Lecture 1 (Cholera).ppt 507.ppt

MPG 507 Bacterial Pathogenesis
& Molecular Epidemiology
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Lecture 1: Cholera (Vibrio cholerae)
Microbiology Department
Primeasia University

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Morphology
– The vibrio cholerae is a short, slightly curved rod about 1.5 μm x 0.2-0.4 μm in size
– motile, with a single polar flagellum.
– stain readily with aniline dyes and are Gram negative and non-acid fast
Cultural characteristics
– The cholera vibrions is strongly aerobic.
– It grows within a temperature range of 16-40 °C (optimum 37 °C).
– Growth is better in an alkaline medium the range of pH being 6.4-9.6 (optimum 8.2)
– It grows well on ordinary media.
On nutrient agar, after overnight growth, colonies are moist, translucent, round disks,
about 1-2 mm in diameter, with a bluish tinge in transmitted light.
In peptone water, growth occurs in about six hours as a fine surface pellicle, which on
shaking breaks up into membranous pieces.
Resistance
– Cholera vibrios are susceptible to heat, drying and acids.
– It resists high alkalinity.
– They are destroyed at 55 °C in 15 minutes.
Morphology & Cultural characteristics

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Vibrio spp. (Family Vibrionaceae) Associated with Human Disease
Epidemiology of Vibrio cholerae
• Cholera recognized for more than two millennia with sporadic disease and epidemics
• Endemic in regions of Southern and Southeastern Asia; origin of pandemic cholera outbreaks
• Generally in communities with poor sanitation
• Seven pandemics (possible beginning of 8th) since 1817 attributable to increased world travel
• Cholera spread by contaminated water and food
• Human carriers and environmental reservoirs

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The AB5 toxins are six-component protein complexes secreted by
certain pathogenic bacteria known to cause human diseases such as cholera, dysentery,
and hemolytic–uremic syndrome.
One component is known as the A subunit, and the remaining five components are B subunits.
All of these toxins share a similar structure and mechanism for entering targeted host cells.
The B subunit is responsible for binding to receptors to open up a pathway for the A subunit to
enter the cell. The A subunit is then able to use its catalytic machinery to take over the host cell's
regular functions.
Cholera toxin (also known as choleragen and sometimes abbreviated to CTX, Ctx or CT)
is AB5 multimeric protein complex secreted by the bacterium Vibrio cholera. CTX is responsible
for the massive, watery diarrhea characteristic of cholera infection. It is a member of the Heat-
labile enter toxin family.

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Classification
Classification
=According to their biological properties, V. cholerae is divided into 2 biovars:
1. classical,
2. El-Tor.
The classical and El Tor vibrios share the same O-Ag and is agglutinated by O1-
antiserum (O-1 serogroup).
=According to structure of the O1-Ag species V. cholerae is subdivided into 3 serotypes:
– Ogawa (AB)
– Inaba (AC)
– Hikojima (ABC)
=All isolates from epidemic cholera (till 1992) belonged to serogroup
0-1. Other vibrio isolates which were not agglutinated by the 0-1 antiserum came to be
called nonagglutinable or NAG vibrios. (nonpathogenic and hence also called non-
cholera vibrios (NCV).

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O1 Serogroup
- 2 Biotypes: El Tor and Classical
- V. cholerae O1 are further divided into 2
major subserotypes (Inaba and Ogawa).
- The basis for subtyping is 3 antigenic
determinants of the O antigen structure of
their LPS.
These serotypes are differentiated in
agglutination and vibriocidal antibody
tests on the basis of their dominant
heat-stable lipopolysaccharide somatic
antigens.
- The serotypes share one determinant known
as the A antigen.
- In addition, Inaba strains express the C
antigen whereas Ogawa strains express the
B antigen .

