Comparative Genomics Final Results

1. Ben • Dan • Deepak • Esha • Kelley • Pramod • Raghav • Smruthy • Vartika • Will Comparative GenomicsFinal 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. Genes common/unique to V.vulnificus and V.navarrensis

4. SPECIATION??

5. Whole genome super matrix tree VN3 100% Bootstrap support >0.98 posterior probability support 97.13 VN2 95.58 VN4 98.35 VN1 82.64 VV1 98.60 VV4 98.88 VV2 97.76 81.94 98.92 VV3 VV5 Vp1 0.02 Aligned using Clustal-omega. A concatenated alignment was generated and a bootstrapped (100) maximum likelihood phylogenetic tree was generated using the Jones-Taylor-Thornton model of evolution and an assumption of a constant rate of change. The tree was rooted with Vibrio parahaemolyticus as an outgroup. All nodes had 100% bootstrapping support and >0.98 posterior probability support (8 chains for 20,000 generations sampling at every 100th generation). ANI support for each node is shown. ANI values for internal nodes were calculated by taking the average ANI for all pairs of genomes representing the bifurcation.

6. VV1 12 VV2 VN4 VN2 11 Vibrio_vulnificus_CMCP6 Vibrio_vulnificus_YJ016 VN1 74 VV4 95 VV5 VN3 VV3 Vibrio_parahaemolyticus 0.015 0.010 0.005 0.000 16S rRNA Tree • 16S is not informative for differentiating closely related Vibrio species. • Full length 16S rRNA sequences were assembled by mapping to the reference . • Aligned using PyNAST • Bootstrapped ML tree was generated using MEGA • Rooted using V. parahaemolyticus

7. PATHOGENECITY??

8. Approach I Approach II Gene Predictions Annotated Dataset Reference Strains Annotation Files from NCBI Existence of Toxins Presence Absence OrthoMCL Machinery for Incorporation Machinery for Incorporation Generation of presence-absence matrix Test for group significance (ANOSIM test) Yes No Different combos of files Potentially Pathogenic Unlikely Pathogenic Correlation with Pathway (KEGG) ID genes associated with groups (SIMPER test) Connecting the dots Pathogenic or Putatively Pathogenic Heatmaps in R to view gene profiles

9. Approach II (contd) Gene files Pre-Processing Filter Fasta BlastDb Blast Parser All v/s All Blast Markov Clustering Upload parsed data to Database Find Protein Pairs Cluster of Orthologs

10. Gene Profiles for All Strains V. fischeri14 Non-human pathogens • V. fischeri11 V.splendidus32 V. cholerae61 V.cholerae95 V.parahaemolyticus33 Pathogens • V.parahaemolyticus0P V.vulnificusYJ016 V.vulnificusCMCP06 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 Presence Absence 60 70 80 90 100 Group Average dendrogram generated from a simple matching resemblance matrix. Similarity

11. Gene Profiles for All Strains path vul nonpath nav VV05 VV04 VV02 VN04 VV03 2D Stress: 0.09 VN01 VV01 VN02 VN03 VVYJ VVCM VC61 VS32 VC95 VP33 VP0P VF14 VF11 MNDS plot generated from a simple matching resemblance matrix. The dendrogram is a bit misleading about the relationship between V. splendidus and V. fischeri.

12. ANOSIM Statistical Test • ANOSIM is a nonparametric method that tests whether two or more groups of samples are significantly different. • R statistic - A measure of the strength of the difference between two groups. A value closer to +1 signifies more dissimilarity between the groups • Significance Level - tests the significance of the difference. Analogous to p-value.

13. Genes significantly different between V. vulnificusand V. navarrensis V. fischeriES114 • V. fischeriMJ11 V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 Presence Absence 60 70 80 90 100 Similarity

14. Genes significantly different between V. vulnificusand V. navarrensis V. fischeriES114 • V. fischeriMJ11 V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 Presence Hypotheticals / Conserved hypotheticals Absence 60 70 80 90 100 Similarity

15. Antibiotic res Toxin related Slime biosyn TonB Vibriobactin related Missing from V. navarrensis Type 1 & 2 Secretion Simple sugar uptake rtxC Chemotaxis V. fischeriES114 Biofilm CPS • V. fischeriMJ11 Pilli LPS PTS V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 Unique to V. navarrensis VN04 VN03 VN02 n-acetyl transferase acetyl transferase glucokinase HemeBiosynthesis / Iron acquisition Adhesin Chemotaxis Presence Absence 60 70 80 90 100 Similarity

16. Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups V. fischeriES114 • V. fischeriMJ11 V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 Presence Absence 60 70 80 90 100 A SIMPER test was performed to identify genes that lead to differences between Pathogens (V. cholerae, V. parahaemolyticus, V. vulnificus) and Non-Pathogens (V. fischeri, V. splendidus). Genes were supported by relative abundance in Pathogens, then by relative abundances in non-pathogens. Genomes are arranged based on the clustering pattern identified from the entire gene profile. Similarity

17. Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups Transporters, transcription factors, hemolysins, exonucleases, carbohydrate metabolism (enormous gene variation) V. fischeriES114 • V. fischeriMJ11 V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 Presence Absence A SIMPER test was performed to identify genes that lead to differences between Pathogens (V. cholerae, V. parahaemolyticus, V. vulnificus) and Non-Pathogens (V. fischeri, V. splendidus). Genes were supported by relative abundance in Pathogens, then by relative abundances in non-pathogens. Genomes are arranged based on the clustering pattern identified from the entire gene profile. 60 70 80 90 100 Similarity

18. Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups V. fischeriES114 • V. fischeriMJ11 V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 Presence A SIMPER test was performed to identify genes that lead to differences between Pathogens (V. cholerae, V. parahaemolyticus, V. vulnificus) and Non-Pathogens (V. fischeri, V. splendidus). Genes were supported by relative abundance in Pathogens, then by relative abundances in non-pathogens. Genomes are arranged based on the clustering pattern identified from the entire gene profile. Absence 60 70 80 90 100 Similarity

19. A subset of Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups V. fischeriES114 • V. fischeriMJ11 V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 Presence Absence In yellow: Genes related to type 1 secretion, chemotaxis, permeases, proteases, and LPS synthesis (capsid polysaccharides, lipoproteins, exopolysacharrides) 60 70 80 90 100 Similarity

20. A subset of Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups V. fischeriES114 • V. fischeriMJ11 V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 Presence Absence 60 70 80 90 100 Mostly hypotheticals (40), response regulators, glutathione synthase, starvation proteins Similarity

21. A subset of Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups V. fischeriES114 • V. fischeriMJ11 V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 Presence Absence 60 70 80 90 100 Hypotheticals (153), transcription factors (21), urease operon (10), lipoproteins (16), chemotaxis (8), zinc uptake (3), sideophore synthesis & uptake (6 – 2 operons), luciferase operon (3 genes) Similarity

22. Genes significantly different between the Clinical and Environmental Strains of V. navarrensis V. fischeriES114 • V. fischeriMJ11 V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 Drives separation VN02 VN03 VN04 Hypotheticals, flagellar proteins Presence Endonucleases (5), Channel proteins (2), chemotaxis genes (5), permeases (2), transcriptional regulators (4), dehydratase (4) Absence 60 70 80 90 100 Similarity

23. A Subset of Genes significantly different between the Clinical and Environmental Strains of V. navarrensis V. fischeriES114 • V. fischeriMJ11 V.spendidusLGP32 V. choleraeO1 biovarE1 Tor N16961 V.choleraeO395 V.parahaemolyticsRIMD_2210633 • V.parahaemolyticsBB220P V.vulnificusYJ016 V.vulnificusCMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN02 VN03 VN04 Presence ATP dep. endonuclease Phage tail collar domain Absence 60 70 80 90 100 Channel proteins Flagellin Similarity Chemotaxis Transcript. regulators Endonucleases dehydratase

24. Previously Discusses Virulence Factors

25. Capsular Polysaccharides • The most important virulence factor for V. vulnificusis its capsular polysaccharide (CPS). • V. vulnificusis an extracellular pathogen that relies on its CPS to avoid phagocytosis by host defense cells and complement (Linkous and Oliver, 1999; Strom and Paranjpye, 2000). • Unencapsulatedmutants are susceptible to bactericidal activity in human serum (Shinoda et al., 1987). • Presence of capsule is related to the colony morphology (Yoshida et al., 1985; Wright et al., 1999).

27. Selected Hemolysins

28. Iron Acquisition • Vibrio vulnificuspathogenecity - increased iron in the host results in increased susceptibility to infection (Weinberg 2000). • As with other invasive bacterial pathogens, iron-scavenging siderophores and proteins that bind host iron-containing proteins were identified in V. vulnificus. • A couple of studies indicated that the protease produced by V. vulnificuscould be involved in acquisition of iron from heme proteins (Nishina et al., 1992; Okujo et al., 1996). • Litwin and Calderwood (1993) cloned the V. vulnificusfur gene, which encodes the central regulator in iron metabolism in many bacteria. • The essential role for vulnibactin in virulence was confirmed by Litwin et al. (1996).V. vulnificusmutant for vuuA, the ferric vulnibactin receptor, could not use vulnibactin and was decreased for virulence in mice.

30. Flagella and Motility

31. The mystery behind RTX toxin • These following are the hits from the annotation for rtx: • RTX toxin – Toxin metabolic process; cytolysis • RTX protein – iron regulated protein • When we BLAST these proteins with NCBI we found the following hits, • M6 family metalloprotease domain protein • Iron regulated protein frpC

32. RTX machinery

33. Some other interesting factors!

34. Conclusions • V. navarrensis is unlikely to be a pathogen to healthy human individuals. • Absence of toxins • Absence of CPS • Presence of hemolysins similar to V. vulnificus • Very different profile from the compared Vibrios. • Vibrio navarrensis is not similar to the non-human pathogenic Vibrios. • Blood and environmental strains of V. navarrensisare very similar. • Differences: LPSsynthesis, Type-I secretion system, Permeases. • We still believe that these will store a similar niche in the environment. • Vibriosare difficult to study owing to their metabolic versatility and wide range of animal hosts.

35. Questions?