1. Vibrio Cholera
Michelle Ross, Kristin Roman, Risa Siegel

2. Micro and Molecular biology
Vibrio Cholera
Micro and Molecular biology

3. Vibrio Cholera Gram-negative Curved rod .5-.8 μm width
μm length
Facultative anaerobe
Single polar flagellum
Chemoorganotroph
Optimal growth degrees

4. V. Cholera Gram-negative
lipopolysaccharide coat which provides protection against hydrophobic compounds
provides a surface for immune recognition

5. Divisions of V. Cholera
Biotype (biovar)
different strains of the same bacterial species
distinguished by a group of phenotypic or genetic traits
Serogroup
bacteria of the same species with different antigenic
determinants on the cell surface
V. Cholera has more than 150 different serogroups, only two of which cause epidemic disease

6. V. Cholera 01 serogroup A antigen B, C antigen Classic
genome: Mb
El Tor (El)
genome: 4 Mb
01 antigen is divided into 3 types: A,B,C
A antigen
made of 3-deoxy-L-glycerotetronic acid
B, C antigen
not been characterized

7. Horizontal Gene Transfer
1. acquisition of VPI
2. lysogenic conversion by phage
3. exchange of genes leads to expression of O-antigen and capsule

8. V. Cholera
the 01 strain and the recent 0139 strain have different antigens expressed in the polysaccharide capsule
the change in structure is thought to have arisen from a recombination event.

9. V. Cholera two circular chromosomes Chromosome 1 is larger (2.96
million base pairs) and carries
many genes for essential cell
functions and housekeeping
Chromosome 2 is smaller (about
1.07 million base pairs and
carries the integron island

10. Chromosome 1
carries many genes for essential cell functions and housekeeping. It also contains important virulence genes, most of which have been acquired by lateral gene transfer from other species
chromosome one carries two bacteriophages.
ONE VIRUS is called the V. cholera pathogenicity island phage
(VPI), which infects and inserts its DNA into the bacterial
chromosome and allows the synthesis of a pilus which
the bacteria uses to attach to the host intestine
SECOND VIRUS is called the cholera-toxin phage (CTX).
The CTX phage inserts itself into chromosome
one and the bacterium is then capable of secreting
a powerful enterotoxin

11. Chromosome 2
The integron region is often found on plasmids and serves as a "gene capture system." This region may contain antibiotic resistance genes.

12. Pathogenesis of V. Cholera
Cholera disease begins with ingestion of contaminated water or food. The bacteria that survive the acidic conditions of the stomach colonize in the small intestine.
The cholera toxin (CT) is responsible for the severe diarrhea characteristic of the disease.
Cholera Toxin
CT is a proteinaceous enterotoxin secreted by
V. Cholera

13. Cholera Toxin Structure Pathway
Composed of a AB subunit. The B subunit forms a pentameric “doughnut” like structure that binds the CT to the receptor on the eukaryotic cells
Pathway
The A subunit contains the enzymatically active portion or the toxin
Proteolytic cleavage of the A subunit results in A1 and A2 peptide units which remain linked by a disulfide bond
Once the A subunit is internalized by the eukaryotic cell, the disulfide bond is reduced

14. Pathway continued
The A1 subunit contains a ADP-ribosyltransferase which covalently modifies the G protein, which regulates adenylate cyclase. Adenylate cyclase mediates the formation of cAMP
The increase in cAMP levels bring about the secretion of chloride and bicarbonate from the mucosal cells into the intestinal lumen
The change in ion concentrations leads to the secretion of large amounts of water into the lumen, known as diarrhea

15. Toxin Pathway Cartoon

16. Genetics of Cholera Toxin
Genes encoding CT
ctxAB - recognized to be the genome of a filamentous phage CTXΦ (ctxA and ctxB)
Transcription of ctxAB is regulated by several proteins
CTXΦ genome can integrate into the host genome at a specific site, attRS
The CTX genetic element also has a “core” region carrying several phage morphogenesis genes
These entire CTX gene set is flanked repeated sequences, the attRS1 site
The entire genetic element is 6.9kb
The receptor for CTXΦ is Toxin-Coregulated Pili (TCP)

17. Toxin-Coregulated Pili
Efficient colonization of V. cholera in the small intestine requires the expression of TCP’s
TCP’s are expressed on the surface of V. cholera
TCP’s are long laterally associated filaments
The major pilin subunit is TcpA
Genes for TCP production are clustered on the pathogenicity island located on chromosome 2

18. Regulation of Virulence Factors
Expression of CT & TCP have been shown in vitro to be strongly influenced by changes in cultural conditions
ie. temperature, pH, osmolarity, &
growth medium composition
CT & TCP are regulated via a cascade in
which ToxR and TcpP control expression
of ToxT, which is a transcriptional activator
directly controlling several virulence genes
ToxR & TcpP are inner-membrane proteins
which interact with other transmembrane
regulatory proteins
ToxR/S proteins are required for transcription of toxT gene and are also important for ctx transcription and regulation other outer membrane proteins

19. Recap of phage movement
V. cholerae did not always cause disease. Infection with the CTX phage gives the bacterium its toxinogenicity. The phage recognizes a pilus on the surface of the bacterium and uses it to enter the cell. Once inside the cell, the CTX phage integrates into the chromosome and the lysogen expresses cholera toxin.
The CTX phage has received special attention because it is the first filamentous phage found to transfer toxin genes to its host. The important lesson from this discovery is that many different types of phage may carry virulence factors, and transfer of virulence genes by phage may be a major mechanism of evolution of new bacterial diseases.