1887

Abstract

A high-throughput screening (HTS) assay was developed for identifying compounds with inhibitory effect on aphA, one of the key regulators positively controlling Vibrio cholerae pathogenesis. An inhibitory effect on aphA was expected to lead to attenuation in the secretion of the major pathogenicity factors of V. cholerae, cholera toxin and toxin co-regulated pilus. The plasmid construct pAKSB was developed with a kanamycin resistance (Km) gene under the control of the aphA -like promoter for conferring a Km phenotype under aphA -expressing conditions. The HTS assay was performed to identify compounds with inhibitory effect on the growth of O139 V. cholerae MO10 carrying the construct pAKSB in growth medium containing Km (30 g ml), but not in its absence. Of 20 338 compounds screened, six compounds were identified to inhibit the pAKSB-induced Km phenotype and these compounds caused transcriptional inhibition of aphA in V. cholerae O139 strain MO10 as well as variant V. cholerae O1 El Tor strain NM06-058. Of the three most active substances, compound 53760866 showed lowest half-maximal cytotoxicity in a eukaryotic cell viability assay and was characterized further. Compound 53760866 caused reduction in cholera toxin secretion and expression of TcpA in vitro. The in vitro virulence attenuation corroborated well in a suckling mouse model in vivo, which showed reduction of colonization by V. cholerae NM06-058 when co-administered with 53760866. The screening method and the compounds may lead to new preventive strategies for cholera by reducing the pathogenicity of V. cholerae .

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2016-07-01
2019-12-06
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References

