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