1887

Microbiology Society logo

Volume 164, Issue 2

OpaR and RpoS are positive regulators of a virulence factor PrtA in Free

Abstract

PrtA is an extracellular serine protease of and has haemolytic and cytotoxic activities. Many extracellular proteases have been shown to be required for nutrient intake and the infection mechanism of vibrios. In this study, we report that OpaR, a quorum sensing regulator, and RpoS, a general stress response regulator, play important roles in the PrtA regulation pathway. Extracellular protease activity was highest during the late-log growth of no.93 (VP93). The absence of PrtA distinctly decreased the extracellular protease activity. Deletion of or alone reduced PrtA-specific activity of VP93. Quantitative reverse-transcriptase PCR and Western blot analysis suggested that OpaR and RpoS promote PrtA expression at the transcriptional level and affect the amount of extracellular PrtA. A luciferase assay revealed that OpaR regulates on the promoter region. Electrophoretic mobility shift assays indicated that the purified His-OpaR was able to bind specifically to two sequences (PrtA-1 and PrtA-2) of the promoter region. Footprinting analysis showed that OpaR regulates by binding to the promoter region of at positions –269 to –246 and –88 to –68 from the translational start site. Together, the results suggest that PrtA was upregulated by two global regulators, OpaR and RpoS.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): extracellular serine protease PrtA , OpaR , RpoS , Vibrio parahaemolyticus and virulence factor
© 2018 The Authors
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000591
2018-02-01
2024-04-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/164/2/221.html?itemId=/content/journal/micro/10.1099/mic.0.000591&mimeType=html&fmt=ahah

