Download Comparitive Genomics- Final Results

Comparative Genomics
Final Results
Ben
Dan
Deepak
Esha
Kelley
Pramod
Raghav
Smruthy
Vartika
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??
Whole genome super matrix tree
VN3
97.13
100% Bootstrap support
>0.98 posterior probability support
VN2
95.58
VN4
98.35
82.64
VN1
VV1
98.60
98.88
97.76
81.94
98.92
VV4
VV2
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.
16S rRNA Tree
12
VV1
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 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
PATHOGENECITY??
Approach I
Approach II
Annotated
Dataset
Gene Predictions
Reference Strains
Annotation Files
from NCBI
Existence
of Toxins
Presence
Absence
Machinery for
Incorporation
Machinery for
Incorporation
Yes
Correlation with
Pathway
(KEGG)
Connecting the dots
Pathogenic
or
Putatively Pathogenic
OrthoMCL
Potentially
Pathogenic
Generation of
presence-absence
matrix
Test for group
significance
(ANOSIM test)
No
Unlikely
Pathogenic
ID genes
associated with
groups
(SIMPER test)
Heatmaps in R to view
gene profiles
Different
combos of
files
Approach II (contd)
Gene
files
Blast
Parser
Upload
parsed data
to Database
PreProcessing
All v/s All
Blast
Find Protein
Pairs
Filter Fasta
BlastDb
Markov
Clustering
Cluster of
Orthologs
Gene Profiles for All Strains
V. fischeri 14
V. fischeri 11
V. splendidus 32
Non-human
pathogens
V. cholerae 61
V. cholerae 95
V. parahaemolyticus 33
V. parahaemolyticus 0P
Pathogens
V. vulnificus YJ016
V. vulnificus CMCP06
VV01
VV02
VV03
VV04
VV05
VN01
VN04
VN03
VN02
60
70
80
90
Similarity
100
Group Average dendrogram generated from a simple matching resemblance matrix.
Presence
Absence
Gene Profiles for All Strains
VV05
VV04
VN04
VN02
VV02
VN01
VV01
VN03
2D Stress: 0.09
VVYJ
VVCM
VC61
VC95
VV03
path
vul
nonpath
nav
VS32
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.
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
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN04
VN03
VN02
60
70
80
90
Similarity
100
Presence
Absence
Genes significantly different between V. vulnificus and V. navarrensis
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN04
VN03
VN02
60
70
80
90
Similarity
Presence
Absence
100
Hypotheticals / Conserved hypotheticals
Presence
Absence
Missing from
V. navarrensis
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN04
VN03
VN02
60
70
80
90
Similarity
100
Unique to
V. navarrensis
n-acetyl transferase
acetyl transferase
glucokinase
Heme Biosynthesis
/ Iron acquisition
Adhesin
Chemotaxis
Genes significantly enriched in a priori defined “Pathogens” and
“Non-pathogens” Groups
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN04
VN03
VN02
60
70
80
90
Similarity
100
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.
Genes significantly enriched in a priori defined “Pathogens” and
“Non-pathogens” Groups
Transporters, transcription factors, hemolysins, exonucleases, carbohydrate metabolism (enormous gene variation)
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN04
VN03
VN02
60
70
80
90
Similarity
100
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.
Genes significantly enriched in a priori defined “Pathogens” and
“Non-pathogens” Groups
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN04
VN03
VN02
60
70
80
90
Similarity
100
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.
