Comparative GenomicsFinal Results
BenDan
DeepakEsha
KelleyPramodRaghav
SmruthyVartika
Will
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??
VN3
VN2
VN4
VN1
VV1
VV4
VV2
VV3
VV5
Vp1
0.02
100% Bootstrap support >0.98 posterior probability support
98.35
97.13
95.58
98.60
97.76
82.64
81.94
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.
98.88
98.92
Whole genome super matrix tree
VV1
VV2
VN4
VN2
Vibrio_vulnificus_CMCP6
Vibrio_vulnificus_YJ016
VN1
VV4
VV5
VN3
VV3
Vibrio_parahaemolyticus
95
74
11
12
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
VN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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
VV01
VV02VV03
VV04
VV05
VF11VF14
VS32
VN01VN02
VN03
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
10090807060Similarity
PresenceAbsence
VN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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
10090807060Similarity
VN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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
Bio
film
Che
mot
axis
CPS
Pilli
PTS
rtxC
Vib
riob
actin
rela
ted
Type
1 &
2 S
ecre
tion
Slim
e bi
osyn
TonB
Toxi
n re
late
dAnt
ibio
tic
res
Sim
ple
suga
r upt
ake
LPS
n-acetyl transferaseacetyl transferaseglucokinaseHeme Biosynthesis / Iron acquisitionAdhesin Chemotaxis
Missing from V.
navarrensis
Unique to V.
navarrensis
PresenceAbsence
10090807060Similarity
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
VN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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 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.
10090807060Similarity
PresenceAbsence
Transporters, transcription factors, hemolysins, exonucleases, carbohydrate metabolism (enormous gene variation)
VN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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 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.
10090807060Similarity
PresenceAbsence
VN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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
10090807060Similarity
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
VN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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
A subset of Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups
Mostly hypotheticals (40), response regulators, glutathione synthase, starvation proteins
10090807060Similarity
PresenceAbsence
VN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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
A subset of Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups
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)
10090807060Similarity
PresenceAbsence
VN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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 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
10090807060Similarity
Drives separationVN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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
10090807060Similarity
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
VN02
VN03
VN04
VN01
VV05
VV04
VV03
VV02
VV01
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
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
VV2
VV3
VV4
VV5
VN1
VN2
VN3
VN4
Capsular Polysaccharide
Involved in subunit transport and flanked by
direct repeat DNA sequence
wzb
wzc
Capsular polysaccharide
biosynthesis
LPS Biosynthesis
Capsular polysaccharide biosynthesis
Capsular polysaccharide biosynthesis
system
Serum resistance genes Serum resistance trkA
Selected Hemolysins
Class Function GeneVV1
VV2
VV3
VV4
VV5
VN1
VN2
VN3
VN4
Hemolysins HlyA (E1 Tor haemolysin)
family
vvhA
vvhB
Similar to hemolysin III of
B.sereushlyIII
Hemolysins vllY
Virulence gene regulation
hlyU
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.
Class Function GeneVV1
VV2
VV3
VV4
VV5
VN1
VN2
VN3
VN4
Iron acquisition
Central regulator in iron metabolism
fur
Ferric vulnibactin receptor
vuuA
Vulnibactin utilization protein
viuB
Siderophore synthaseVulnibactin synthase
Flagella and Motility
Class Function GeneVV1
VV2
VV3
VV4
VV5
VN1
VN2
VN3
VN4
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
VV2
VV3
VV4
VV5
VN1
VN2
VN3
VN4
RTX
toxin rtxA
ATP Binding cassette
transporter for rtxA
rtxB
Essential acyclase of rtxA rtxC
unknown function in transport rtxD
Type 1 Secretion System
Outer membrane protein
tolC
ABC transporter hlyB
Membrane fusion protein
hlyD
Type IV Pilus Adherence (Present)
RTX machinery
Some other interesting factors!
Function GeneVV1
VV2
VV3
VV4
VV5
VN1
VN2
VN3
VN4
Heme receptor hupA
DNA binding transcriptional regulator hupB
Metalloprotease vvpE
Hypothetical protein
vvp15
vvp22
vvp28
Adherence to human epithelial cells pilD
Relating to loss in cytotoxic activity
purH
pyrH
Relating to decreased expression of Hemolysins
toxR
toxS
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?
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