*M37: Mobile Genetic Elements and Bacterial Virulence

Question 1

Role of Mobile Genetic Elements in Bacterial Virulence:

1. The repertoire of _ genes carried by a pathogen dictates what type of disease develops.
   a. This repertoire varies considerably among different _ (sometimes even among different _).
2. Differences in virulence gene repertoires result from the presence of many virulence genes on _, including _, _, and _.
   a. Mobile genetic elements are typically restricted to certain _.
3. Advantages of having virulence genes on mobile genetic elements include:
   a. By acquiring different sets of mobile genetic elements, a single pathogen can _.
   b. By locating virulence genes on genetic elements that are mobile, new virulence genes can be rapidly _ within a bacterial population.

Answer 1

1. virulence

a. pathogens
strains of a single pathogen

2. mobile genetic elements, including plasmids, phages, and transposons
   a. pathogens
   a. cause different diseases
   b. transferred

Question 2

Role of Mobile Genetic Elements in Bacterial Virulence:

4. Real or potential disadvantages of having virulence genes on mobile genetic elements include:
   a. Extrachromosomal mobile genetic elements tend to be _ (virulence genes can be _).
   b. Since virulence is often multifactorial, it might be necessary for a single bacterial cell to acquire and maintain _, if each mobile element carried only a single _.
5. Solutions to those problems include:

a. Some mobile genetic elements can stably _ onto the _
i) Phages - lysogeny
ii) Transposons can integrate

b. Many mobile genetic elements have acquired multiple (complementary) virulence genes.

Answer 2

a. unstable
lost

b. many mobile genetic elements
virulence gene

a. integrate
chromosome

Question 3

Lysogeny and Diphtheria:

1. Like human cells, bacteria are susceptible to infection with viruses (called phages).
   a. Outcomes of phage infection include:
   i) Lytic infection: progeny virus is produced, but this infection is lethal for bacterial host cell.
   ii) Lysogenic infection: no progeny virus is produced and the infection is nonlethal for the bacterial host, but allows phage genes to be stably maintained in the lysogenized bacterium.

Answer 3

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Question 4

Lysogeny and Diphtheria:

2. The connection between lysogeny and diphtheria:
   a. Symptoms of diphtheria are caused by diphtheria toxin.
   b. Gene encoding diphtheria toxin (the tox gene) is carried by a phage (b-phage), which can lysogenically infect Corynebacterium diphtheriae (a gram-positive bacterium).
   c. This lysogenic infection leads to stable expression of diphtheria toxin.
   d. Expression of diphtheria toxin is tightly regulated in response to the bacterium’s growth environment:
   i) Expression only occurs under low-iron conditions.
   ii) Regulation involves an iron-binding repressor called DtxR (Diphtheria Toxin Repressor). DtxR is encoded by the dtxR gene that is present in the C. diphtheriae chromosome (not encoded on the phage).

Answer 4

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Question 5

Case study: An 18 month old female develops a fever of 39.7°C, with anorexia and lethargy. She is brought to the emergency room by her grandmother. Physical examination indicates a clear chest and enlarged cervical lymph nodes. The pediatrician notes the presence of a thick gray adherent pseudomembrane over the tonsils and throat.

Samples of this pseudomembrane were cultured by the Micro lab. The cultures grew gram-positive, club-shaped rods which were arranged in palisades or in “Chinese letter” formations. The patient was given penicillin but still dies later that day.

Later discussion with her parents revealed that they belonged to a religion that forbids immunizations.

Cause: _

Answer 5

Corynebacterium diphtheriae

Question 6

Corynebacterium diphtheriae:
Biological characteristics:

Corynebacterium diphtheriae is a gram-positive, club-shaped rod. It often contains polyphosphate granules and is somewhat resistant to environmental factors (e.g., drying).

C. diphtheriae grows best under aerobic conditions and is relatively fastidious. Selective media containing tellurite salts are used for isolation.

