Comparative Genomics Final Results Ben Dan Deepak Esha Kelley Pramod Raghav Smruthy Vartika Will.

Comparative GenomicsFinal Results

BenDan

DeepakEsha

KelleyPramodRaghav

SmruthyVartika

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?”

Genes common/unique to V.vulnificus and V.navarrensis

SPECIATION??

100% Bootstrap support >0.98 posterior probability support

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.

Whole genome super matrix tree

Vibrio_vulnificus_CMCP6

Vibrio_vulnificus_YJ016

Vibrio_parahaemolyticus

0.0000.0050.0100.015

• 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

16S rRNA Tree

PATHOGENECITY??

Annotated Dataset

Existence of

Toxins

Machinery for Incorporation

Presence Absence

Machinery for Incorporation

Potentially Pathogenic

Yes No

Correlation with Pathway(KEGG)

Pathogenicor

Putatively Pathogenic

Connecting the dots

Unlikely Pathogenic

Gene Predictions

OrthoMCL

Generation of presence-absence

matrix

Heatmaps in R to view gene profiles

Reference Strains

Annotation Files from NCBI

Test for group significance

(ANOSIM test)

ID genes associated with

groups(SIMPER test)

Different combos of files

Approach I Approach II

Gene files

Pre-Processing

Filter Fasta

BlastDbAll v/s All

BlastBlast

Parser

Markov Clustering

Find Protein Pairs

Upload parsed data to Database

Cluster of Orthologs

Approach II (contd)

Gene Profiles for All Strains

V. vulnificus CMCP06

V. vulnificus YJ016

V. parahaemolyticus 0P

V. parahaemolyticus 33

V. cholerae 95

V. cholerae 61

V. splendidus 32

V. fischeri 11

V. fischeri 14

10090807060Similarity

Non-human pathogens

Pathogens

Group Average dendrogram generated from a simple matching resemblance matrix. PresenceAbsence

pathvulnonpathnav

VC61VC95

VP33VP0P

VVCMVVYJ

VV02VV03

VF11VF14

VN01VN02

VN042D Stress: 0.09

MNDS plot generated from a simple matching resemblance matrix. The dendrogram is a bit misleading about the relationship between V. splendidus and V. fischeri.

Gene Profiles for All Strains

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.

Groups R statistic Significance Level %

Pathogenic, V. vulnificus 0.487 0.6

Pathogenic, Non-Pathogenic 0.37 6

Pathogenic, V. navarrensis 0.712 1

V. vulnificus, Non-Pathogenic 1 1.8

V. vulnificus, V. navarrensis 1 0.8

Non-Pathogenic, V. navarrensis 1 2.9

Genes significantly different between V. vulnificus and V. navarrensis

PresenceAbsence

V. vulnificus CMCP6

V. vulnificus YJ016

V. parahaemolytics BB220P

V. parahaemolytics RIMD_2210633

V. cholerae O395

V. cholerae O1 biovarE1 Tor N16961

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

Genes significantly different between V. vulnificus and V. navarrensis

10090807060Similarity Hypotheticals / Conserved hypotheticals

PresenceAbsenceVN02

V. vulnificus CMCP6

V. vulnificus YJ016

V. cholerae O395

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

V. vulnificus CMCP6

V. vulnificus YJ016

V. cholerae O395

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

n-acetyl transferaseacetyl transferaseglucokinaseHeme Biosynthesis / Iron acquisitionAdhesin Chemotaxis

Missing from V.

navarrensis

Unique to V.

navarrensis

PresenceAbsence

Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups

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.

PresenceAbsence

V. vulnificus CMCP6

V. vulnificus YJ016

V. cholerae O395

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

PresenceAbsence

Transporters, transcription factors, hemolysins, exonucleases, carbohydrate metabolism (enormous gene variation)

V. vulnificus CMCP6

V. vulnificus YJ016

V. cholerae O395

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

PresenceAbsence

V. vulnificus CMCP6

V. vulnificus YJ016

V. cholerae O395

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

A subset of Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups

In yellow: Genes related to type 1 secretion, chemotaxis, permeases, proteases, and LPS synthesis (capsid polysaccharides, lipoproteins,

exopolysacharrides)

PresenceAbsence

V. vulnificus CMCP6

V. vulnificus YJ016

V. cholerae O395

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

Mostly hypotheticals (40), response regulators, glutathione synthase, starvation proteins