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- O1 cholera ferment sucrose and mannose and they produce acid but not gas.
- V. cholera also possesses lysine and ornithine decarboxylase.
- Freshly isolated agar-grown vibrios of the El Tor biotype, in contrast to classical V. cholerae,
produce a cell-associated mannose-sensitive hemagglutinin which is found active in chicken
erythrocytes.
- Strains of the El Tor biotype, however, produce less cholera toxin, but appear to colonize
intestinal epithelium better than vibrios of the classical variety.
- Also, they seem some what more resistant to environmental factors. Thus, El Tor strains have
a higher tendency to become endemic and exhibit a higher infection-to-case ratio than the
classical biotype.
Other antigens
O139 Serogroup
- In 1993, the emergence of an entirely new serogroup (O139) was the cause an epidemic in
Bangladesh.
- O139 organisms produce a polysaccharide capsule but do not produce O1 LPS or O1 antigen.
- Toxigenic O139 cholera arose through the acquisition of a large block of genes encoding the
O139 antigen by O1 El Tor.
Non-O1, Non-O139 Serogroup
- Most are CT (cholera toxin) negative and are not associated with epidemic disease.

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Molecular Biology of Vibrio cholerae
• The species V. cholerae can be sub-classified into 200 serogroups based on the O antigen of LPS
(lipopolysaccharide).
– Only O1 and O139 strains have been implicated in the cholera syndrome.

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Genomic Structure
The cholera genome contains 2 circular
chromosomes.
- The genome is approximately 4.0Mb, in
which the classical strain is divided between
a 2.4Mb large chromosome and a 1.6 Mb
small chromosome.
- In the El Tor strain, the large chromosome
contains 2.96Mb and the small chromosome
contains 1.07Mb
Genomic Structure: Mobile Elements (PLASMIDS)
- Although several plasmids have been isolated, none appear to be involved in
pathogenesis.
- A 4.7Kb cryptic plasmid is present in all ctx-positive strains.
- A 6.8Kb plasmid (P factor) is capable of mobilizing chromosomal genes but less efficiently
than the F factor in E. Coli.

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1. Exotoxin (choleragen, cholera enterotoxin, cholera toxin, CT, or CTX).
2. Endotoxin- The lipopolysaccharide O antigen (LPS, endotoxin). This apparently plays no
role in the pathogenesis of cholera but is responsible for the immunity induced by killed
vaccines.
3. Adherence factors (pili)
4. Proteolytic enzymes (gelatinase, mucinase)
Virulence Factors
Virulence Factors Associated with Vibrio cholerae O1 and O139

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Cholera
• Cholera is an acute diarrhoeal disease caused V. cholerae serogroup O1 or O139.
Signs and symptoms
– The usual incubation period is 2 to 5 days, although it can be as short as several hours.
– Severe cholera is characterised by a sudden onset of profuse, watery diarrhoea
accompanied by nausea and vomiting.
– If left untreated, this can rapidly lead to serious dehydration, electrolyte imbalance and
circulatory collapse.
– Over 50% of the most severe cases die within a few hours; with prompt, effective treatment,
mortality is less than 1%.
– Cholera may be asymptomatic or mild in healthy individuals, with diarrhoea as the only
symptom.

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Cholera Toxin
- Ingestion of only 5μg of purified toxin resulted in production of 1-6L of diarrheal stool.
- CT elicits vigorous mucosal immune responses in the absence of a conventional adjuvant.
- Direct immunomodulatory effects of CT on leukocytes include induction of CD25 and class II MHC on B
cells, apoptosis of CD8+ T cells, and activation of macrophages with release of IL-10.
Structure
- Structurally & functionally similar to ETEC LT.
- CT is a prototype A/B subunit toxin, consisting of 1 A subunit and 5 B subunits.
1. The B subunit weighs 11.6kDa each and multimerize to form a pentameric ring.
2. The A subunit contains an intracellular ADP-ribosyltransferase activity.
- The crystal structure of CT revealed that the A and B subunits are connected through the C-
terminus of the A2 subunit, which is inserted through the central pore of the B pentamer.
- The mature A subunit is proteolytically cleaved to produce a 21.8kDa A1 polypeptide, which
contains the intracellular enzymatic activity, and a 5.4kDa A2 polypeptide
- CT must be assembled for activity, as neither the A nor B subunit individually can cause secretory
diarrhea.
- CT holotoxin is assembled in the periplasmic space.
- The subunits are exported individually into the periplasm through the
cytoplasmic membrane via the general secretion pathway; both the
A and B protein subunits contain
normal sequences at their N-terminus.
The catalytic portion of cholera toxin performs a single function: it seeks out the G proteins used for
cellular signaling and attaches an ADP molecule to them. This converts the G-protein into a
permanently active state, so it sends a never-ending signal.

