Vibrio Cholera
Michelle Ross, Kristin Roman, Risa Siegel
Vibrio Cholera
Micro and Molecular biology
Vibrio Cholera
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Gram-negative
Curved rod
.5-.8 μm width
1.4-2.6 μm length
Facultative anaerobe
Single polar flagellum
Chemoorganotroph
Optimal growth 20-30 degrees
V. Cholera
Gram-negative
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lipopolysaccharide coat which provides protection
against hydrophobic compounds
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provides a surface for immune recognition
Divisions of V. Cholera
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Biotype (biovar)
different strains of the same bacterial species
distinguished by a group of phenotypic or
genetic traits
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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
V. Cholera
01 serogroup
Classic
genome: 3.2-3.6 Mb
El Tor (El)
genome: 4 Mb
01 antigen is divided into 3
types: A,B,C
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A antigen
made of 3-deoxy-Lglycerotetronic acid
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B, C antigen
not been characterized
Horizontal Gene Transfer
1. acquisition of VPI
2. lysogenic conversion by phage
3. exchange of genes leads to expression of O-antigen and capsule
V. Cholera
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the 01 strain and the
recent 0139 strain have
different antigens
expressed in the
polysaccharide capsule
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the change in structure is
thought to have arisen
from a recombination
event.
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
Chromosome 1
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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
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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
Chromosome 2
The integron region is often found on plasmids and
serves as a "gene capture system." This region may
contain antibiotic resistance genes.
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
Cholera Toxin
Structure
• 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
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
Toxin Pathway Cartoon
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)
Toxin-Coregulated Pili
Efficient colonization of V. cholera in the small
intestine requires the expression of TCP’s
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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
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
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.