Answer 6

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Question 7

Corynebacterium diphtheriae:
Reservoir and transmission:

a. There are two forms of diphtheria: nasopharyngeal and cutaneous. Both forms are transmitted by aerosol droplets: if inhaled, you get the nasopharyngeal form, if droplets settle on skin at site of a cut or ulcer, you get the cutaneous form.
b. Humans are the only reservoir. Note that nontoxigenic C. diphtheriae can be present in throats of healthy people. Can the immunized be healthy carriers of toxigenic strains?

Answer 7

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Question 8

Corynebacterium diphtheriae:
Pathogenesis:

a. Pathogenesis is relatively simple: colonization and toxin production.
b. After colonization of nasopharynx or skin, C. diphtheriae starts producing diphtheria toxin.
c. About 1-7 days later, localized necrosis develops. Initial symptoms are relatively nonspecific: malaise, sore throat, enlarged cervical lymph nodes (from inflammation).
d. With time, this necrosis leads to formation of a pseudomembrane in the throat; the pseudomembrane contains bacteria, fibrin, necrotic epithelial cells, PMNs, and RBCs.

Initially, the pseudomembrane can be easily wiped off, but it becomes much more adherent with time. As the pseudomembrane enlarges, it can block respiration and cause death (especially in children).

e. C. diphtheriae cells remain on the mucosal epithelium; they are not invasive. However, with time, some diphtheria toxin is absorbed and enters the circulation where it can damage internal organs (e.g. the heart), causing death.

Answer 8

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Question 9

Corynebacterium diphtheriae:

Virulence factors:

a. Adhesins
b. Diphtheria toxin: The toxin is responsible for the disease. Toxin inhibits host cell protein synthesis by ADP-ribosylating elongation factor 2.

Diagnosis:

a. Must be made initially on clinical grounds.
b. Lab confirms diagnosis: isolation and culture, followed by toxin testing.
7. Prevention and treatment:
a. Treatment must include antitoxin (to inactivate preformed toxin) plus antimicrobials (to kill the bacterium).
b. Can be prevented by immunization (part of DPT regimen).

Answer 9

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Question 10

Pathogenicity Islands:

1. Some mobile genetic elements carry entire sets of virulence genes.
   a. This allows a bacterium to acquire, in a single event, most or all of the virulence genes it needs to cause disease.
2. Some plasmids carry sets of virulence genes.
   a. Example: ETEC strains carry their enterotoxin genes and CFA adhesin genes on a single plasmid.

Answer 10

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Question 11

Pathogenicity Islands:

To overcome stability problems, mobile genetic elements with sets of virulence genes often integrate into the chromosome.

a. This results in the clustering of virulence genes in one specific region of the bacterial chromosome. These regions are referred to as pathogenicity islands.
i) Pathogenicity islands are thought to have been transferred from other isolates or even other species. Thus they often have a DNA base composition that is different from the rest of the bacterium’s organism chromosome.
ii) Pathogenicity islands have been found in both Gram + and Gram - bacteria.
iii) A single bacterial cell can carry more than one pathogenicity island.
iv) Type III secretion systems of Gram-negative bacteria are on pathogenicity islands.

Answer 11

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Question 12

Pathogenicity Islands:

To overcome stability problems, mobile genetic elements with sets of virulence genes often integrate into the chromosome.

b. Transfer and integration of virulence factors can also involve lysogenic infection by a phage as described for diphtheria. Another example is toxigenic V. cholerae which is lysogenized with a phage (the CTXf phage) that carries the genes that encode for the production of cholera toxin and other accessory enterotoxins.
i) Experiments have shown transfer of CTXf between V. cholerae isolates, converting the recipient to virulence.
ii) The receptor for CTXf phage is the TCP (toxin-coregulated pilus) adhesion. The TCP is required for V. cholerae colonization of the small intestine.

Answer 12

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Question 13

Case study: A 47 year-old American businessman returned from a trip to Peru. On his return he suddenly starts vomiting and develops severe diarrhea. This diarrhea is voluminous, but painless and watery. After he becomes lightheaded, his wife rushes him to the emergency room, where he presents with a very weak pulse. He is afebrile.

