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Comparative Genomics Preliminary Results

Ben Dan Deepak Esha Kelly Pramod Raghav Smruthy Vartika Will. Comparative Genomics Preliminary Results. Questions to be Addressed. Sixteen strains clustered with V. navarrensis type strain LMG15976 16S rRNA , pyrH , recA and rpoA Four formed a distinct cluster

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Comparative Genomics Preliminary Results

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  1. Ben • Dan • Deepak • Esha • Kelly • Pramod • Raghav • Smruthy • Vartika • Will Comparative GenomicsPreliminary Results

  2. Questions to be Addressed • 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?” • V. navarrensisstrains 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?”

  3. SPECIATION??

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

  5. ANI Results : WHOLE GENOME

  6. ANI : whole genome tree VN4 VN1 VN3 VN2 VV3 VV2 VV1 VV4 VV5 75 80 85 90 95 100 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. VN4 VN1 VN2 VN3 VV5 VV1 VV2 VV4 VV3 Vp1 0.02 SuperMatrixWhole 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

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

  10. VN1 50 VN4 93 VN2 VN3 39 VV5 VV1 95 VV2 31 VV3 22 VV4 26 VP1 SupertreeWhole Genome Tree 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. navarrensisbelong 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

  12. PATHOGENECITY??

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

  14. Ctx + zot + ace  form a V. cholerae virulence cassette • Ctxgenes code for cholera toxin • zotcauses decrease in intestinal tissue resistance by modifying intercellular tight junctions. • ace causes increases in fluid secretion in ligatedillealloops

  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 hemolysinsextend to other cells such as neutrophils, mast cells and enhances virulence by causing tissue damage.

  17. Four defined classes of Hemoylsins Experimental evidence suggests “Hemolysinsare involved in disease pathogenesis” (Zhang et.al 2005)

  18. (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-siderophoresre-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-Bonifacioet 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 Mshapilus plays role in adherence • PilA and pil D plays role in V. vulnificus adherence to human epithelial cells

  21. SECRETION SYSTEMS

  22. SECRETION SYSTEMS • 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

  23. PathogenicityIslands • 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 pathogenicityisland • These pathogenic islands are present in V. choleraestrains • Presence of these islands have been used to elucidate the pathogenicity of Vibrio species like V.mimicus.

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

  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 RTX MACHINERY Hemolysin Check CTX RELATED MACHINERY Х Х Х

  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. navarrensisis 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

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