhibitory mechanism of a synthetic ToxT inhibitor, virstatin, which inhibits ToxT dimerization. Understanding the mechanisms for inhibition of virulence could lead to better cholera therapeutics.

A small unstructured region in Vibrio cholerae ToxT mediates the response to positive and negative effectors and ToxT proteolysis.

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. The production of the virulence factors that are required for human disease is controlled by a complex network of transcriptional and posttranscriptional regulators. ToxT is the transcription regulator that directly controls the production of the two major virulence factors, toxin-coregulated pilus (TCP) and cholera toxin (CT). The solved crystal structure of ToxT revealed an unstructured region in the N-terminal domain between residues 100 and 110. This region and the surrounding amino acids have been previously implicated in ToxT proteolysis, resistance to inhibition by negative effectors, and ToxT dimerization. To better characterize this region, site-directed mutagenesis was performed to assess the effects on ToxT proteolysis and bile sensitivity. This analysis identified specific mutations within this unstructured region that prevent ToxT proteolysis and other mutations that reduce inhibition by bile and unsaturated fatty acids. In addition, we found that mutations that affect the sensitivity of ToxT to bile also affect the sensitivity of ToxT to its positive effector, bicarbonate. These results suggest that a small unstructured region in the ToxT N-terminal domain is involved in multiple aspects of virulence gene regulation and response to human host signals.

A metalloprotease secreted by the type II secretion system links Vibrio cholerae with collagen.

Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program.

Conjugated Linoleic Acid Reduces Cholera Toxin Production In Vitro and In Vivo by Inhibiting Vibrio cholerae ToxT Activity.

The severe diarrheal disease cholera is endemic in over 50 countries. Current therapies for cholera patients involve oral and/or intravenous rehydration, often combined with the use of antibiotics to shorten the duration and intensity of the disease. However, as antibiotic resistance increases, treatment options will become limited. Linoleic acid has been shown to be a potent negative effector of V. cholerae virulence that acts on the major virulence transcription regulator protein, ToxT, to inhibit virulence gene expression. ToxT activates transcription of the two major virulence factors required for disease, cholera toxin (CT) and toxin-coregulated pilus (TCP). A conjugated form of linoleic acid (CLA) is currently sold over the counter as a dietary supplement and is generally recognized as safe by the U.S. Food and Drug Administration. This study examined whether CLA could be used as a new therapy to reduce CT production, which, in turn, would decrease disease duration and intensity in cholera patients. CLA could be used in place of traditional antibiotics and would be very unlikely to generate resistance, as it affects only virulence factor production and not bacterial growth or survival.

Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters.

The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription.

Zebrafish as a natural host model for Vibrio cholerae colonization and transmission.

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival.

Advances in cholera research: from molecular biology to public health initiatives.

The aquatic bacterium Vibrio cholerae is the etiological agent of the diarrheal disease cholera, which has plagued the world for centuries. This pathogen has been the subject of studies in a vast array of fields, from molecular biology to animal models for virulence activity to epidemiological disease transmission modeling. V. cholerae genetics and the activity of virulence genes determine the pathogenic potential of different strains, as well as provide a model for genomic evolution in the natural environment. While animal models for V. cholerae infection have been used for decades, recent advances in this area provide a well-rounded picture of nearly all aspects of V. cholerae interaction with both mammalian and non-mammalian hosts, encompassing colonization dynamics, pathogenesis, immunological responses, and transmission to naïve populations. Microbiome studies have become increasingly common as access and affordability of sequencing has improved, and these studies have revealed key factors in V. cholerae communication and competition with members of the gut microbiota. Despite a wealth of knowledge surrounding V. cholerae, the pathogen remains endemic in numerous countries and causes sporadic outbreaks elsewhere. Public health initiatives aim to prevent cholera outbreaks and provide prompt, effective relief in cases where prevention is not feasible. In this review, we describe recent advancements in cholera research in these areas to provide a more complete illustration of V. cholerae evolution as a microbe and significant global health threat, as well as how researchers are working to improve understanding and minimize impact of this pathogen on vulnerable populations.