References

  1. Deepanjali A, Kumar HS, Karunasagar I, Karunasagar I. Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the southwest coast of India. Appl Environ Microbiol 2005; 71:3575–3580 [View Article][PubMed]
     [Google Scholar]
  2. McCarter L. The multiple identities of Vibrio parahaemolyticus. J Mol Microbiol Biotechnol 1999; 1:51–57[PubMed]
     [Google Scholar]
  3. Su YC, Liu C. Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol 2007; 24:549–558 [View Article][PubMed]
     [Google Scholar]
  4. Broberg CA, Calder TJ, Orth K. Vibrio parahaemolyticus cell biology and pathogenicity determinants. Microbes Infect 2011; 13:992–1001 [View Article][PubMed]
     [Google Scholar]
  5. Syngkon A, Elluri S, Koley H, Rompikuntal PK, Saha DR et al. Studies on a novel serine protease of a ΔhapAΔprtV Vibrio cholerae O1 strain and its role in hemorrhagic response in the rabbit ileal loop model. PLoS One 2010; 5:e13122 [View Article][PubMed]
     [Google Scholar]
  6. Sikora AE. Proteins secreted via the type II secretion system: smart strategies of Vibrio cholerae to maintain fitness in different ecological niches. PLoS Pathog 2013; 9:e1003126 [View Article][PubMed]
     [Google Scholar]
  7. Benitez JA, Silva AJ. Vibrio cholerae hemagglutinin (HA)/protease: an extracellular metalloprotease with multiple pathogenic activities. Toxicon 2016; 115:55–62 [View Article][PubMed]
     [Google Scholar]
  8. Silva AJ, Pham K, Benitez JA. Haemagglutinin/protease expression and mucin gel penetration in El Tor biotype Vibrio cholerae. Microbiology 2003; 149:1883–1891 [View Article][PubMed]
     [Google Scholar]
  9. Liang W, Pascual-Montano A, Silva AJ, Benitez JA. The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio cholerae. Microbiology 2007; 153:2964–2975 [View Article][PubMed]
     [Google Scholar]
  10. Lim MS, Kim JA, Lim JG, Kim BS, Jeong KC et al. Identification and characterization of a novel serine protease, VvpS, that contains two functional domains and is essential for autolysis of Vibrio vulnificus. J Bacteriol 2011; 193:3722–3732 [View Article][PubMed]
     [Google Scholar]
  11. Yu MS, Lee CY. Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli. Microbiology 1999; 145:143–150 [View Article][PubMed]
     [Google Scholar]
  12. Lee CY, Cheng MF, Yu MS, Pan MJ. Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus. FEMS Microbiol Lett 2002; 209:31–37 [View Article][PubMed]
     [Google Scholar]
  13. Miller MB, Bassler BL. Quorum sensing in bacteria. Annu Rev Microbiol 2001; 55:165–199 [View Article][PubMed]
     [Google Scholar]
  14. Ng WL, Bassler BL. Bacterial quorum-sensing network architectures. Annu Rev Genet 2009; 43:197–222 [View Article][PubMed]
     [Google Scholar]
  15. Makino K, Oshima K, Kurokawa K, Yokoyama K, Uda T et al. Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae. Lancet 2003; 361:743–749 [View Article][PubMed]
     [Google Scholar]
  16. Pompeani AJ, Irgon JJ, Berger MF, Bulyk ML, Wingreen NS et al. The Vibrio harveyi master quorum-sensing regulator, LuxR, a TetR-type protein is both an activator and a repressor: DNA recognition and binding specificity at target promoters. Mol Microbiol 2008; 70:76–88 [View Article][PubMed]
     [Google Scholar]
  17. McCarter LL. OpaR, a homolog of Vibrio harveyi LuxR, controls opacity of Vibrio parahaemolyticus. J Bacteriol 1998; 180:3166–3173[PubMed]
     [Google Scholar]
  18. Zhang Y, Qiu Y, Tan Y, Guo Z, Yang R et al. Transcriptional regulation of opaR, qrr2-4 and aphA by the master quorum-sensing regulator OpaR in Vibrio parahaemolyticus. PLoS One 2012; 7:e34622 [View Article][PubMed]
     [Google Scholar]
  19. Bishop RE, Leskiw BK, Hodges RS, Kay CM, Weiner JH. The entericidin locus of Escherichia coli and its implications for programmed bacterial cell death. J Mol Biol 1998; 280:583–596 [View Article][PubMed]
     [Google Scholar]
  20. Lange R, Hengge-Aronis R. Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol Microbiol 1991; 5:49–59 [View Article][PubMed]
     [Google Scholar]
  21. Hengge-Aronis R. A role for the sigma S subunit of RNA polymerase in the regulation of bacterial virulence. Adv Exp Med Biol 2000; 485:85–93 [View Article][PubMed]
     [Google Scholar]
  22. Loewen PC, Hu B, Strutinsky J, Sparling R. Regulation in the rpoS regulon of Escherichia coli. Can J Microbiol 1998; 44:707–717 [View Article][PubMed]
     [Google Scholar]
  23. Dong T, Yu R, Schellhorn H. Antagonistic regulation of motility and transcriptome expression by RpoN and RpoS in Escherichia coli. Mol Microbiol 2011; 79:375–386 [View Article][PubMed]
     [Google Scholar]
  24. Bullock WO, Fernandez JM, Short JM. Xl1-Blue - a high-efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. Biotechniques 1987; 5:376–379
     [Google Scholar]
