Iron Differentially Regulates Gene Expression and Extracellular Secretion of Vibrio vulnificus Cytolysin‐Hemolysin

Kim

et al. 2013

Molecular Microbiology

SummaryVvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since haemolysis by VvhA may provide iron for bacterial growth and pathogenicity, we investigated the expression of VvhA to elucidate the regulatory roles of Fur, a major transcription factor controlling ironhomeostasis. Fur repressed the transcription of vvhBA operon via binding to the promoter region. However, haemolysin content and haemolytic activity were lowered in cell-free supernatant of fur mutant. This discrepancy between the levels of vvhA transcript and VvhA protein in fur mutant was caused by exoproteolytic activities of the elastase VvpE and another metalloprotease VvpM, which were also regulated by Fur. vvpE gene expression was repressed by Fur via binding to the Fur-box homologous region. Regulation of VvpM expression by Fur did not occur at the level of vvpM transcription. In vitro proteolysis assays showed that both proteases efficiently degraded VvhA. In addition, the extracellular levels of VvhA were higher in culture supernatants of vvpE or vvpM mutants than in the wild type. Thus this study demonstrates that Fur regulates haemolysin production at the transcription level of the vvhBA operon and at the post-translation level by regulating the expressions of two VvhA-degrading exoproteases, VvpE and VvpM.

Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus: repression of vvhA transcription by Fur and proteolysis of VvhA by Fur‐repressive exoproteases

Kim

et al. 2013

Molecular Microbiology

“…Similarly, the expressions of other V. vulnificus iron-uptake systems are also known to be under the negative control of Fur or iron (30~33). Furthermore, in a previous study, we found that Fur negatively regulated the expression of vvhBA encoding a cytolysin/hemolysin, which destroys a variety of cells, including red blood cells, and causes the release of intracellular iron, including hemoglobin (20).…”

Section: Vulnificusmentioning

confidence: 92%

Coordinate Regulation ofVibrio vulnificusHeme Receptor HupA Expression by Cyclic AMP-receptor Protein and Ferric Uptake Regulator

et al. 2012

Vibrio vulnificus causes rapid progressing fulminant infections in susceptible individuals, especially those with elevated serum iron levels. This ferrophilic bacterium can directly acquire iron from heme-containing proteins, such as, hemoglobin, via its heme receptor protein HupA. This study was undertaken to determine the roles of cyclic AMP-receptor protein (Crp) as an activator and of ferric uptake regulator (Fur) as a repressor in regulating hupA expression at various iron and glucose concentrations. Under severely iron-deficient conditions, hupA expression in the absence of Crp was induced albeit at low levels and repressed by the addition of iron. In contrast, hupA expression in the presence of Crp was increased by the addition of iron. Under moderately iron-deficient and iron-sufficient conditions, iron addition repressed hupA expression in the presence of Fur, but not in the absence of Fur. Glucose addition repressed hupA expression in the presence of Fur but not in the absence of Fur. Furthermore, a mutation in cyaA encoding adenylate cyclase required for cAMP synthesis repressed hupA expression, and this repression was prevented by the exogenous addition of cAMP. These results indicate that hupA expression is under the coordinate control of cAMP or Crp, which responds to glucose availability, and of Fur, which responds to iron availability, and that Crp is not essential for the constitutional expression of hupA, but is required for the optimal expression of hupA, whereas Fur is essential for the prevention of hupA over-expression.

“…Infection with V. vulnificus typically shows rapid progression and mortality rates greater than 50% (6,7). In Gram-negative bacteria, the inner membrane protein complex TonB plays a crucial role in the uptake of iron (8,9), which is an important micronutrient for numerous biological processes (10)(11)(12). TonB complexes transduce the proton motive force (PMF) of the cytoplasmic membrane to energize iron-siderophore complex transport through a specific TonB-dependent transporter (TBDT) across the outer membrane (OM) (9,13,14).…”

mentioning

confidence: 99%

All Three TonB Systems Are Required for Vibrio vulnificus CMCP6 Tissue Invasiveness by Controlling Flagellum Expression

et al. 2016

e TonB systems actively transport iron-bound substrates across the outer membranes of Gram-negative bacteria. Vibrio vulnificus CMCP6, which causes fatal septicemia and necrotizing wound infections, possesses three active TonB systems. It is not known why V. vulnificus CMCP6 has maintained three TonB systems throughout its evolution. The TonB1 and TonB2 systems are relatively well characterized, while the pathophysiological function of the TonB3 system is still elusive. A reverse transcription-PCR (RT-PCR) study showed that the tonB1 and tonB2 genes are preferentially induced in vivo, whereas tonB3 is persistently transcribed, albeit at low expression levels, under both in vitro and in vivo conditions. The goal of the present study was to elucidate the raison d'être of these three TonB systems. In contrast to previous studies, we constructed in-frame single-, double-, and triple-deletion mutants of the entire structural genes in TonB loci, and the changes in various virulence-related phenotypes were evaluated. Surprisingly, only the tonB123 mutant exhibited a significant delay in killing eukaryotic cells, which was complemented in trans with any TonB operon. Very interestingly, we discovered that flagellum biogenesis was defective in the tonB123 mutant. The loss of flagellation contributed to severe defects in motility and adhesion of the mutant. Because of the difficulty of making contact with host cells, the mutant manifested defective RtxA1 toxin production, which resulted in impaired invasiveness, delayed cytotoxicity, and decreased lethality for mice. Taken together, these results indicate that a series of virulence defects in all three TonB systems of V. vulnificus CMCP6 coordinately complement each other for iron assimilation and full virulence expression by ensuring flagellar biogenesis. Vibrio vulnificus is a halophilic estuarine pathogen that causes fatal septicemia and necrotizing wound infections in patients suffering from hepatic diseases with high levels of circulating iron or who are immunocompromised (1-5). Infection with V. vulnificus typically shows rapid progression and mortality rates greater than 50% (6, 7). In Gram-negative bacteria, the inner membrane protein complex TonB plays a crucial role in the uptake of iron (8, 9), which is an important micronutrient for numerous biological processes (10-12). TonB complexes transduce the proton motive force (PMF) of the cytoplasmic membrane to energize iron-siderophore complex transport through a specific TonB-dependent transporter (TBDT) across the outer membrane (OM) (9,13,14). This system's known biological roles had been restricted to iron complexes (15, 16) and vitamin B 12 (14), but recent experimental evidence of the TonB-energized transport of nickel and various carbohydrates suggests that the number and variety of TonB-dependent substrates have been underestimated (17)(18)(19). Unlike the single TonB system in Escherichia coli (8), the genomes of Vibrio species carry multiple TonB systems. Interestingly, three TonB systems were first report...