A subset of Genes significantly enriched in a priori defined “Pathogens”
and “Non-pathogens” Groups
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN04
VN03
VN02
60
70
80
90
Similarity
100
Presence
Absence
In yellow: Genes related to type 1 secretion, chemotaxis, permeases, proteases,
and LPS synthesis (capsid polysaccharides, lipoproteins, exopolysacharrides)
A subset of Genes significantly enriched in a priori defined “Pathogens” and
“Non-pathogens” Groups
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN04
VN03
VN02
60
70
80
90
Similarity
100
Presence
Absence
Mostly hypotheticals (40), response regulators, glutathione synthase, starvation proteins
A subset of Genes significantly enriched in a priori defined “Pathogens”
and “Non-pathogens” Groups
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN04
VN03
VN02
60
70
80
90
Similarity
100
Presence
Absence
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)
Genes significantly different between the
Clinical and Environmental Strains of V. navarrensis
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN02
Drives separation
VN03
VN04
60
70
80
90
Similarity
100
Endonucleases (5), Channel proteins (2),
chemotaxis genes (5), permeases (2),
transcriptional regulators (4), dehydratase (4)
Hypotheticals, flagellar
proteins
Presence
Absence
60
A Subset of Genes significantly different between the
Clinical and Environmental Strains of V. navarrensis
V. fischeri ES114
V. fischeri MJ11
V. spendidus LGP32
V. cholerae O1 biovarE1 Tor N16961
V. cholerae O395
V. parahaemolytics RIMD_2210633
V. parahaemolytics BB220P
V. vulnificus YJ016
V. vulnificus CMCP6
VV01
VV02
VV03
VV04
VV05
VN01
VN02
VN03
VN04
70
80
90
Similarity
100
ATP dep. endonuclease
Channel proteins
Chemotaxis
Endonucleases
Phage tail collar domain
Flagellin
Transcript. regulators
dehydratase
Presence
Absence
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, hlyA
Experimental 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 II
RTX 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
Capsular
Polysaccharide
Function
Involved in subunit transport
and flanked by direct repeat
DNA sequence
Gene
wzb
wzc
LPS Biosynthesis
Capsular
polysaccharide
biosynthesis
Serum resistance
genes
Capsular polysaccharide
biosynthesis
Capsular
polysaccharide
biosynthesis
system
Serum resistance
trkA
V V V V V V V V V
V V V V V N N N N
1 2 3 4 5 1 2 3 4
Selected Hemolysins
Class
Hemolysins
Function
HlyA (E1 Tor
haemolysin) family
Gene
vvhA
vvhB
Similar to hemolysin
III of B.sereus
hlyIII
Hemolysins
vllY
Virulence gene
regulation
hlyU
V V V V V V V V V
V V V V V N N N N
1 2 3 4 5 1 2 3 4
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
Iron acquisition
Function
Gene
Central regulator in iron
metabolism
fur
Ferric vulnibactin receptor
vuuA
Vulnibactin utilization
protein
viuB
Siderophore synthase
Vulnibactin
synthase
V V V V V V V V V
V V V V V N N N N
1 2 3 4 5 1 2 3 4
Flagella and Motility
Class
Flagella and
Motility
Function
Gene
Encodes the
flagellar basal body
flgC
Encodes flagellar
hook protein
flgE
Involved in flagellar
biosynthesis
fliP
V V V V V V V V V
V V V V V N N N N
1 2 3 4 5 1 2 3 4
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
RTX machinery
Class
RTX
Type 1 Secretion
System
Type IV Pilus
Function
Gene
toxin
rtxA
ATP Binding
cassette transporter
for rtxA
rtxB
Essential acyclase of
rtxA
rtxC
unknown function
in transport
rtxD
Outer membrane
protein
tolC
ABC transporter
hlyB
Membrane fusion
protein
hlyD
Adherence
(Present)
V
V
1
V
V
2
V
V
3
V
V
4
V
V
5
V
N
1
V
N
2
V
N
3
V
N
4
Some other interesting factors!
Function
Gene
Heme receptor
hupA
DNA binding transcriptional
regulator
hupB
Metalloprotease
vvpE
vvp15
Hypothetical protein
Adherence to human epithelial
cells
vvp22
vvp28
pilD
Relating to loss in cytotoxic
activity
purH
Relating to decreased
expression of Hemolysins
toxR
Autoinducer II production
luxS
pyrH
toxS
V
V
1
V
V
2
V
V
3
V
V
4
V
V
5
V
N
1
V
N
2
V
N
3
V
N
4
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?