  25. de Lorenzo V, Eltis L, Kessler B, Timmis KN. Analysis of Pseudomonas gene products using lacIq/Ptrp-lac plasmids and transposons that confer conditional phenotypes. Gene 1993; 123:17–24 [View Article][PubMed]
     [Google Scholar]
  26. Philippe N, Alcaraz JP, Coursange E, Geiselmann J, Schneider D. Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria. Plasmid 2004; 51:246–255 [View Article][PubMed]
     [Google Scholar]
  27. Schäfer A, Kalinowski J, Simon R, Seep-Feldhaus AH, Pühler A. High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria. J Bacteriol 1990; 172:1663–1666 [View Article][PubMed]
     [Google Scholar]
  28. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248–254 [View Article][PubMed]
     [Google Scholar]
  29. Wang C, Lee J, Deng Y, Tao F, Zhang LH. ARF-TSS: an alternative method for identification of transcription start site in bacteria. Biotechniques 2012; 52:1–3 [View Article][PubMed]
     [Google Scholar]
  30. Zianni M, Tessanne K, Merighi M, Laguna R, Tabita FR. Identification of the DNA bases of a DNase I footprint by the use of dye primer sequencing on an automated capillary DNA analysis instrument. J Biomol Tech 2006; 17:103–113[PubMed]
     [Google Scholar]
  31. Hengge-Aronis R. Signal transduction and regulatory mechanisms involved in control of the sigma(S) (RpoS) subunit of RNA polymerase. Microbiol Mol Biol Rev 2002; 66:373–395 [View Article][PubMed]
     [Google Scholar]
  32. Gode-Potratz CJ, McCarter LL. Quorum sensing and silencing in Vibrio parahaemolyticus. J Bacteriol 2011; 193:4224–4237 [View Article][PubMed]
     [Google Scholar]
  33. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010; 11:119 [View Article][PubMed]
     [Google Scholar]
  34. Gode-Potratz CJ, Kustusch RJ, Breheny PJ, Weiss DS, McCarter LL. Surface sensing in Vibrio parahaemolyticus triggers a programme of gene expression that promotes colonization and virulence. Mol Microbiol 2011; 79:240–263 [View Article][PubMed]
     [Google Scholar]
  35. Kernell Burke A, Guthrie LT, Modise T, Cormier G, Jensen RV et al. OpaR controls a network of downstream transcription factors in Vibrio parahaemolyticus BB22OP. PLoS One 2015; 10:e0121863 [View Article][PubMed]
     [Google Scholar]
  36. Wang H, Wu JH, Ayala JC, Benitez JA, Silva AJ. Interplay among cyclic diguanylate, HapR, and the general stress response regulator (RpoS) in the regulation of Vibrio cholerae hemagglutinin/protease. J Bacteriol 2011; 193:6529–6538 [View Article][PubMed]
     [Google Scholar]
  37. Chaparian RR, Olney SG, Hustmyer CM, Rowe-Magnus DA, van Kessel JC. Integration host factor and LuxR synergistically bind DNA to coactivate quorum-sensing genes in Vibrio harveyi. Mol Microbiol 2016; 101:823–840 [View Article][PubMed]
     [Google Scholar]
  38. van Kessel JC, Ulrich LE, Zhulin IB, Bassler BL. Analysis of activator and repressor functions reveals the requirements for transcriptional control by LuxR, the master regulator of quorum sensing in Vibrio harveyi. MBio 2013; 4:e00378 [View Article][PubMed]
     [Google Scholar]
  39. Burden S, Lin YX, Zhang R. Improving promoter prediction for the NNPP2.2 algorithm: a case study using Escherichia coli DNA sequences. Bioinformatics 2005; 21:601–607 [View Article][PubMed]
     [Google Scholar]
  40. Shahmuradov IA, Mohamad Razali R, Bougouffa S, Radovanovic A, Bajic VB. bTSSfinder: a novel tool for the prediction of promoters in cyanobacteria and Escherichia coli. Bioinformatics 2017; 33:334–340 [View Article][PubMed]
     [Google Scholar]
  41. Browning DF, Busby SJ. Local and global regulation of transcription initiation in bacteria. Nat Rev Microbiol 2016; 14:638–650 [View Article][PubMed]
     [Google Scholar]
  42. de Avila E Silva S, Echeverrigaray S, Gerhardt GJ. BacPP: bacterial promoter prediction–a tool for accurate sigma-factor specific assignment in enterobacteria. J Theor Biol 2011; 287:92–99 [View Article][PubMed]
     [Google Scholar]
  43. Bendtsen KM, Erdossy J, Csiszovszki Z, Svenningsen SL, Sneppen K et al. Direct and indirect effects in the regulation of overlapping promoters. Nucleic Acids Res 2011; 39:6879–6885 [View Article][PubMed]
     [Google Scholar]
  44. Watnick PI, Butterton JR, Calderwood SB. The interaction of the Vibrio cholerae transcription factors, Fur and IrgB, with the overlapping promoters of two virulence genes, irgA and irgB. Gene 1998; 209:65–70 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000591
Loading
OpaR and RpoS are positive regulators of a virulence factor PrtA in Vibrio parahaemolyticus
Microbiology 164, 221 (2018); https://doi.org/10.1099/mic.0.000591
/content/journal/micro/10.1099/mic.0.000591
/content/journal/micro/10.1099/mic.0.000591
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error