1. Comparative Genomics Preliminary Results Ben Dan Deepak Esha Kelly Pramod Raghav Smruthy Vartika Will

2. Questions to be Addressed 1.Sixteen strains clustered with V. navarrensis type strain LMG15976 16S rRNA, pyrH, recA and rpoA Four formed a distinct cluster V. vulnificus  Closest relative to both lineages of V. navarrensis “Is it a different species or biotype?” 2. V. navarrensis strains isolated from various sources. nav_2423 (VN1) : Blood nav_2462 (VN2) : Surface Wound nav_2541 (VN3) : Sewage nav_2756 (VN4) : Water “Is Vibrio navarrensis pathogenic?”

4. Draft Genome Gene Predictions Translated Genes ANI Dendrogram Identifying Core Genome OrthoMCL Custom Script Multiple Alignment Super Tree Super Matrix Super Matrix Consensus Tree Strategy for Defining Species New Species?? PHYLIP MEGA PAUP Mr Bayes Clustal Ω MUSCLE

5. ANI Results : WHOLE GENOME VN1VN2VN3VN4VV1VV2VV3VV4 VN1 VN2 96.10 VN3 97.7296.11 VN4 98.0595.3295.28 VV1 77.9377.9777.5378.02 VV2 76.8976.1376.4376.1198.36 VV3 77.5076.3877.0476.8098.7298.74 VV4 76.1076.3176.2275.9898.4198.1098.72 VV5 76.0676.4476.0776.1697.2596.8797.2397.28

6. ANI : whole genome tree VV5 VV4 VV1 VV2 VV3 VN2 VN3 VN1 VN4 1009590858075 ANI Similarity Group Average Dendrogram generated using ANI distance

7. Correlation b/w ANI tree and Three - gene tree VN1 VN4 VN2 VN3 VV1 VV3 VV4 VV2 VV5

8. SuperMatrix Whole Genome Tree Neighbor-joining tree constructed using Jones-Taylor-Thornton distance 1398 orthologous genes were used to generate this tree Protein sequences were aligned using clustal-omega, and were concatenated VN4 VN1 VN2 VN3 VV5 VV1 VV2 VV4 VV3 Vp1 0.02

9. SuperMatrix Whole Genome Tree (BOOTSTRAP) VN1 VN4 VN2 VN3 VV1 VV3 VV4 VV2 VV5 Vp1 100 60 100

10. Supertree Whole Genome Tree VN1 VN4 VN2 VN3 VV5 VV1 VV2 VV3 VV4 39 50 93 26 22 31 95 VP1 Supertree generated by taking a majority rule consensus tree from 1398 gene trees Orthologous genes were aligned using clustal-omega Distances were calculated assuming Jones-Taylor-Thornton distance, and generated using the Neighbor-Joining method Values indicate the percentage of trees where this tree topology was observed

11. Preliminary Conclusions Tentative results suggest that all four strains in V. navarrensis belong to same species. The super matrix NJ tree confirms the topology of the tree by CDC. However, the consensus supertree indicates weak support for two clades of V. navarrensis. – This will be confirmed shortly

13. CTX – Cholera Toxin It is in the genome of lysogenic bacteriophage (CTXPhi) and is carried by Vibrio cholerae Causes the major symptoms of infection: diarrhea, vomiting and cramps.

14. Ctx + zot + ace  form a V. cholerae virulence cassette Ctx genes code for cholera toxin zot causes decrease in intestinal tissue resistance by modifying intercellular tight junctions. ace causes increases in fluid secretion in ligated illeal loops

15. repeat-in-toxin (RTX) family Group of related proteins found in gram negative bacteria Has a broad range of distribution and activities RTX – rtx A gene encodes for the RTX toxin which is related with septicemia and gastroenteritis

16. HEMOLYSINS Exotoxins that lyse erythrocyte membranes by formation of pores with the liberation of iron binding proteins (transferrin, lactoferrin and hemoglobin). The pore forming activity of hemolysins extend to other cells such as neutrophils, mast cells and enhances virulence by causing tissue damage.

