MSHA-like pili of non-toxigenic Vibrio cholerae strains

Aim. In this study, we set out to identify the homologues of genes from the msh-cluster in the genomes of non-toxigenic V cholerae, to perform the bioinformatics analysis of their products, as well as to study the adhesive properties of strains containing altered genes. Materials and methods. We analysed 17 clinical strains of non-O1/non-O139 V cholerae and 2 strains of the O1 serogroup isolated from water bodies. Genes belonging to the msh-cluster were identified in the whole genomes using the BLASTN 2.2.29 and BioEdit 7.2.5 programs. Gene translation, comparative analysis of their nucleotide sequences and the amino acid sequences of deduced products were performed using the Vector NTI Advance 11 (Invitrogen). Results and discusssion. In 18 out of the 19 studied genomes we identified gene clusters responsible for production of adhesion pili (mshH-Q) represented by diverse alleles, the majority of which differed from the prototype genes of the msh-cluster in nucleotide composition but had the same localization and arrangement. Only one strain had a cluster that was close to that of the prototype. A bioinformatics analysis of their deduced products indicated that the amino acid sequence of the major MshA pilus subunit is homologous to the prototype only in a short N-terminal region (1-41) while sharing no similarities with the rest of the sequence. Nevertheless, this protein, similar to VcfA described by Kuroki H. et al. (2001) and designated by us as MshA-like, retained a putative pilus domain. A similar pattern was observed in the minor subunits designated as MshC-like. Other minor subunits also retained their characteristic domains. All of the strains agglutinated human erythrocytes (group O) and chicken erythrocytes, and in isolates harboring modified mshA-like and mshC-like genes the reaction was not inhibited by mannose. Since most of the studied strains were isolated from hospitalized patients, it is possible that in non-toxigenic V. cholerae lacking the pathogenicity island VPI, MSHA-like pili may serve as a colonization factor of the human intestine, in contrast to VPI-positive strains. The obtained information provides a basis for experimental verification of this assumption.

1. Chiavelli D.A., Marsh J.W., Taylor R.K. The mannose-sensitive hemagglutinin of Vibrio cholerae promotes adherence to zooplankton. Appl. Environ. Microbiol. 2001; 67(7):3220-5.

2. Wang J., Yan M., Gao H., Lu X., Kan B. Vibrio cholerae colonization of soft-shelled turtles. Appl. Environ. Microbiol. 2017; 83(14):e00713-17. DOI: 10.1128/AEM.00713-17.

3. List C., Grutsch A., Radler C., Cakar F., Zingl F.G., Schild-Prufert K., Schild S. Genes activated by Vibrio cholerae upon expo-sure to Caenorhabditis elegans reveal the mannose-sensitive hemag-glutinin to be essential for colonization. mSphere 2018; 3(3):e00238-18. DOI: 10.1128/mSphereDirect.00238-18.

4. Attridge S.R., Manning P.A., Holmgren J., Jonson G. Relative significance of mannose-sensitive hemagglutinin and toxin-coregulated pili in colonization of infant mice by Vibrio cholerae El Tor. Infect. Immun. 1996; 64(8):3369-73.

5. Thelin K.H., Taylor R.K. Toxin-coregulated pilus, but not mannose-sensitive hemagglutinin, is required for colonization by Vibrio cholerae O1 El Tor biotype and O139 strains. Infect. Immun. 1996; 64f7):2853—6.

6. Tacket C.O., Taylor R.K., Losonsky G., Lim Y, Nataro J.P., Kaper J.B., Levine M.M. Investigation of the roles of toxin-coregulated pili and mannose-sensitive hemagglutinin pili in the patho-genesis of Vibrio cholerae о139 infection. Infect. Immun. 1998; 6б(2):692-5.

