ABSTRACT
AIMS: This work investigates the maintenance of viability and potential virulence of Vibrio parahaemolyticus in a viable but nonculturable population (VBNC) state by reverse transcription-polymerase chain reaction (RT-PCR). METHODS AND RESULTS: Housekeeping genes, 16S-23S rDNA and rpoS, as well as virulence genes, tdh1 and tdh2, were selected and detected by PCR in a pathogenic strain of V. parahaemolyticus (Vp4). Their expression was then studied by RT-PCR in V. parahaemolyticus Vp4 cultivated in rich medium at 37 degrees C. The 16S-23S rDNA and rpoS, tdh1, tdh2 genes were transcripted at the mid-logarithmic, stationary and late stationary phases, corresponding to various physiological states. The expression of these genes was also studied by RT-PCR in a VBNC population of V. parahaemolyticus Vp4 in artificial seawater (ASW). The effect of temperature (washing of bacterial culture and microcosms) on the attaining VBNC bacteria was first considered. Washing of V. parahaemolyticus Vp4, collected at the mid-logarithmic phase, at 10 or 4 degrees C before inoculation in ASW at 4 degrees C allowed bacteria entered the VBNC state between 22 and 31 days. The 16S-23S rDNA and rpoS gene were expressed in the VBNC bacteria whereas no expression of the tdh1 and tdh2 genes was observed in the same populations. CONCLUSION: The two selected housekeeping genes, 16S-23S rDNA and rpoS, proved to be good viability markers for V. parahaemolyticus Vp4 in culturable and VBNC states. These first data indicated that the pathogenic strain Vp4 would not maintain the expression of the virulence genes, tdh1 and tdh2, in VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of RT-PCR for investigating the maintenance or not of viability and potential virulence in VBNC V. parahaemolyticus will facilitate further study to evaluate the potential risk presented by this pathogen in the environment.
Subject(s)
RNA, Bacterial/analysis , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Vibrio parahaemolyticus/pathogenicity , Bacterial Proteins/genetics , Bacterial Toxins , Cell Survival/genetics , Colony Count, Microbial/methods , Culture Media , DNA, Ribosomal/analysis , Gene Expression Regulation, Bacterial/genetics , Hemolysin Proteins/genetics , RNA, Ribosomal, 16S/analysis , RNA, Ribosomal, 23S/analysis , Sigma Factor/genetics , Vibrio parahaemolyticus/genetics , Vibrio parahaemolyticus/growth & development , VirulenceABSTRACT
The HB-19 pseudopeptide 5[Kpsi(CH2N)PR]-TASP[psi(CH2N) indicating a reduced peptide bond], which binds the cell surface-expressed nucleolin, is a potent inhibitor of HIV infection. Here, by using primary T lymphocyte cultures and an experimental cell model to monitor HIV entry, we show that HB-19 inhibits in a dose-dependent manner both T lymphocyte- and macrophage-tropic HIV isolates. Similar positively charged control pseudopeptides have no effect on HIV infection even at high concentrations. These observations, and the fact that HB-19 has no effect on SIV-mac and HIV-1 pseudotyped with VSV envelope glycoproteins, confirm the specific nature of this inhibitor against the entry process mediated by the HIV envelope glycoproteins. Finally, association of low doses of HB-19 with beta-chemokines or AZT results in an increased inhibitory effect on HIV infection. HB-19 has no inhibitory effect when added to cells a few hours after HIV entry. On the other hand, in HB-19-pretreated cells, the inhibitory effect persists for several hours, even after washing cells to remove away the unbound pseudopeptide. Under such conditions, the attachment of HIV particles to cells is inhibited as efficiently as by neutralizing monoclonal antibodies directed against the V3 loop. In view of its specific mode of action on various HIV isolates, HB-19 represents a potential anti-HIV drug.