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- Once in the periplasm, both subunits must undergo modification by the periplasmic enzyme DsbA,
which is responsible for disulphide bond formation.
- Again, once the holotoxin is secreted from the bacterium, the A subunit must be cleaved to
generate separate A1 and A2 peptides for maximum toxin activity.
- The biological activity of CT is dependent on binding of the holotoxin B pentamer to specific
receptors on the eukaryotic cell.
- The B oligomer binds with high affinity exclusively to GM1 ganglioside.
Function
- B subunit binds the holotoxin to a eukaryotic cell surface receptor. It attach to the ganglioside
receptors on the surface of jejunal epithelial cells (small intestine).
- A (active) unite causes prolonged activation of cellular adenylate cyclate and accumulation of
cAMP, leading to outpouring into the small intestinal lumen, of large quantities of water and
electrolytes and the consequent watery diarrhea.
- After cleavage, the A1 and A2 polypeptides remain linked by a disulphide bond.
- Reduction of disulfide bond in A-subunit activates A1 fragment that activate ADP-ribosylates
guanosine triphosphate (GTP)-binding protein (Gs) by transferring ADP-ribose from nicotinamide
adenine dinucleotide (NAD)
- ADP-ribosylated GTP-binding protein activates adenyl cyclase leading to an increased cyclic AMP
(cAMP) level and hyper secretion of fluids and electrolytes.

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Infectious Dose
- 106-1011 CFU
- Why such a high dosage?
Series of changes as moves from aquatic environment to intestine
Temperature, acidity
Acidic environment of stomach
Intestinal environment
Bile salts, organic acids, complement inhibit bacteria growth
Must penetrate mucous lining of intestinal epithelial cells
Mechanism of action of cholera enterotoxin
(a) Cholera toxin approaches target cell surface.
(b) B subunits bind to oligosaccharide of GM1 ganglioside receptor in small intestine.
(c) Conformational alteration of holotoxin occurs, allowing the presentation of the A subunit to cell
surface.
(d) The A subunit enters the cell.
(e) The disulfide bond of the A subunit is reduced by intracellular glutathione, freeing A1 and A2.
(f) NAD is hydrolyzed by A1, yielding ADP-ribose and nicotinamide.
(g) One of the G proteins of adenylate cyclase is ADP-ribosylated, inhibiting the action of GTPase and
locking adenylate cyclase in the "on" mode.

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Pathogenesis
To establish disease, V. cholerae must be ingested in
contaminated food or water and survive passage
through the gastric barrier of the stomach.
On reaching the lumen of the small intestine, the
bacteria must overcome the clearing mechanism of
the intestine (peristalsis), penetrate the mucous
layer and establish contact with the epithelial cell
layer.
Colonization of the intestinal microvilli and the
subsequent production and release of cholera
toxin, lead to the purging diarrhea.
This complex progression of events appears to involve
tightly regulated differential gene expression by the
bacteria.
– This is because expression of intestinal
colonization factors is unlikely to be of
advantage to the bacterium in its salt/fresh
water environment niche.

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Pathogenesis: Mechanism of Action
- Adenylate cyclase (AC) is activated normally by a
regulatory protein (GS) and GTP; however
activation is normally brief because another
regulatory protein (Gi) hydrolyzes GTP.
Cholera
- Enzymatically, fragment A1 catalyzes the transfer
of the ADP-ribosyl moiety of NAD to a component
of the adenylate cyclase system.
- The A1 fragment catalyzes the attachment of
ADP-Ribose (ADPR) to the regulatory protein
forming Gs-ADPR from which GTP cannot be
hydrolyzed.
- Since GTP hydrolysis is the event that inactivates
the adenylate cyclase, the enzyme remains
continually activated.
NORMAL CONDITION
CHOLERA

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- Thus, the net effect of the toxin is to cause cAMP to be produced at an
abnormally high rate which stimulates mucosal cells to pump large amounts of Cl-
into the intestinal contents.