PresenceAbsence

V. vulnificus CMCP6

V. vulnificus YJ016

V. cholerae O395

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

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)

PresenceAbsence

V. vulnificus CMCP6

V. vulnificus YJ016

V. cholerae O395

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

Genes significantly different between the Clinical and Environmental Strains of V. navarrensis

Endonucleases (5), Channel proteins (2), chemotaxis genes (5), permeases

(2), transcriptional regulators (4), dehydratase (4)

Hypotheticals, flagellar proteins

PresenceAbsence

Drives separationVN02

V. vulnificus CMCP6

V. vulnificus YJ016

V. cholerae O395

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

A Subset of Genes significantly different between the Clinical and Environmental Strains of V. navarrensis

ATP dep. endonuclease

ChemotaxisEndonucleases

Channel proteins

Phage tail collar domainFlagellinTranscript. regulatorsdehydratase

PresenceAbsence

V. vulnificus CMCP6

V. vulnificus YJ016

V. cholerae O395

V. spendidus LGP32

V. fischeri MJ11

V. fischeri ES114

Previously Discusses Virulence Factors

Virulence Factor

Description

RTX Toxin rtxA gene encodes for the RTX toxin which is related with septicemia and gastroenteritis

Hemolysins Exotoxins that lyse erythrocyte membranes by formation of pores with the liberation of iron binding proteins (transferrin,

lactoferrin and hemoglobin).Four defined classes of Hemoylsins: TDH, TLH, δ – VPH, hlyAExperimental evidence suggests Hemolysins are involved in

disease pathogenesis.

Siderophores Low molecular weight compounds that have high affinity for iron molecules.

Studies show the association of siderophores with virulence in Vibrios.

Attachment Factors

Toxin Co-regulated Pilus (TCP) and Type IV pilus

Secretion Systems

CTX is associated with Type IIRTX is associated with Type I

Capsular Polysaccharides

•The most important virulence factor for V. vulnificus is its capsular polysaccharide (CPS).

•V. vulnificus is 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).

•Unencapsulated mutants 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).

Class Function GeneVV1

Capsular Polysaccharide

Involved in subunit transport and flanked by

direct repeat DNA sequence

Capsular polysaccharide

biosynthesis

LPS Biosynthesis

Capsular polysaccharide biosynthesis

system

Serum resistance genes Serum resistance trkA

Selected Hemolysins

Hemolysins HlyA (E1 Tor haemolysin)

family

Similar to hemolysin III of

B.sereushlyIII

Hemolysins vllY

Virulence gene regulation

Iron Acquisition

•Vibrio vulnificus pathogenecity - 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. vulnificus could be involved in acquisition of iron from heme proteins (Nishina et al., 1992; Okujo et al., 1996).

•Litwin and Calderwood (1993) cloned the V. vulnificus fur 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. vulnificus mutant for vuuA, the ferric vulnibactin receptor, could not use vulnibactin and was decreased for virulence in mice.

Iron acquisition

Central regulator in iron metabolism

Ferric vulnibactin receptor

Vulnibactin utilization protein

Siderophore synthaseVulnibactin synthase

Flagella and Motility

Encodes the flagellar basal

bodyflgC

Encodes flagellar hook protein flgE

Involved in flagellar

biosynthesisfliP

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

Class Function Gene VV1

toxin rtxA

ATP Binding cassette

transporter for rtxA

Essential acyclase of rtxA rtxC

unknown function in transport rtxD

Type 1 Secretion System

Outer membrane protein

ABC transporter hlyB

Membrane fusion protein

Type IV Pilus Adherence (Present)

RTX machinery

Some other interesting factors!

Function GeneVV1

Heme receptor hupA

DNA binding transcriptional regulator hupB

Metalloprotease vvpE

Hypothetical protein

Adherence to human epithelial cells pilD

Relating to loss in cytotoxic activity

Relating to decreased expression of Hemolysins

Autoinducer II production luxS

Conclusions

1. 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

2. Very different profile from the compared Vibrios.

3. Vibrio navarrensis is not similar to the non-human pathogenic Vibrios.

4. Blood and environmental strains of V. navarrensis are very similar.• Differences: LPS synthesis, Type-I secretion system, Permeases.

5. We still believe that these will store a similar niche in the environment.

6. Vibrios are difficult to study owing to their metabolic versatility and wide range of animal hosts.

Questions?

Comparative Genomics Final Results Ben Dan Deepak Esha Kelley Pramod Raghav Smruthy Vartika Will.

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