Transcriptional Regulation Of The Genes Associated With Quorum Sensing System By AphA And OpaR In Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that naturallyoccurs in coastal areas, bottom sediments and marine animals, such as fishes, shrimp,clams, shellfish, and crabs. It is a human pathogen that causes food-borne acutegastroenteritis, often associated with the consumption of raw or undercookedcontaminated seafood. Clinical symptoms of V. parahaemolyticus infections includefever, nausea, vomiting, abdominal cramping, and watery diarrhea. Severe cases candevelop into fatal septicemia and cause death without timely and effective treatment.The major virulence-associated determinants of V. parahaemolyticus includethermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), type IIIsecretion system (T3SS), capsular polysaccharide, and so on. Specifically, TDH is thedirect cause of Kanagawa phenomenon. In recent years, V. parahaemolyticus has beenrecognized as a leading cause of bacterial gastroenteritis in costal regions worldwide.The quorum sensing (QS) systems, widely distributed in bacteria, act through thecomplex signal transduction cascades involving the cell density-dependent synthesis,release, and detection of signal molecules called autoinducers. QS is employed bybacteria to regulate a diverse array of physiological activities including biofilmformation, virulence and drug resistance, which are beneficial for their survival andpathogenesis. QS systems are also widely distributed in Vibrio species, among whichVibrio harveyi has been used as a model for QS studies. At low cell density (LCD),low concentrations of autoinducers lead to phosphorylation of LuxO (LuxO-P), andthen it activates expression of the five qrr genes encoding sRNAs Qrr1-5. The QrrsRNAs promote the translation of AphA, while inhibit the translation of LuxR. AphAfurther regulates the downstream celluar pathways, such as the virulence and biofilmformation. At high cell density (HCD), high concentrations of autoinducers reversethe phosphate flow in the circuit, leading to the dephosphorylation of LuxO.Dephosphorylated LuxO is inactive as a regulator, leading to the cessation of QrrsRNA production. In the absence of the Qrr sRNAs, AphA is not produced, but the translation of LuxR occurs. LuxR further regulates the expression of downstreamtarget genes. Thus, AphA and LuxR represent the master QS regulators operating atLCD and HCD respectively, through which QS systems effectively control the biofilmformation and the expression of virulence determinants.All the components of the V. harveyi QS system can be annotated to be intact inthe genome V. parahaemolyticus. For example, AphA and OpaR of V.parahaemolyticus are the master QS regulators operating at LCD and HCD,respectively, and they share high homology (≥90%) at amino acid sequences with theorthologous AphA and LuxR in V. harveyi. However, the signal transduction cascadesof V. parahaemolyticus QS system and the functions of AphA and OpaR are stillpoorly understood so far. In present study, the known or predicted binding sites ofAphA and LuxR orthologs were collected, respectively. Then, we used a web toolnamed RSAT (http://rsat.ulb.ac.be/rsat/) to generate the AphA and OpaR consensusthat manifested as a PFM, in which each row and column represented a position and anucleotide, respectively. Two20bp sequences, TATTGATAAA-TTTATCAATA andATATGCA-N6-TGCATAT, were obtained as the OpaR and AphA box, respectively.The key genes of signal transduction cascades of V. parahaemolyticus QS systemwere predicted using the OpaR and AphA boxes, the results showed that thetranscription of aphA, opaR, and qrr2-4is probably under direct control of OpaR,while aphA, opaR, and qrr4may be directly regulated by AphA. In this study, wedisclosed the transcriptional regulation mechanism of aphA, opaR, and qrr2-4byOpaR and AphA using the electrophoretic mobility shift assay (EMSA), DNase Ifootprinting, LacZ fusion experiment, primer extension, and qRT-PCR assays. Whenthe bacteria were cultivated on the HI-0.5%S agar plate to enter the HCD phase,OpaR repressed the transcription of qrr2-4、aphA, and opaR in an direct manner.When the bacteria were allowed to be grown in DifcoTMMarine broth at37°C withshaking at200rpm to enter the LCD conditions (with an OD600value of0.1to0.15),AphA repressed the transcription of qrr4、aphA, and opaR through direct AphA-targetpromoter DNA association, while it inhibited the qrr2-3transcription in an indirectmanner. Considering that the transcription mechanism of above targets by OpaR andAphA is consistent with that of the homologous genes by the orthologous regulatorsin V. harveyi, the results presented here indicated that the signal transduction cascadesof QS systems were conserved in V. parahaemolyticus and V. harveyi.During the natural survival and infection processes, V. parahaemolyticus will encounter multiple adverse environmental conditions, such as temperature changing,pH and osmotic pressure. V. parahaemolyticus can sense and response to these diverseenvironmental signals, then tightly control expression of virulence-related genes forsuccessful survival. It has been established that ToxR is a virulence regulator, as itregulates the expression of major virulence determinants of V. parahaemolyticusincluding thermostable direct hemolysin (TDH) and T3SS effectors, and alsoregulates ompU encoding a porin protein. However, the regulation mechanism ofToxR in V. parahaemolyticus is still not elucidated by now. VPA0606is a member ofthe AraC family of transcriptional regulators and is responsible for the biofilmformation of V. parahaemolyticus, but the molecular mechanism is still unclear.Moreover, it also may regulate the expression of virulence determinants in V.parahaemolyticus. In V. parahaemolyticus, vpa0607and vpa0606transcribed in thesame direction with a71bp intergenic-region, and the RT-PCR results indicated thatthey constituted an operon, vpa0607-0606, and transcribed as a single primary RNA.The QS dependent transcription of toxR and vpa0607revealed that the expression ofthese two genes should be regulated by OpaR and AphA, respectively. In this article,we employed the Heart Infusion broth to cultivate the bacteria, and investigated theregulation mechanism of toxR and vpa0607by OpaR and AphA in V.parahaemolyticus.The primer extension and Western blot results showed that the maximumexpression of opaR and vpa0606is at an OD600value of0.6to0.8, while aphA andtoxR is at0.05to0.2and0.2to0.4, respectively. Then, we harvested the cultivatedbacterial cells at the corresponding maximum expression time points to study thetranscriptional regulation mechanism using classical molecular experiments, such asprimer extension, LacZ fusion. The results showed that when at an OD600value of0.05to0.5, AphA repressed the transcription of aphA and opaR through directAphA-target promoter DNA association, while it inhibited and activated the toxR andvpa0607-0606transcription in an indirect manner, respectively; when at an OD600value of0.6to0.8, OpaR directly repressed the transcription of aphA, opaR andvpa0607-0606, while it inhibited the toxR transcription indirectly. Based on the factthat AphA is an activator of vpa0607-0606operon, we concluded that the highestvpa0607-0606transcription should be at LCD, and the succedent primer extensionassay result confirmed this hypothesis. However, the above results have shown thatvpa0607and vpa0606consititue a single operon, and the maximum expression of VPA0606is occured at HCD. Actually, vpa0607encodes a deduced protein of668amino acids with a conserved RNB domain, which is the catalytic domain ofribonuclease II with the function of hydrolysis phosphodiester bond from3’ to5’-endof mRNA. Thus, we assumed that only at the low levels of the VPA0607proteinconditions, such as at HCD, the basis transcribed vpa0607-0606can be translated intoVPA0606protein, and this hypothesis needs to be verified by further experiments.In summary, our results not only indicated that the signal transduction cascadesof QS systems are conserved between V. parahaemolyticus and V. harveyi, but alsoconfirmed that ToxR and VPA0606are all involved in QS system regulation network.The cell density-dependent expression of AphA, OpaR, ToxR and VPA0606playedmajor roles at the different growth stages, respectively, and ultimately tightlyregulated the expression of virulence factors and biofilm formation. This study laid asolid theoretical foundation for the future study, and also helped us to understand thepathogenesis of the V. parahaemolyticus.