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- H2O, Na+ and other electrolytes follow due to the osmotic and electrical gradients caused by
the loss of Cl-.
- The lost H2O and electrolytes in mucosal cells are replaced from the blood.
- Thus, the toxin-damaged cells become pumps
for water and electrolytes causing the diarrhea,
loss of electrolytes, and dehydration that are
characteristic of cholera.
- Normally, the epithelial cells of the inner lining
of the intestines (lumen) transfer sodium and
chloride ions from the inside of the intestines
to the blood stream.
- The "B" subunit of cholera toxin is bound by a
host receptor (like a specific "landing pad")
allowing the "A" subunit to enter the cell.
- Once inside the cell the "A" subunit causes a change
in the regulation of the cells genes and as a result,
the flow of ions and water is reversed.

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Mechanism of Action

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2. Two other possible adhesins in V. cholerae are
- a surface protein that agglutinates red blood cells (hemagglutinin) and
- a group of outer membrane proteins which are products of the acf (accessory colonization
factor) genes. acf mutants have been shown to have reduced ability to colonize the intestinal tract.
It has been suggested that V. cholerae might use these nonfimbrial adhesins to mediate a tighter
binding to host cells than is attainable with fimbriae alone.
V. cholerae produces a protease originally called mucinase that degrades different types of protein
including fibronectin, lactoferrin and cholera toxin itself. Its role in virulence is not known but it
probably is not involved in colonization since mutations in the mucinase gene (designated hap for
hemagglutinin protease) do not exhibit reduced virulence.
It has been suggested that the mucinase might contribute to detachment rather than attachment.
Possibly the vibrios would need to detach from cells that are being sloughed off of the mucosa in
order to reattach to newly formed mucosal cells.

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Colonization of the Small Intestine
• There are several characteristics of pathogenic V. cholerae that are important determinants of
the colonization process.
• These include adhesins, neuraminidase, motility, chemotaxis and toxin production.
• If the bacteria are able to survive the gastric secretions and low pH of the stomach, they are well
adapted to survival in the small intestine. V. cholerae is resistant to bile salts and can penetrate
the mucus layer of the small intestine, possibly aided by secretion of neuraminidase and
proteases (mucinases).
• They withstand propulsive gut motility by their own swimming ability and chemotaxis directed
against the gut mucosa.
Adherence mediators
1. Specific adherence of V. cholerae to the intestinal mucosa is probably mediated by long
filamentous fimbriae that form bundles at the poles of the cells.
- These fimbriae have been termed Tcp pili (for toxin coregulated pili), because expression of
these pili genes is co-regulated with expression of the cholera toxin genes.
- Not much is known about the interaction of Tcp pili with host cells, and the host cell receptor for
these fimbriae has not been identified.
- Tcp pili share amino acid sequence similarity with N-methylphenylalanine pili of Pseudomonas
and Neisseria.

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In Vibrio cholerae, the production of virulence factors is regulated at several levels.
- V. cholerae enterotoxin is a product of ctx genes.
ctxA encodes the A subunit of the toxin, and
ctxB encodes the B subunit.
The genes are part of the same operon.
The transcript (mRNA) of the ctx operon has two ribosome binding sites (rbs)-
one upstream of the A coding region and
another upstream of the B coding region.
The rbs upstream of the B coding region is at least seven-times stronger than the rbs of the A coding
region.
In this way the organism is able to translate more B proteins than A proteins, which is required to
assemble the toxin in the appropriate 1A: 5B proportion.
The components are assembled in the periplasm after translation. Any extra B subunits can be excreted
by the cell, but A must be attached to 5B in order to exit the cell. Intact A subunit is not enzymatically
active, but must be nicked to produce fragments A1 and A2 which are linked by a disulfide bond.
Once the cholera toxin has bound to the GM1 receptor on host cells, the A1 subunit is released from the
toxin by reduction of the disulfide bond that links it to A2, and enters the cell by an unknown
translocation mechanism.
One hypothesis is that the 5 B subunits form a pore in the host cell membrane through which the A1 unit
passes.
Genetic Organization and Regulation of Virulence Factors in V. cholerae

