Comparative Genomics:

Vibrio spp.

ICGEB Workshop Group 4 Albert Auguste, Reia Guppy, Amalia Hosein, Andrés López, Maria Jose Muňoz, Silvia Vasquez, Jerome Foster

Study organism

 Vibrio spp. of anaerobic bacteria (curved rods)  > 70 species, across 4 families  symbiotic, communalistic, pathogenic  at least 12 are important human pathogens (Vibrionaceae)

Study organism

 Pathogenic species are major causative agents of several clinical illnesses in humans (gastroenteritis , wound infections w/ septicaemia)  Transmission usually by consumption of raw/uncooked shellfish, or exposure to contaminated or warm sea water  E.g. V. cholerae, V. parahaemolyticus, V. vulnificus

Study organism

 Invasive infections such as that of V. cholerae may rapidly progress to death  Patients w/ liver malfunction are at high morbidity & mortality risk (e.g. V. vulnificus)  CDC, 2008: 131 per 100,00 lab-confirmed infections of Vibrio spp. in the U.S.

Study organism

 Other Vibrio spp. are marine pathogens e.g.

 V. Harveyi - pathogen of fish and invertebrates, including sharks, sea bass, seahorses, lobster, and shrimp  V. splendidus inflicts disease and death in many marine species including fish, oysters, mussels, and scallops  Non-pathogenic include V. fischeri,

V. logei

Vibrio genome organization

Project Objectives

 Compare nonpathogenic vs. pathogenic Vibrio

spp.

 similarities?

  unique traits?

genome organization?

Methodology

 Pairwise comparison between Vibrio spp. using ACT (Artemis Comparison Tool; Carver et al., 2005 ) 

V. fischeri as a “reference” species for all

comparisons   basal in taxonomy & phylogeny* of Vibrio spp to other available complete genomes (based on 16s rRNA; Thompson et al., 2009) non-pathogenic in both humans & marine life

Methodology

 Dataset of eight (8) complete & published Vibrio genomes each with 2 chromosomes ( I and II)  WebACT used to generate sequence & blast comparisons (blastn only*)  ACT used on local server for actual comparative analyses

Methodology

 Total of 14 ACT runs (7 x 2 chromosomes), V. fischeri

(MJ11) vs.

      

V. cholerae (M66_2) V. cholerae (MJ_1236) V. harveyi (ATCC_BAA_1116) V. parahaemolyticus (RIMD 2210633) V. vulnificus (YJ016) V. Sp. Ex25 V. splendidus (LGP32)

R ESULTS

ACTs for Chromosome I

V. harveyi V. sp Ex25 V. cholerae MJ66 V. splendidus V. vulnificus V. cholerae MJ1236 V. parahaemolyticus

ACTs for Chromosome II

V. harveyi V. sp Ex25 V. cholerae MJ66 V. vulnificus V. cholerae MJ 1236 V. parahaemolyticus V.splendidus

Selected genes for comparison

 TCP – encodes pili protein (pathogenicity island)  toxR – virulence regulation  rtxA – modulation of toxicity  ompU – membrane protein assoc. with virulence  Pili– genes associated with pili formation (responsible for attachment to epithelium)  PutA – part of PutAB operon (osmotic tolerance)

Strain

Vibrio fischeri V. Cholerae (MJ1236) V. harveyi** V. parahaemolyticus V. vulnificus V. sp Ex25 V. splendidus V. cholerae (MJ66)

-

TCP

1 1 2 8 8 9

Pili

5 8 6 8

ompU Genes rtxA

1 1 1 1 1 1 1 1* 1 5 1 1 -

toxR

1 1 1 TABLE 1. Select genes for pathogenesis and virulence (chromosome 1) -

PutA

-

Strain

Vibrio fischeri V. Cholerae (MJ1236) V. harveyi** V. parahaemolyticus V. vulnificus V. sp Ex25 V. splendidus V. cholerae (MJ66)

-

TCP

1 1 2 8 8 9

Pili

5 8 6 8

ompU Genes rtxA

1 1 1 1 1 1 1 1* 1 5 1 1 -

toxR

1 1 1 TABLE 1. Select genes for pathogenesis and virulence (chromosome 1) -

PutA

-

Strain

Vibrio fischeri V. Cholerae (MJ1236) V. harveyi** V. parahaemolyticus V. vulnificus V. sp Ex25 V. splendidus V. cholerae (MJ66)

-

TCP

1 1 2 8 8 9

Pili

5 8 6 8

ompU Genes rtxA

1 1 1 1 1 1 1 1* 1 5 1 1 -

toxR

1 1 1 TABLE 1. Select genes for pathogenesis and virulence (chromosome 1) -

PutA

-

Strain

Vibrio fischeri V. Cholerae (MJ1236) V. harveyi** V. parahaemolyticus V. vulnificus V. Sp Ex25 V. splendidus V. cholerae (MJ66)

-

TCP

2 3 1` 2 -

Pili

1 1 -

ompU Genes rtxA

2 -

toxR

1 -

PutA

1 1 TABLE 2. Select genes for pathogenesis and virulence (chromosome 2)

Strain

Vibrio fischeri V. Cholerae (MJ1236) V. harveyi** V. parahaemolyticus V. vulnificus V. Sp Ex25 V. splendidus V. cholerae (MJ66)

-

TCP

2 3 1` 2 -

Pili

1 1 -

ompU Genes rtxA

2 -

toxR

1 -

PutA

1 1 TABLE 2. Select genes for pathogenesis and virulence (chromosome 2)

CDSs

(at 100% Identity)

showing reassortment (V. Fischeri

vs

V. Cholerae M66)

Chrom. 2: Bifunctional protein PutA, absent in

V. sp Ex25 (possibly reassorted & pseudogeneized?)

Limitations

 Only pairwise comparisons possible  time constraints  Inexperience with software e.g.

  CDS key queries - “PutA” vs “puta” Tblastx run error (WebACT) – only Blastn used

C ONCLUSIONS  Seems to be numerous reassortments among Vibrio spp. in comparison to V. fischeri  Genomes very similar but “messy” (reassortments, duplicated stretches of sequence)  Common gene products across all species, e.g. pilus associated genes  Toxin genes maintained across pathogenic species  Group needs more practice in analyzing this kind of data (steep learning curve)!!

A CKNOWLEDGEMENTS  All the module instructors (esp. Dr. Pain!)  Organizers @ UWI, UNU-BIOLAC & ICGEB