comparative genomics preliminary results ben dan deepak esha kelly pramod raghav smruthy vartika...
TRANSCRIPT
Comparative GenomicsPreliminary Results
BenDan
DeepakEshaKelly
PramodRaghav
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?”
SPECIATION??
Draft GenomeGene
PredictionsTranslated
Genes
ANI ANI
Dendrogram
Dendrogram
Identifying Core
Genome
OrthoMCLCustom Script
Multiple Alignment
Super TreeSuperMatrix
Consensus Tree
Strategy for Defining Species
New Species?? PHYLIP
MEGA
PAUP
Mr Bayes
ClustalΩ
MUSCLE
ANI Results : WHOLE GENOME
VN1 VN2 VN3 VN4 VV1 VV2 VV3 VV4
VN1
VN2 96.10
VN3 97.72 96.11
VN4 98.05 95.32 95.28
VV1 77.93 77.97 77.53 78.02
VV2 76.89 76.13 76.43 76.11 98.36
VV3 77.50 76.38 77.04 76.80 98.72 98.74
VV4 76.10 76.31 76.22 75.98 98.41 98.10 98.72
VV5 76.06 76.44 76.07 76.16 97.25 96.87 97.23 97.28
ANI : whole genome tree
VV5
VV4
VV1
VV2
VV3
VN2
VN3
VN1
VN4
1009590858075ANI Similarity
Group Average Dendrogram generated using ANI distance
Correlation b/w ANI tree and Three - gene tree
VN1
VN4
VN2
VN3VV1
VV3VV4
VV2
VV5
SuperMatrix Whole 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
VN4
VN1
VN2
VN3
VV5
VV1
VV2
VV4
VV3
Vp1
0.02
SuperMatrix Whole Genome Tree (BOOTSTRAP)
VN1
VN4
VN2
VN3
VV1
VV3
VV4
VV2
VV5
Vp1
100
100
100
60
100
100
100
Supertree Whole Genome Tree
VN1
VN4
VN2
VN3
VV5
VV1
VV2
VV3
VV4
39
50
93
26
22
31
95
VP1
Supertree generated by taking a majority rule consensus tree from 1398 gene treesOrthologous 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
Preliminary Conclusions
• Tentative results suggest that all four strains in V. navarrensis belong 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
PATHOGENECITY??
CTX – Cholera Toxin
• It is in the genome of lysogenic bacteriophage (CTXPhi) and is carried by Vibrio cholerae
• Causes the major symptoms of infection: diarrhea, vomiting and cramps.
• Ctx + zot + ace form a V. cholerae virulence cassette
• Ctx genes code for cholera toxin
• zot causes decrease in intestinal tissue resistance by modifying intercellular tight junctions.
• ace causes increases in fluid secretion in ligated illeal loops
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
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 hemolysins extend to other cells such as neutrophils, mast cells and enhances virulence by causing tissue damage.
Four defined classes of Hemoylsins
Experimental evidence suggests“Hemolysins are involved in disease pathogenesis”
(Zhang et.al 2005)
TDH family(Thermostab
le Direct Hemolysin)
HlyA family (El Tor
Hemolysin)
TLH family(Thermolabile Hemolysin)
δ – VPH family
(Thermostable
Hemolysin)
Bacterial Taxon Hemolysin Family
Specific Hemolysin
V. cholerae O1 HlyA HlyA
TLH Lecithinase LEC
δ – VPH Vc- δTH
V. cholerae non-O1 HlyA HlyA
TDH NAG-TDH
V. vulnificus HlyA VVH
TLH VPL
- hly III
- vll Y
V. parahaemolyticus TLH TLH or LDH
δ – VPH δ – VPH
TDH Vp-TRH/Vp-TDH
(Zhang et.al 2005)
Siderophores
• Low molecular weight compounds that have high affinity for iron molecules.
• Chelates iron ions in the environment whereupon the ferri-siderophores re-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-Bonifacio et al 2013)
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 Msha pilus plays role in adherence
– PilA and pil D plays role in V. vulnificus adherence to human epithelial cells
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
SECRETION SYSTEMS
Pathogenicity Islands
• Vibrio seventh pandemic islands VSP1 ,VSP 2
• Genes encode hypothetical functions presumed to be necessary for evolutionary fitness
VPI 2
• A 57.3 kb gene cluster encoding genes for neuraminidase (nanH) and amino sugar metabolism, which has the characteristic features of a pathogenicity island
• These pathogenic islands are present in V. cholerae strains
• Presence of these islands have been used to elucidate the pathogenicity of Vibrio species like V.mimicus.
Annotated Dataset
Existence of
Toxins
Machinery for Incorporation
(Pili/Attachment
Factors)
Presence Absence
Machinery for Incorporation
(Pili/Attachment
Factors)
Potentially Pathogenic
Unlikely Pathogenic
Yes No
Correlation with Pathway(KEGG)
Pathogenicor
Putatively Pathogenic
Connecting the dots
Strategy for Pathogenicity
Blood isolate VN1 Hemolysin Check
Hemolysins (HlyU,vvhA,hlyIII)
Siderophores (TonB-dependent siderophore receptor)
Serum resistance genes (BrkB)
Effect(probably septicemia)
Presence of RTX (RTX toxin, rtxD one of the genes in RTX cluster)
Attachment (Type IV pilus)
Secretion System (Type I)
Effect(Septicemia and Gastroenteritis??)
Blood isolate VN1 RTX MACHINERY
Blood isolate VN1 CTX RELATED MACHINERY
Presence of CTX (absent)
Attachment factors(TCP)
Secretion System (Type II)
Effect
Х
Х
Preliminary Results : VV2Haemolysins (TLH, TDH, vllY)
Siderophores (TonB-dependent siderophore receptor)
Serum resistance genes (BrkB)
Effect( Septicemia)
Х
Presence of RTX(Rtx1 b, RTX Toxin, rtxC)
Attachment(Type IV pilus)
Secretion System (Type I)
Effect (Septicemia and Gastroenteritis )
Presence of CTX (absent)
Attachment factors(TCP)Secretion System (Type II)
Effect
Х
Х
Hemolysin Check
RTX MACHINERY CTX RELATED MACHINERY
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. navarrensis is able to integrate plasmid containing rtx gene cluster.
• CTX - Absent lack the TCP genes
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