  1. Anthouard R., DiRita V. J..( 2013;). Small-molecule inhibitors of toxT expression in Vibrio cholerae. . MBio 4: e00403-13. [CrossRef] [PubMed]
    [Google Scholar]
  2. Brocklehurst C..( 2014;). Scaling up rural sanitation in India. . PLoS Med 11: e1001710. [CrossRef] [PubMed]
    [Google Scholar]
  3. Cámara M., Hardman A., Williams P., Milton D..( 2002;). Quorum sensing in Vibrio cholerae. . Nat Genet 32: 217–218. [CrossRef] [PubMed]
    [Google Scholar]
  4. Clatworthy A. E., Pierson E., Hung D. T..( 2007;). Targeting virulence: a new paradigm for antimicrobial therapy. . Nat Chem Biol 3: 541–548. [CrossRef] [PubMed]
    [Google Scholar]
  5. Dörwald F. Z..( 2012;). Lead Optimization for Medicinal Chemists: Pharmacokinetic Properties of Functional Groups and Organic Compounds. Weinheim:: Wiley;.[Crossref]
    [Google Scholar]
  6. Gerth K., Pradella S., Perlova O., Beyer S., Müller R..( 2003;). Myxobacteria: proficient producers of novel natural products with various biological activities--past and future biotechnological aspects with the focus on the genus Sorangium. . J Biotechnol 106: 233–253.[PubMed] [Crossref]
    [Google Scholar]
  7. Ghosh A., Ramamurthy T..( 2011;). Antimicrobials & cholera: are we stranded?. Indian J Med Res 133: 225–231.[PubMed]
    [Google Scholar]
  8. Ghosh-Banerjee J., Senoh M., Takahashi T., Hamabata T., Barman S., Koley H., Mukhopadhyay A. K., Ramamurthy T., Chatterjee S. et al.( 2010;). Cholera toxin production by the El Tor variant of Vibrio cholerae O1 compared to prototype El Tor and classical biotypes. . J Clin Microbiol 48: 4283–4286. [CrossRef] [PubMed]
    [Google Scholar]
  9. Hammer B. K., Bassler B. L..( 2003;). Quorum sensing controls biofilm formation in Vibrio cholerae. . Mol Microbiol 50: 101–104.[PubMed] [Crossref]
    [Google Scholar]
  10. Heidelberg J. F., Eisen J. A., Nelson W. C., Clayton R. A., Gwinn M. L., Dodson R. J., Haft D. H., Hickey E. K., Peterson J. D. et al.( 2000;). DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. . Nature 406: 477–483. [CrossRef] [PubMed]
    [Google Scholar]
  11. Hung D. T., Shakhnovich E. A., Pierson E., Mekalanos J. J..( 2005;). Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. . Science 310: 670–674. [CrossRef] [PubMed]
    [Google Scholar]
  12. Iwanaga M., Yamamoto K..( 1985;). New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor. . J Clin Microbiol 22: 405–408.[PubMed]
    [Google Scholar]
  13. Jobling M. G., Holmes R. K..( 1997;). Characterization of hapR, a positive regulator of the Vibrio cholerae HA/protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene. . Mol Microbiol 26: 1023–1034.[PubMed] [Crossref]
    [Google Scholar]
  14. Kaper J. B., Morris J. G., Levine M. M..( 1995;). Cholera. . Clin Microbiol Rev 8: 48–86.[PubMed] [Crossref]
    [Google Scholar]
  15. Kitaoka M., Miyata S. T., Unterweger D., Pukatzki S..( 2011;). Antibiotic resistance mechanisms of Vibrio cholerae. . J Med Microbiol 60: 397–407. [CrossRef] [PubMed]
    [Google Scholar]
  16. Kovacikova G., Skorupski K..( 1999;). A Vibrio cholerae LysR homolog, AphB, cooperates with AphA at the tcpPH promoter to activate expression of the ToxR virulence cascade. . J Bacteriol 181: 4250–4256.[PubMed]
    [Google Scholar]
  17. Kovacikova G., Skorupski K..( 2002;). Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter. . Mol Microbiol 46: 1135–1147.[PubMed] [Crossref]
    [Google Scholar]
  18. Lin W., Kovacikova G., Skorupski K..( 2007;). The quorum sensing regulator HapR downregulates the expression of the virulence gene transcription factor AphA in Vibrio cholerae by antagonizing Lrp- and VpsR-mediated activation. . Mol Microbiol 64: 953–967. [CrossRef] [PubMed]
    [Google Scholar]
  19. Lisurek M., Rupp B., Wichard J., Neuenschwander M., von Kries J. P., Frank R., Rademann J., Kühne R..( 2010;). Design of chemical libraries with potentially bioactive molecules applying a maximum common substructure concept. . Mol Divers 14: 401–408. [CrossRef] [PubMed]
    [Google Scholar]
  20. Miller M. B., Skorupski K., Lenz D. H., Taylor R. K., Bassler B. L..( 2002;). Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae. . Cell 110: 303–314. [CrossRef] [PubMed]
    [Google Scholar]
  21. Mondal S. I., Khadka B., Akter A., Roy P. K., Sultana R..( 2014;). Computer based screening for novel inhibitors against Vibrio cholerae using NCI diversity set-II: an alternative approach by targeting transcriptional activator ToxT. . Interdiscip Sci 6: 108–117. [CrossRef] [PubMed]
    [Google Scholar]
  22. Mosmann T..( 1983;). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. . J Immunol Methods 65: 55–63.[PubMed] [Crossref]
    [Google Scholar]
  23. Nandy R. K., Mukhopadhyay S., Ghosh A. N., Ghose A. C..( 1999;). Antibodies to the truncated (short) form of ‘O’ polysaccharides (TFOP) of Vibrio cholerae O139 lipopolysaccharides protect mice against experimental cholera induced by encapsulated O139 strains and such protection is mediated by inhibition of intestinal colonization of vibrios. . Vaccine 17: 2844–2852.[PubMed] [Crossref]
    [Google Scholar]
  24. Patra T., Koley H., Ramamurthy T., Ghose A. C., Nandy R. K..( 2012;). The Entner-Doudoroff pathway is obligatory for gluconate utilization and contributes to the pathogenicity of Vibrio cholerae. . J Bacteriol 194: 3377–3385. [CrossRef] [PubMed]
    [Google Scholar]
  25. Raju R. M., Goldberg A. L., Rubin E. J..( 2012;). Bacterial proteolytic complexes as therapeutic targets. . Nat Rev Drug Discov 11: 777–789. [CrossRef] [PubMed]
    [Google Scholar]
  26. Raychoudhuri A., Mukhopadhyay A. K., Ramamurthy T., Nandy R. K., Takeda Y., Nair G. B..( 2008;). Biotyping of Vibrio cholerae O1: time to redefine the scheme. . Indian J Med Res 128: 695–698.[PubMed]
    [Google Scholar]
  27. Rutherford S. T., van Kessel J. C., Shao Y., Bassler B. L..( 2011;). AphA and LuxR/HapR reciprocally control quorum sensing in vibrios. . Genes & Development 25: 397–408. [CrossRef] [PubMed]
    [Google Scholar]
  28. Sasse F., Steinmetz H., Schupp T., Petersen F., Memmert K., Hofmann H., Heusser C., Brinkmann V., von Matt P. et al.( 2002;). Argyrins, immunosuppressive cyclic peptides from myxobacteria. I. Production, isolation, physico-chemical and biological properties. . J Antibiot 55: 543–551.[PubMed] [Crossref]
    [Google Scholar]
  29. Sergeev G., Roy S., Jarek M., Zapolskii V., Kaufmann D. E., Nandy R. K., Tegge W..( 2014;). High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae. . BMC Microbiol 14: 49. [CrossRef] [PubMed]
    [Google Scholar]
  30. Shakhnovich E. A., Hung D. T., Pierson E., Lee K., Mekalanos J. J..( 2007a;). Virstatin inhibits dimerization of the transcriptional activator ToxT. . Proc Natl Acad Sci U S A 104: 2372–2377.[Crossref]
    [Google Scholar]
  31. Shakhnovich E. A., Sturtevant D., Mekalanos J. J..( 2007b;). Molecular mechanisms of virstatin resistance by non-O1/non-O139 strains of Vibrio cholerae. . Mol Microbiol 66: 1331–1341.
    [Google Scholar]
  32. Tegge W., Bautsch W., Frank R..( 2007;). Synthesis of cyclic peptides and peptide libraries on a new disulfide linker. . J Pept Sci 13: 693–699. [CrossRef] [PubMed]
    [Google Scholar]
  33. Weissman K. J., Müller R..( 2009;). A brief tour of myxobacterial secondary metabolism. . Bioorg Med Chem 17: 2121–2136. [CrossRef] [PubMed]
    [Google Scholar]
  34. WHO( 2010;). Cholera vaccines: WHO position paper. . Wkly Epidemiol Rec 85: 117–128.[PubMed]
    [Google Scholar]
  35. WHO( 2014;). Cholera, 2013. . Wkly Epidemiol Rec 89: 345–355.[PubMed]
    [Google Scholar]
  36. Zahid M. S., Awasthi S. P., Asakura M., Chatterjee S., Hinenoya A., Faruque S. M., Yamasaki S..( 2015;). Suppression of virulence of toxigenic Vibrio cholerae by Anethole through the cyclic AMP (cAMP)-cAMP receptor protein signaling system. . PLoS One 10: e0137529. [CrossRef] [PubMed]
    [Google Scholar]
  37. Zhang J. H., Chung T. D., Oldenburg K. R..( 1999;). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. . J Biomol Screen 4: 67–73.[PubMed] [Crossref]
    [Google Scholar]
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