17. Four defined classes of Hemoylsins Experimental evidence suggests “Hemolysins are involved in disease pathogenesis” (Zhang et.al 2005) TDH family (Thermostable Direct Hemolysin) HlyA family (El Tor Hemolysin) TLH family (Thermolabile Hemolysin) δ – VPH family (Thermostable Hemolysin)

18. Bacterial TaxonHemolysin FamilySpecific Hemolysin V. cholerae O1HlyA TLHLecithinase LEC δ – VPHVc- δTH V. cholerae non-O1HlyA TDHNAG-TDH V. vulnificusHlyAVVH TLHVPL -hly III -vll Y V. parahaemolyticusTLHTLH or LDH δ – VPH TDHVp-TRH/Vp-TDH (Zhang et.al 2005)

19. Siderophores Low molecular weight compounds that have high affinity for iron molecules. Chelates iron ions in the environment whereupon the ferri-siderophores re- enter the bacterial cells by means of specific cell-surface receptors. The iron is then released for incorporation into bacterial proteins. Studies show the association of siderophores with virulence in Vibrios. (Natividad-Bonifacio et al 2013)

20. Attachment Factors Toxin Co-regulated Pilus (TCP) – Toxin Co-regulated Pilus (TCP) has been identified as a critical colonization factor in both animal models and humans for V. cholerae – The major colonization factor of V. cholerae is TCP, which consists of TcpA subunits encoded by the tcpA gene located in the tcp gene cluster. TCP is also the receptor for CTXΦ Type IV pilus – Type IV pilus plays a role in adherence and colonization to mammalian host. Like Msha pilus plays role in adherence – PilA and pil D plays role in V. vulnificus adherence to human epithelial cells

21. SECRETION SYSTEMS

22. Six distinct secretion systems have been shown to mediate protein export through the inner and outer membranes of Gram-negative bacteria. Ctx (Cholera Toxin) is associated with type II secretion system Rtx (Repeat-in Toxin) is associated with type I secretion system SECRETION SYSTEMS

23. Pathogenicity Islands Vibrio seventh pandemic islands VSP1,VSP 2 Genes encode hypothetical functions presumed to be necessary for evolutionary fitness

24. VPI 2 A 57.3 kb gene cluster encoding genes for neuraminidase (nanH) and amino sugar metabolism, which has the characteristic features of a pathogenicity island These pathogenic islands are present in V. cholerae strains Presence of these islands have been used to elucidate the pathogenicity of Vibrio species like V.mimicus.

25. Annotated Dataset Existence of Toxins Machinery for Incorporation (Pili/Attachment Factors) Machinery for Incorporation (Pili/Attachment Factors) PresenceAbsence Machinery for Incorporation (Pili/Attachment Factors) Machinery for Incorporation (Pili/Attachment Factors) Potentially Pathogenic Unlikely Pathogenic Yes No Correlation with Pathway (KEGG) Pathogenic or Putatively Pathogenic Pathogenic or Putatively Pathogenic Connecting the dots Strategy for Pathogenicity

26. Blood isolate VN1 Hemolysin Check

27. Blood isolate VN1 RTX MACHINERY

28. Blood isolate VN1 CTX RELATED MACHINERY Х Х

29. Preliminary Results : VV2 Х Х Х Hemolysin Check RTX MACHINERY CTX RELATED MACHINERY

30. Preliminary Conclusions (Connecting the dots) VN1  potentially pathogenic? Presence of hemolysins (vvhA along with its regulator HlyU), Siderophores (required for Fe III solubilization and iron uptake) and a serum resistance genes  indicates signs of causing septicemia. RTX toxin seems to be non functional but presence of rtx toxin and rtxD gene indicates that V. navarrensis is able to integrate plasmid containing rtx gene cluster. CTX - Absent lack the TCP genes

31. WHAT NEXT??  Calculate ANI from gene prediction results.  Validate tree topology using ML and Bayesian.  Try and solve mystery of the missing RTX genes.  Detail analysis of immune evasion systems  Environment v/s clinical strains comparison  All v/s All within our nine strains