HlyU acts as an H‐NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen <i>Vibrio vulnificus</i> CMCP6

Liu, Moqing; Naka, Hiroaki; Crosa, Jorge H.

doi:10.1111/j.1365-2958.2009.06664.x

Cited by 52 publications

(86 citation statements)

References 62 publications

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“…However, lacZ fusion experiments demonstrated that HlyU regulation must occur by derepression, rather than by positive regulation, of the rtxA1 operon. That work also demonstrated that rtxA1 expression is normally silenced by the master regulator H-NS and that HlyU acts as a derepressor by displacing H-NS from its binding sites, thus relieving the repression of rtxA1 (13).…”

mentioning

confidence: 84%

“…We also showed that the HlyU protein plays an essential role in its regulation. In its normal state, rtxA1 expression is repressed by the H-NS protein, and HlyU acts as a derepressor by binding to a region upstream of the rtxA1 operon promoter, resulting in the removal of H-NS and allowing rtxA1 expression and bacterial invasion to occur (13). However, an intimate knowledge of the intrinsic steps involved in the biological mechanism of action of HlyU is limited to predictions based on the recently solved crystal FIG.…”

Section: Discussionmentioning

confidence: 99%

“…In our previous work (13), using a DNase I footprint assay, we identified the binding DNA sequence for HlyU in the promoter region of the rtxA1 operon. However, lacZ fusion experiments demonstrated that HlyU regulation must occur by derepression, rather than by positive regulation, of the rtxA1 operon.…”

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confidence: 99%

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Homodimerization and Binding of Specific Domains to the Target DNA Are Essential Requirements for HlyU To Regulate Expression of the Virulence Gene rtxA1 , Encoding the Repeat-in-Toxin Protein in the Human Pathogen Vibrio vulnificus

2011

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The virulence gene rtxA1, encoding the repeat-in-toxin protein, plays an essential role in the pathogenesis of Vibrio vulnificus infections. Expression of this gene is controlled by the HlyU regulator by direct contact of the DNA upstream of the rtxA1 toxin operon acting as a derepressor of the H-NS protein. The crystal structure suggests that HlyU forms a homodimer in vitro. However, knowledge of the biological implications of these findings in vivo is limited. In this work, we endeavored to dissect, using genetic and biochemical approaches, the domains of this protein that are essential for homodimer formation and the interaction of HlyU with the target DNA. We identified that residues L18, N22, R25, S54, Q55, L57, W59, R61, K70, and Y77 are essential for the HlyU protein binding to the DNA and that amino acids L17 and L91 are important for HlyU dimerization. We also determined that HlyU homodimer formation is an essential requirement for binding to the upstream region of the rtxA1 operon and is the key feature in relieving the H-NS repression of rtxA1 transcription.Vibrio vulnificus is an opportunistic human pathogen capable of causing fatal primary septicemia after ingestion of contaminated seafood or necrotizing fasciitis after contamination of wounds (4,19,24). Septicemia occurs mainly in patients who are immunocompromised, suffering from hemochromatosis, or have other underlying liver disorders, such as cirrhosis and alcoholic liver disease (8,25). The common theme in most of these patients is that iron is present at a level higher than the physiological level (27). During V. vulnificus infections, the bacterium reaches the intestine and then invades the bloodstream by crossing the intestinal mucosal barrier of the host, which results in a systemic septicemia (9). Using an in vivoinduced antigen technology (IVIAT) to study blood from hemochromatosis patients, it was possible to identify antibodies against several V. vulnificus proteins, among them, the HlyU protein, that is a homologue of the hemolysin gene regulator in Vibrio cholerae (7). It was further demonstrated that this protein is essential for virulence of V. vulnificus (7,12). HlyU belongs to the ArsR/SmtB protein family (26). Members of this family are mostly metal-regulatory transcriptional repressors that normally repress the expression of operons linked to concentrations of stress-inducing heavy metal ions (3,14,15). However, sequence analysis shows that HlyU does not possess a metal-binding domain (21). In our previous work, we demonstrated that HlyU regulates the expression of the RTX (RTX stands for repeats in toxin) toxin rtxA1 gene by direct contact of the DNA upstream of the rtxA1 toxin operon (12). RTX toxin is the largest single-polypeptide toxin known, and it is proposed to form a pore in the eukaryotic cell plasma membrane for translocation of the central portion of the secreted toxin to the cytosol (22). Our lab and others demonstrated that the RtxA1 protein is responsible not only for cytotoxicity but that it also plays an essent...

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confidence: 84%

Section: Discussionmentioning

confidence: 99%

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Homodimerization and Binding of Specific Domains to the Target DNA Are Essential Requirements for HlyU To Regulate Expression of the Virulence Gene rtxA1 , Encoding the Repeat-in-Toxin Protein in the Human Pathogen Vibrio vulnificus

2011

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“…For example, nucleoid-associated protein (H-NS) binds DNA at lower temperatures blocking the transcription of multiple genes, whereas higher temperatures cause loosening in the DNA structure allowing transcription to occur. H-NS suppresses virulence-associated genes, such as RTX and CTX, in V. vulnificus (Liu et al, 2009) and V. cholerae (Stonehouse et al, 2011), respectively. The concurrent upregulation of H-NS (ZP_05887985.1) and RTX toxin (ZP_05887531.1) on CPI-1 in the Vc450 proteome at 27 1C could be the result of a conserved Vc450 H-NS unable to regulate a more recently acquired RTX toxin.…”

