Mutation in the relA Gene of Vibrio cholerae Affects In Vitro and In Vivo Expression of Virulence Factors

Abstract

The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relA VCH ) was cloned and sequenced. The relA VCH gene encodes a 738-aminoacid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A ⌬relA::kan allele was generated by replacing ϳ31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.Vibrio cholerae is a facultative anaerobic gram-negative bacterium and the causative agent of the severe diarrheal disease cholera. In addition to residing temporarily in the intestinal lumen of humans during the diseased state, V. cholerae has its natural niche in the aquatic environment, residing in the freeliving aquatic flora found in estuarine areas and in association with crustaceans and mollusks (25). The strains of V. cholerae that cause epidemic cholera belong to serogroups O1 and O139 (3, 4, 28, 41, 50). The O1 serogroup is again divided into two biotypes, classical and El Tor (28). Strains other than O1 and O139 are known as non-O1/non-O139 vibrios.A pathogen in its natural environment and host-associated state is subjected to a plethora of stresses, such as fluctuations in pH, salinity, osmolarity, oxygen tension, temperature, and nutritional availability. These offer selective pressure to a bacterium, eliciting various adaptive responses for its survival. The adaptive response to nutritional stre...

RelA regulates virulence and intracellular survival of Francisella novicida

“…In this study, we have confirmed the role of FTN_1518 in F. novicida U112 as a (p)ppGpp synthetase, and demonstrated that disruption of this gene results in abolition of ppGpp production under conditions of amino acid starvation. This is similar to the situation in Pseudomonas aeruginosa (Erickson et al, 2004) and V. cholerae (Haralalka et al, 2003), in which mutation of the relA gene alone results in reduced (p)ppGpp accumulation. Unlike other organisms such as E. coli, in which both relA and spoT genes need to be inactivated (Xiao et al, 1991), RelA alone appears to be required for (p)ppGpp synthesis in F. novicida during amino acid starvation.…”

“…For example, in Legionella pneumophila, (p)ppGpp triggers a switch between the avirulent replicative phase and the virulent motile phase (Hammer & Swanson, 1999), while in M. tuberculosis, (p)ppGpp accumulation is important for the transition into latency (Ojha et al, 2000;Primm et al, 2000). In addition, the expression of specific virulence genes has been shown to be modified by RelA: in Vibrio cholerae, inactivation of RelA results in decreased expression of cholera toxin and the toxin-coregulated pilus (Haralalka et al, 2003), and a Salmonella enterica spoT/relA mutant has reduced expression of HilA, the central regulator of the Salmonella pathogenicity island (SPI) 1 and SPI 2 virulence genes (Pizarro-Cerda & Tedin, 2004).…”

Role ofluxSinBacillus anthracisgrowth and virulence factor expression

“…QS has been implicated in the regulation of virulence gene expression for a number of pathogenic bacteria. 24,28,30,[41][42][43][44][45][46][47][48] To identify genes regulated by the luxS/AI-2 QS system and CO 2 /bicarbonate, total RNA was isolated from B. anthracis strains 34F 2 and 34F 2 ∆luxS grown in BHI in the presence of 0.8% sodium bicarbonate. CO 2 /bicarbonate is critical for activation of toxin gene expression in B. anthracis.…”

Comparative proteogenomic analysis of the Leptospira interrogans virulence-attenuated strain IPAV against the pathogenic strain 56601

“…Previous studies demonstrated that the ability of bacteria to synthesize (p)ppGpp and mount a stringent response is an essential physiological adaptation required for stages of pathogenesis, such as initial growth of adherent bacteria [38], toxin production [39], motility [40] and expression of other virulence regulators [41]. Comparative analysis revealed that the IPAV spoT gene (LIF_A2491) has two missense mutations at the amino-acid residues, Val293 and Lys407, resulting in replacement by Ala and Glu, respectively.…”