Vibrio CholeraVibrio Cholera

Michelle Ross, Kristin Roman, Risa Michelle Ross, Kristin Roman, Risa SiegelSiegel

Vibrio CholeraVibrio CholeraMicro and Molecular biologyMicro and Molecular biology

Vibrio CholeraVibrio Cholera

• Gram-negativeGram-negative• Curved rodCurved rod• .5-.8 .5-.8 μμm widthm width• 1.4-2.6 1.4-2.6 μμm lengthm length• Facultative anaerobeFacultative anaerobe• Single polar flagellumSingle polar flagellum• Chemoorganotroph Chemoorganotroph • Optimal growth 20-30 Optimal growth 20-30

degreesdegrees

V. CholeraV. Cholera

Gram-negativeGram-negative

• lipopolysaccharide coat which provides lipopolysaccharide coat which provides protection protection against hydrophobic against hydrophobic compounds compounds

• provides a surface for immune provides a surface for immune recognitionrecognition

Divisions of V. CholeraDivisions of V. Cholera

o Biotype (biovar)Biotype (biovar)different strains of the same bacterial speciesdifferent strains of the same bacterial speciesdistinguished by a group of phenotypic or distinguished by a group of phenotypic or genetic traitsgenetic traits

o SerogroupSerogroup

bacteria of the same species with different bacteria of the same species with different antigenic antigenic

determinants on the cell surfacedeterminants on the cell surface

V. CholeraV. Cholera has more than 150 different serogroups, has more than 150 different serogroups, only two of which cause epidemic diseaseonly two of which cause epidemic disease

V. CholeraV. Cholera01 serogroup01 serogroup

01 antigen is divided 01 antigen is divided into 3 types: A,B,Cinto 3 types: A,B,C

• A antigenA antigenmade of 3-deoxy-L-made of 3-deoxy-L-glycerotetronic acidglycerotetronic acid

• B, C antigenB, C antigen not been not been

characterizedcharacterized

ClassicClassicgenome: 3.2-3.6 Mbgenome: 3.2-3.6 Mb

El Tor (El)El Tor (El)genome: 4 Mbgenome: 4 Mb

Horizontal Gene TransferHorizontal Gene Transfer

1. acquisition of VPI1. acquisition of VPI

2. lysogenic conversion by phage2. lysogenic conversion by phage

3. exchange of genes leads to expression of O-antigen and capsule3. exchange of genes leads to expression of O-antigen and capsule

V. CholeraV. Cholera

• the 01 strain and the the 01 strain and the recent 0139 strain recent 0139 strain have different have different antigens expressed in antigens expressed in the polysaccharide the polysaccharide capsulecapsule

• the change in the change in structure is thought to structure is thought to have arisen from a have arisen from a recombination event.recombination event.

V. CholeraV. Cholera

Chromosome 1 is larger (2.96Chromosome 1 is larger (2.96

million base pairs) and million base pairs) and carriescarries

many genes for essential cell many genes for essential cell

functions and housekeeping functions and housekeeping

Chromosome 2 is smaller Chromosome 2 is smaller (about (about

1.07 million base pairs and 1.07 million base pairs and

carries the integron islandcarries the integron island

two circular two circular chromosomeschromosomes

Chromosome 1Chromosome 1• carries many genes for essential cell functions and housekeeping. It carries many genes for essential cell functions and housekeeping. It

also contains important virulence genes, most of which have been also contains important virulence genes, most of which have been acquired by lateral gene transfer from other species acquired by lateral gene transfer from other species

• chromosome one carries chromosome one carries two bacteriophagestwo bacteriophages. .

ONE VIRUSONE VIRUS is called the V. cholera pathogenicity island phage is called the V. cholera pathogenicity island phage

((VPIVPI), which infects and inserts its DNA into the bacterial), which infects and inserts its DNA into the bacterial

chromosome and allows the synthesis of a chromosome and allows the synthesis of a piluspilus which which

the bacteria uses to attach to the host intestine the bacteria uses to attach to the host intestine

SECOND VIRUS is called the cholera-toxin phage (SECOND VIRUS is called the cholera-toxin phage (CTXCTX). ).

The CTX phage inserts itself into chromosome The CTX phage inserts itself into chromosome

one and the bacterium is then capable of secreting one and the bacterium is then capable of secreting

a powerful a powerful enterotoxinenterotoxin

Chromosome 2Chromosome 2

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

Pathogenesis of Pathogenesis of V. CholeraV. Cholera

Cholera disease begins with ingestion of Cholera disease begins with ingestion of contaminated water or food. The bacteria that contaminated water or food. The bacteria that survive the acidic conditions of the stomach survive the acidic conditions of the stomach colonize in the small intestine. colonize in the small intestine.

The cholera toxin (CT) is responsible for the severe The cholera toxin (CT) is responsible for the severe diarrhea characteristic of the disease.diarrhea characteristic of the disease.

Cholera ToxinCholera Toxin

CT is a proteinaceous enterotoxin secreted by CT is a proteinaceous enterotoxin secreted by

V. CholeraV. Cholera

Cholera ToxinCholera Toxin

StructureStructure• Composed of a AB subunit. Composed of a AB subunit.

