Real-Time Reverse Transcription-PCR for Transcriptional Expression Analysis of Virulence and Housekeeping Genes in Viable but NonculturableVibrio parahaemolyticusafter Recovery of Culturability

Abstract

A real-time reverse transcription-PCR method was developed to determine whether the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus induced the expression of virulence genes coding for the thermostable direct hemolysin and for type III secretion system 2 (TTSS2). The culturability of clinical strain Vp5 of V. parahaemolyticus in artificial seawater at 4°C was monitored, and the VBNC state was obtained. One day after entry into the VBNC state, temperature upshifts to 20 and 37°C allowed the recovery of culturability. Standard curves for the relative quantification of expression of the housekeeping genes rpoS, pvsA, fur, and pvuA; the tdh2 gene; and the TTSS2 genes (VPA1354, VPA1346, and VPA1342) were established. The levels of expression of the pvsA and pvuA genes were stable and were used to normalize the levels of expression of the other genes. No transcriptional induction of the selected virulence genes under the temperature conditions used to recover the culturability of the VBNC bacteria was observed. The study results demonstrate that the recovery of culturability of VBNC cells of pathogenic V. parahaemolyticus is restricted to regrowth, without correlation with the induction of virulence gene expression. Disease induction would depend mainly on host-pathogen interactions that allow the expression of the virulence genes. This is the first time that the use of mRNA to detect viable cells was evaluated by computing the half-lives of multiple mRNA species under conditions inducing the VBNC state.Vibrio parahaemolyticus is a marine bacterium of which some strains generate food-borne outbreaks of disease characterized by acute gastroenteritis. The thermostable direct hemolysin (TDH) and TDH-related hemolysin were previously considered to be the main factors at the origin of these enterotoxic phenomena. Recently, the genome sequencing of a clinical strain of V. parahaemolyticus, RIMD 2210633, has revealed several other factors of virulence, including genes for two type III secretion systems (TTSS), TTSS1 and TTSS2, present on chromosomes 1 and 2, respectively (18). TTSS1 has been described as a cytotoxic system, and TTSS2 has been described as an enterotoxic system (25). Under environmental stresses, such as a temperature downshift, V. parahaemolyticus appears to be fairly inactive metabolically and enters into a dormant state, namely, the viable but nonculturable (VBNC) state. The VBNC cells are able to respond to some environmental stimuli, such as a temperature upshift, and to become metabolically active and culturable. The recovery of culturability by the VBNC cells of V. cholerae, V. vulnificus, and V. parahaemolyticus has been demonstrated to cause in vivo pathogenicity (1,6,23). Two scenarios may explain the virulence: a high number of cells (a high infectious dose) without significant regulation of the virulence genes and/or genetic up-regulation of virulence genes under such conditions.To determine if cells induce the expression of virulence genes after the recovery o...

Virulence gene expression of Vibrio parahaemolyticus in the viable but nonculturable state

“…They acknowledged a contradiction with Coutard et al (2005), attributing the discrepancies to experimental factors (e.g., assay sensitivity, VBNC induction period) and biological variations (strain, gene copy number, regulation of expression). Interestingly, Coutard et al (2007b) conducted another study using a different strain, Vp5, and detected the presence (but not upregulation) of virulence genes, tdh2 and escU. The latter gene encodes inner membrane proteins of T3SS2.…”

“…The latter gene encodes inner membrane proteins of T3SS2. Interestingly, the VBNC state was reached after merely 15 days following inoculation at 4°C in ASW (Coutard et al, 2007b …”

“…The gene, pvsA, was reported to have the most stable transcriptional expression levels among four housekeeping gene candidates (rpoS, pvsA, fur, and pvuA) in RT-PCR (Coutard et al, 2007b). Analysis for the stability of gene expression was evaluated with by geNorm software, resulting with pvsA and pvuA having the most stable transcriptional expression genes and a high level of regulation of the rpoS gene.…”

“…Involved in the iron uptake pathway, pvsA, is responsible for the biosynthesis and transport of siderophore vibrioferrin in V. parahaemolyticus (Tanabe et al, 2003). Used as a positive control for the reverse transcription process and qRT-PCR, 1 ng of MS2 RNA (Roche Diagnostics, Indianapolis, Indiana) was included in most qRT-PCR runs to amplify oco gene (Coutard et al, 2007b). The two virulence genes, tdh2 and escU, were the target genes.…”

“…Each qRT-PCR reaction was performed in 20 μL running on 7300 Real-Time PCR system (Applied Biosystems). The thermal cycling conditions were adapted from (Coutard et al, 2007b): a denaturation program (95°C for 10 min), an amplification program repeated 40 times (95°C for 15 s and 60°C for 1 min), and a melting-curve program (55°C to 95°C). Positive control (MS2) and negative controls (no templates and RNA from treatment cells) were included in each run.…”