Section: Temperature-dependent Regulationmentioning

confidence: 99%

Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus

Kimes

Grim

Johnson³

et al. 2011

The ISME Journal

230

200

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Sea surface temperatures (SST) are rising because of global climate change. As a result, pathogenic Vibrio species that infect humans and marine organisms during warmer summer months are of growing concern. Coral reefs, in particular, are already experiencing unprecedented degradation worldwide due in part to infectious disease outbreaks and bleaching episodes that are exacerbated by increasing SST. For example, Vibrio coralliilyticus, a globally distributed bacterium associated with multiple coral diseases, infects corals at temperatures above 27 1C. The mechanisms underlying this temperature-dependent pathogenicity, however, are unknown. In this study, we identify potential virulence mechanisms using whole genome sequencing of V. coralliilyticus ATCC (American Type Culture Collection) BAA-450. Furthermore, we demonstrate direct temperature regulation of numerous virulence factors using proteomic analysis and bioassays. Virulence factors involved in motility, host degradation, secretion, antimicrobial resistance and transcriptional regulation are upregulated at the higher virulent temperature of 27 1C, concurrent with phenotypic changes in motility, antibiotic resistance, hemolysis, cytotoxicity and bioluminescence. These results provide evidence that temperature regulates multiple virulence mechanisms in V. coralliilyticus, independent of abundance. The ecological and biological significance of this temperature-dependent virulence response is reinforced by climate change models that predict tropical SST to consistently exceed 27 1C during the spring, summer and fall seasons. We propose V. coralliilyticus as a model Gram-negative bacterium to study temperature-dependent pathogenicity in Vibrio-related diseases.

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“…HlyU, which was identified by in vivo-induced antigen technology and is essential for V. vulnificus virulence in mice (11,19), is required for the expression of vvhA and rtxA1 and binds directly to a region upstream of the operon where rtxA1 is located (19,20).…”

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confidence: 99%

Regulation of Cytotoxicity by Quorum-Sensing Signaling in Vibrio vulnificus Is Mediated by SmcR, a Repressor of hlyU

Shao

Lin

et al. 2011

J Bacteriol

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Cytotoxicity is an important virulence determinant in the pathogenesis of Vibrio vulnificus, and two cytotoxins, RTX (encoded by rtxA1) and cytolysin/hemolysin (encoded by vvhA), have been identified in this organism. We showed that the quorum-sensing regulator LuxO controlled the cytotoxicity of this organism: a ⌬luxO mutant exhibited low cytotoxicity, whereas a constitutively activated luxO mutant, luxO(D47E), remained highly cytotoxic. The cytotoxicity of the ⌬luxO mutant was restored when smcR, a Vibrio harveyi luxR homologue repressed by luxO, was further deleted. SmcR then was shown to repress the expression of both rtxA1 and vvhA. A DNA library of V. vulnificus was screened in Escherichia coli for clones that upregulated vvhA in the presence of SmcR, and hlyU, which has been shown to positively regulate rtxA1 and vvhA, was identified. We demonstrated that SmcR repressed the expression of hlyU and bound to a region upstream of hlyU in V. vulnificus. The deletion of hlyU resulted in the loss of cytotoxicity and reduced cytolysin/hemolysin production in the ⌬smcR mutant. The ⌬smcR ⌬hlyU mutant regained cytotoxicity and cytolysin/hemolysin activity when hns, which has been shown to repress the transcription of rtxA1 and interfere with hlyU, was further removed. Collectively, our data suggest that SmcR mediates the regulation of cytotoxicity by quorum-sensing signaling in V. vulnificus by repressing hlyU, an activator of rtxA1 and vvhA.Vibrio vulnificus, a Gram-negative marine bacterium, is an opportunistic pathogen causing septicemia and wound infection in humans; it has a high mortality rate, particularly in those who suffer from chronic liver diseases or are immunocompromised. This organism produces two major cytotoxins, cytolysin/hemolysin (encoded by vvhA) and RTX (repeats in toxin; encoded by rtxA1), that are implicated in its virulence (19,29). The cytolysin/hemolysin, which is an extracellular product, is lethal for mice at submicrogram levels. It lyses erythrocytes, increases vascular permeability (resulting in extensive extracellular edema in guinea pig skin), damages capillary endothelial cells, and causes mild inflammatory cell infiltration (7,8). The RTX toxin, which forms pores on cell membranes only after the contact of the bacterium with the host cell (12, 13), is required for V. vulnificus virulence in mice by promoting bacterial colonization at the infection site and subsequent invasion into the bloodstream (12, 15).Several regulators of vvhA and rtxA1 have been identified (1, 19). HlyU, which was identified by in vivo-induced antigen technology and is essential for V. vulnificus virulence in mice (11,19), is required for the expression of vvhA and rtxA1 and binds directly to a region upstream of the operon where rtxA1 is located (19,20).Quorum-sensing (QS) signaling, which is widely used by bacteria to communicate with each other, regulates the virulence genes in a variety of microorganisms, including Vibrio species (23, 30). In Vibrio cholerae, the signals transduced from at least three QS ...

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HlyU acts as an H‐NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen Vibrio vulnificus CMCP6

Cited by 52 publications

References 62 publications

Homodimerization and Binding of Specific Domains to the Target DNA Are Essential Requirements for HlyU To Regulate Expression of the Virulence Gene rtxA1 , Encoding the Repeat-in-Toxin Protein in the Human Pathogen Vibrio vulnificus

Homodimerization and Binding of Specific Domains to the Target DNA Are Essential Requirements for HlyU To Regulate Expression of the Virulence Gene rtxA1 , Encoding the Repeat-in-Toxin Protein in the Human Pathogen Vibrio vulnificus

Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus

Regulation of Cytotoxicity by Quorum-Sensing Signaling in Vibrio vulnificus Is Mediated by SmcR, a Repressor of hlyU

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