The B subunit forms a The B subunit forms a pentameric “doughnut” like pentameric “doughnut” like structure that binds the CT to structure that binds the CT to the receptor on the eukaryotic the receptor on the eukaryotic cellscells

PathwayPathway• The A subunit contains the The A subunit contains the

enzymatically active portion or enzymatically active portion or the toxinthe toxin

• Proteolytic cleavage of the A Proteolytic cleavage of the A subunit results in Asubunit results in A11 and A and A2 2

peptide units which remain peptide units which remain linked by a disulfide bondlinked by a disulfide bond

• Once the A subunit is Once the A subunit is internalized by the eukaryotic internalized by the eukaryotic cell, the disulfide bond is cell, the disulfide bond is reducedreduced

Pathway continuedPathway continued

The AThe A11 subunit contains a ADP-ribosyltransferase subunit contains a ADP-ribosyltransferase which covalently modifies the G protein, which which covalently modifies the G protein, which regulates adenylate cyclase. Adenylate cyclase regulates adenylate cyclase. Adenylate cyclase mediates the formation of cAMPmediates the formation of cAMP

The increase in cAMP levels bring about the The increase in cAMP levels bring about the secretion of chloride and bicarbonate from the secretion of chloride and bicarbonate from the mucosal cells into the intestinal lumenmucosal cells into the intestinal lumen

The change in ion concentrations leads to the The change in ion concentrations leads to the secretion of large amounts of water into the secretion of large amounts of water into the lumen, known as diarrhealumen, known as diarrhea

Toxin Pathway CartoonToxin Pathway Cartoon

Genetics of Cholera ToxinGenetics of Cholera Toxin

Genes encoding CTGenes encoding CTctxctxAB - recognized to be AB - recognized to be the genome of a the genome of a filamentous phage CTXfilamentous phage CTXΦΦ ((ctxctxA and A and ctxctxB)B)

Transcription of Transcription of ctxctxAB is AB is regulated by several regulated by several proteins proteins

CTXCTXΦΦ genome can integrate genome can integrate into the host genome at a into the host genome at a specific site, specific site, attattRSRS

The CTX genetic element also The CTX genetic element also has a “core” region has a “core” region carrying several phage carrying several phage morphogenesis genesmorphogenesis genes

These entire CTX gene set is These entire CTX gene set is flanked repeated flanked repeated sequences, the attRS1 sitesequences, the attRS1 site

The entire genetic element is The entire genetic element is 6.9kb6.9kb

The receptor for CTXThe receptor for CTXΦΦ is Toxin-Coregulated Pili (TCP) is Toxin-Coregulated Pili (TCP)

Toxin-Coregulated PiliToxin-Coregulated Pili

Efficient colonization of Efficient colonization of V. choleraV. cholera in the small in the small intestine requires the expression of TCP’sintestine requires the expression of TCP’s

• TCP’s are expressed on the surface of TCP’s are expressed on the surface of V. V. choleracholera

• TCP’s are long laterally associated filamentsTCP’s are long laterally associated filaments

The major pilin subunit is TcpAThe major pilin subunit is TcpA

Genes for TCP production are clustered on the Genes for TCP production are clustered on the pathogenicity island located on chromosome 2pathogenicity island located on chromosome 2

Regulation of Virulence FactorsRegulation of Virulence Factors

Expression of CT & TCP have been shown Expression of CT & TCP have been shown in in vitro to be strongly influenced vitro to be strongly influenced by changes in cultural conditionsby changes in cultural conditions

ie. temperature, pH, osmolarity, & ie. temperature, pH, osmolarity, & growth medium compositiongrowth medium composition

CT & TCP are regulated via a cascade in CT & TCP are regulated via a cascade in which ToxR and TcpP control expression which ToxR and TcpP control expression of ToxT, which is a transcriptional activator of ToxT, which is a transcriptional activator directly controlling several virulence genesdirectly controlling several virulence genes

ToxR & TcpP are inner-membrane proteins ToxR & TcpP are inner-membrane proteins which interact with other transmembrane which interact with other transmembrane regulatory proteinsregulatory proteins

ToxR/S proteins are required for transcription of toxT gene and are also ToxR/S proteins are required for transcription of toxT gene and are also important for important for ctxctx transcription and regulation other outer membrane transcription and regulation other outer membrane proteinsproteins

Recap of phage movementRecap of phage movement

V. cholerae did not always cause disease. Infection V. cholerae did not always cause disease. Infection with the CTX phage gives the bacterium its with the CTX phage gives the bacterium its toxinogenicity. The phage recognizes a pilus on the toxinogenicity. The phage recognizes a pilus on the surface of the bacterium and uses it to enter the surface of the bacterium and uses it to enter the cell. Once inside the cell, the CTX phage integrates cell. Once inside the cell, the CTX phage integrates into the chromosome and the lysogen expresses into the chromosome and the lysogen expresses cholera toxin.cholera toxin.

The CTX phage has received special attention because The CTX phage has received special attention because it is the first filamentous phage found to transfer it is the first filamentous phage found to transfer toxin genes to its host. The important lesson from toxin genes to its host. The important lesson from this discovery is that many different types of phage this discovery is that many different types of phage may carry virulence factors, and transfer of may carry virulence factors, and transfer of virulence genes by phage may be a major virulence genes by phage may be a major mechanism of evolution of new bacterial diseases.mechanism of evolution of new bacterial diseases.