Vibrio%20Cholera

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

• Micro and Molecular biology

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Vibrio Cholera

• 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

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V. Cholera

• Gram-negative
• lipopolysaccharide coat which provides
  protection against hydrophobic compounds
• provides a surface for immune recognition

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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

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V. Cholera01 serogroup
Classic genome 3.2-3.6 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

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

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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

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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

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

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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

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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

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Toxin Pathway Cartoon
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Genetics of Cholera Toxin

• Genes encoding CT
• ctxAB - recognized to be the genome of a
  filamentous phage CTXF (ctxA and ctxB)
• Transcription of ctxAB is regulated by several
  proteins
• CTXF 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 CTXF is Toxin-Coregulated Pili
(TCP)
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Toxin-Coregulated Pili

• Efficient colonization of V. cholera in the small
  intestine requires the expression of TCPs
• TCPs are expressed on the surface of V. cholera
• TCPs 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
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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

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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.