Identification and characterization of the functional toxboxes in the Vibrio cholerae cholera toxin promoter.

Following the consumption of contaminated food or water by a human host, the Vibrio cholerae bacterium produces virulence factors, including cholera toxin (CT), which directly causes voluminous diarrhea, producing cholera. A complex regulatory network controls virulence gene expression and responds to various environmental signals and transcription factors. Ultimately, ToxT, a member of the AraC/XylS transcription regulator family, is responsible for activating the transcription of the virulence genes. ToxT-regulated promoters all contain one or more copies of the toxbox, a 13-bp DNA sequence which ToxT recognizes. Nucleotides 2 through 7 of the toxbox sequence are well conserved and contain an invariant tract of four consecutive T nucleotides, whereas the remainder of the toxbox sequence is not highly conserved other than being A/T rich. The binding of ToxT to toxboxes is required to activate the transcription of virulence genes, and toxboxes in several virulence gene promoters have been characterized. However, the toxboxes required for the activation of transcription from the cholera toxin promoter PctxAB have not been identified. PctxAB contains a series of heptad repeats (GATTTTT), each of which matches the 5' end of the toxbox consensus sequence and is a potential binding site for ToxT. Using site-directed mutagenesis and high-resolution copper-phenanthroline footprinting, we have identified the functional toxboxes required for the ToxT activation of PctxAB. Our findings suggest that ToxT binds to only two toxboxes within PctxAB, despite the presence of several other potential ToxT binding sites within the promoter. Both toxboxes are essential for DNA binding and the full activation of ctxAB transcription.

Termination of Vibrio cholerae virulence gene expression is mediated by proteolysis of the major virulence activator, ToxT.

Vibrio cholerae is the causative agent of cholera, a severe diarrhoeal illness. V. cholerae produces two major virulence factors: the cholera toxin, which directly causes diarrhoea, and the toxin-coregulated pilus, which is required for intestinal colonization. Production of these virulence factors is dependent on the major virulence regulator, ToxT. Under virulence-inducing growth conditions, transcription factors ToxR and TcpP initially activate transcription of toxT. However, once ToxT has been expressed, it produces more of itself independent of ToxR and TcpP by activating transcription of the long tcpA operon, within which toxT is located. It is known that V. cholerae terminates virulence gene expression prior to escape from the host, but it is unknown how this ToxT-positive feedback loop is broken, an essential step in terminating virulence gene expression. To better understand how ToxT protein activity is regulated, we monitored ToxT accumulation and activity under virulence-inducing and -repressing growth conditions. Our results suggest that ToxT protein undergoes proteolytic degradation to terminate virulence gene expression. This directed degradation of ToxT supports a model for terminating V. cholerae virulence gene expression late in infection, with both ToxT and TcpP undergoing proteolysis prior to escape from the host.

An adult zebrafish model for adherent-invasive Escherichia coli indicates protection from AIEC infection by probiotic E. coli Nissle.

Adherent-invasive Escherichia coli (AIEC) is an opportunistic pathogen associated with major inflammatory bowel disease, Crohn disease, and ulcerative colitis. Unfavorable conditions push commensal AIEC to induce gut inflammation, sometimes progressing to inflammation-induced colon cancer. Recently, zebrafish have emerged as a useful model to study human intestinal pathogens. Here, a zebrafish model to study AIEC infection was developed. Bath inoculation with AIEC resulted in colonization and tissue disruption in the zebrafish intestine. Gene expression of pro-inflammatory markers including interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNFα), interferon-γ (IFNγ), and S100A-10b (akin to human calprotectin) in the zebrafish intestine was significantly induced by AIEC infection. The probiotic E. coli Nissle 1917 (EcN) was tested as a therapeutic and prophylactic against AIEC infection and reduced AIEC colonization, tissue damage, and pro-inflammatory responses in zebrafish. Furthermore, EcN diminished the propionic-acid-augmented hyperinfection of AIEC in zebrafish. Thus, this study shows the efficacy of EcN against AIEC in an AIEC-zebrafish model.