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Transcription of the ctxAB operon is regulated by a number of environmental signals, including temperature,
pH, osmolarity, and certain amino acids.
Several other V. cholerae genes are co-regulated in the same manner including the tcp operon, which is concerned
with fimbrial synthesis and assembly. Thus the ctx operon and the tcp operon are part of a regulon, the
expression of which is controlled by the same environmental signals.
The proteins involved in control of this regulon expression have been identified as ToxR, ToxS and ToxT.
(i) ToxR is a transmembranous protein with about two-thirds of its amino terminal part exposed to the
cytoplasm. ToxR dimers, but not ToxR monomers, will bind to the operator region of ctxAB operon and
activate its transcription.
(ii) ToxS is a periplasmic protein. It is thought that ToxS can respond to environmental signals, change
conformation, and somehow influence dimerization of ToxR which activities transcription of the operon.
ToxR and ToxS appear to form a standard two-component regulatory system with ToxS functioning as a
sensor protein that phosphorylates and thus converts ToxR to its active DNA binding form.
(iii) ToxT is a cytoplasmic protein that is a transcriptional activator of the tcp operon. Expression of ToxT is
activated by ToxR, while ToxT, in turn, activates transcription of tcp genes for synthesis of tcp pili.
Thus, the ToxR protein is a regulatory protein which functions as an inducer in a system of positive control.
Tox R is thought to interact with ToxS in order to sense some change in the environment and transmit a
molecular signal to the chromosome which induces the transcription of genes for attachment (pili formation)
and toxin production.
It is reasonable to expect that the environmental conditions that exist in the GI tract (i.e., 37o temperature, low pH,
high osmolarity, etc.), as opposed to conditions in the extraintestinal (aquatic) environment of the vibrios, are
those that are necessary to induce formation of the virulence factors necessary to infect.

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Specimens: Watery stool, rectal swab, water, food, vomiting
Microscopy
a. Stained smears by Gram
b. Wet drop smears to determine vibratory motility .
Bacteriological method . It is the most reliable to make diagnosis.
The major steps are:
1. Inoculation of the collected samples into alkaline peptone water and spread a large loop of feces over a
plate of TCBS medium.
2. After incubation for 5 h subculture from first peptone water is transmitted into second alkaline PW and on
the second plate of TCBS agar. Microscopy of wet smears from PW, make a agglutination with O-1
antiserum.
3. After incubation for 12 h grown colonies from TCBS are investigated with agglutination test, microscopy of
stained smear. Suspected colony is transferred onto slant alkaline MPA.
4. Identification of vibrio pure culture (biochemical typing, serological and phage typing)
Serological method: detection vibriocidal antibody or agglutinins.
For rapid diagnosis, the characteristic motility of the vibrio and its inhibition by antiserum can be demonstrated under
the dark field or phase contrast microscope, using cholera stool from acute cases
Laboratory diagnosis

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Prophylaxis
– It includes general measures (purification of water supplies, better provision for sewage
disposal, microbiological control of sewage and drinking water).
– Infected patients should be isolated, their excreta disinfected. Contacts and carriers are
followed up.
Specific measures
– Killed parenteral vaccine – composed of equal number of Inaba and Ogava strains
– Killed oral vaccine – B subunit whole cell vaccine. The vaccine contains cholera toxin B
subunit, heat killed classical vibrio and formalin killed El- Tor vibrio
– Live oral vaccine – recombinant DNA vaccine
Treatment
Rapid fluid replacement with a balanced solution of sugar, electrolytes and water (oral rehydration
salts) should be started urgently. This may be done orally, but severely dehydrated cases may
require intravenous administration. Cases may also be treated with antibiotics, usually a
tetracycline if the organism is sensitive, in order to improve symptoms and decrease the intestinal
excretion of the organism. Patients who are promptly treated should respond rapidly and recover.
Oral rehydration therapy, antibiotics
– Rapid fluid replacement with a balanced solution of sugar, electrolytes and water (oral
rehydration salts) should be started urgently. This may be done orally, but severely
dehydrated cases may require intravenous administration.
– Cases may also be treated with antibiotics, usually a tetracycline if the organism is sensitive,
in order to improve symptoms and decrease the intestinal excretion of the organism.
– Patients who are promptly treated should respond rapidly and recover.
Prophylaxis & Treatment

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