7. Dalsgaard A., Albert M.J., Taylor D.N., Shimada T., Meza R., Serichantalergs O., Echeverria P. Characterization of Vibrio cholerae non-O1 serogroups obtained from an outbreak of diarrhea in Lima, Peru. J. Clin. Microbiol. 1995; 33(10):27l5-22.

8. Eroshenko G.A., Osin A.V., Shchelkanova E.Yu., Smirnova N.I. [A comparative analysis of genomes of virulent and avirulent strains of Vibrio cholerae 0139]. Molecular Genetics, Microbiology and Virology. 2004; (2):11—6.

9. Marsh J.W., Taylor R.K. Genetic and transcriptional analyses of the Vibrio cholerae mannose-sensitive hemagglutinin type 4 pilus gene locus. J. Bacteriol. __1999; 18(4):1110—7.

10. Hsiao A., Toscano K., Zhu J. Post-transcriptional cross-talk between pro- and anti-colonization pili biosynthesis systems in Vibrio cholerae.Mol. Microbiol. 2008; 67(4):849—60. DOI: 10.1111/j.1365-2958.2007.06091.x

11. Nakasone N., Iwanaga M. Pili of Vibrio cholerae non-01. Infect. Immun. 1990; 58(6):1640—6.

12. Yamashiro T., Nakasone N., Iwanaga M. Purification and characterization of pili of a Vibrio oholerae non-01 strain. Infect. Immun. 1993; 61(12):5398—400.

13. Yamashiro T., Iwanaga M. Purification and characterization of a pilus of a Vibrio cholerae strain: a possible colonization factor. Infect. Immun. 1996; 64(12):5233-8.

14. Kuroki H., Toma C., Nakasone N., Yamashiro T., Iwanaga M. Gene analysis of Vibrio cholerae NAGV14 pilus and its distribution. Microbiol. Immunol. 2001; 45(6):417—24. DOI: 10.1111/j.1348-0421.2001.tb02640.x

15. Toma C., Kuroki H., Nakasone N., Ehara M., Iwanaga M. Minor pilin subunits are conserved in Vibrio cholerae type IV pili. FEMSImmunol. Med. Microbiol. 2002; 33(1):35—40.

16. Miyazato T., Toma C., Nakasone N., Yamamoto K., Iwanaga M. Molecular analysis of VcfQ protein involved in Vibrio cholerae type IV pilus biogenesis. J. Med. Microbiol. 2003; 52(Pt 4):283—8. DOI: 10.1099/jmm.0.04967-0.

17. Titova S.V., Monakhova E.V., Alekseeva L.P., Pisanov R.V [Molecular genetic basis of biofilm formation as a component of Vibrio cholerae persistence in the water reservoirs of Russian Federation]. Ecological Genetics 2018; 16(4):23—32. DOI: 10.17816/ecogen16423-32.

18. Arkhangelskaya I.V., Nepomnyaschaya N.B., Monakhova E.V., Vodopyanov A.S., Vodopyanov S.O., Kruglikov VD. [Genetic diversity of the population of Vibrio cholerae non-O1/non-O139 circulating in Rostov region]. Public Health and Life Environment 2015; 3(264):25—8.

19. Hsiao A., Xu X., Kan B., Kulkarni R. V, Zhu, J. Direct regulation by the Vibrio cholerae regulator ToxT to modulate colonization and anticolonization pilus expression. Infect. Immun. 2009; 77(4):1383—8. DOI: 10.1128/iai.01156-08.

20. Marsh J.W., Sun D., Taylor R.K. Physical linkage of the Vibrio cholerae mannose-sensitive hemagglutinin secretory and structural subunit gene loci: identification of the mshG coding sequence. Infect. Immun. 1996; 64(2):460—5.

21. Hsiao A., Liu Z., Joelsson A., Zhu J. Vibrio cholerae virulence regulator-coordinated evasion of host immunity. Proc. Natl. Acad. Sci. USA. 2006(39); 103:14542—7. DOI: 10.1073/pnas.0604650103.