The man is diagnosed as suffering from severe dehydration and alterations in serum electrolytes. When a lab tech examines his stool microscopically, no white blood cells are present.

When the stool is cultured, Gram-negative comma-shaped rods are present.

Cause: _

Answer 13

Vibrio cholerae

Question 14

Vibrio cholerae:
Biologic characteristics:

a. V. cholerae are highly motile, gram-negative, comma-shaped rods.
b. These bacteria are facultative anaerobes but, because they are oxidase-positive, they do not belong to the Enterobacteriaceae.
c. TCBS agar (thiosulfate-citrate-bile salts) is selective for growth and used for identification.
d. V. cholerae, like other Vibrio spp., are halotolerant.
e. V. cholerae can be distinguished from other Vibrio spp. by biochemical tests.
f. V. cholerae are classified by serotyping (based on differences in “O” antigen).
g. O1 and O139 serotypes are associated with epidemic/pandemic spread; other serotypes cause sporadic localized outbreaks.

Answer 14

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Question 15

Vibrio cholerae:

Reservoir and Transmission:

a. Transmission: Cholera typically occurs in large outbreaks that can spread rapidly to produce epidemics. Worldwide spread of cholera has resulted in global outbreaks called pandemics. Transmission of cholera during outbreaks usually occurs via ingestion of food/water contaminated with V. cholerae. Humans are important for transmission; stools of the infected contain very large numbers of infectious bacteria.
b. Reservoir: V. cholerae is native to estuary waters. Asymptomatically colonized people in endemic areas are also likely to be important reservoir.

Answer 15

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Question 16

Vibrio cholerae:
Virulence Factors:

a. Cholera toxin (CT): ADP-ribosylates Gs protein, which regulates intestinal adenylate cyclase. Causes increased cAMP levels in intestines, producing massive intestinal fluid and electrolyte loss.
c. Colonization factors:
i) Toxin-coregulated pilus (TCP): essential adhesin for V. cholerae colonization of the small intestine epithelium.
ii) Other adhesins: V. cholerae has other afimbrial adhesins including: outer membrane proteins, polysaccharide capsule (for O139 strains), and LPS.
iii) Other colonization factors: flagella, which help V. cholerae to move through intestinal mucus to reach the epithelium.
iv) Biofilm production. Protects the bacterium against acid exposure during passage through the stomach.

Answer 16

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Question 17

Vibrio cholerae:
Virulence Factors:

b. Accessory enterotoxins:
i) Zonula occludens toxin (ZOT): affects tight junctions.
ii) Accessory enterotoxin (ACE): may form an ion channel.
d. Regulators of virulence factor production:
i) Many V. cholerae virulence factors are coordinately expressed (e.g. CT and TCP; also see Lecture 34). In V. cholerae the coordinate expression of virulence gene expression is controlled by the ToxR regulon.
ii) Regulation involves TcpP (not the pilus protein) and ToxR, which are membrane –associated regulatory proteins that function together to activate transcription of ToxT.
iii) ToxT, which is another regulatory protein, then binds to the virulence gene promoters (i.e. ctxAB and tcp) to activate their expression.

Answer 17

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Question 18

Vibrio cholerae:
Pathogenesis:

a. The bacterium is ingested and passed through the gastric acid barrier of the stomach. V. cholera survives exposure to gastric acid by a process that is greatly facilitated by biofilm production.
b. Upon entering the small intestine the bacteria move through the intestinal mucus via their flagella. During this process virulence factor expression is turned on. The regulon controlling virulence factor production is called the ToxR regulon. This allows for the production of the “toxin coregulated pilus” (TCP) and other adhesins which facilitate colonization and disease development.
c. During colonization V. cholerae also produces cholera toxin (CT). CT is an enterotoxin that binds to enterocytes and increases cAMP levels in the enterocytes. cAMP binds to protein kinases, which activate channels (e.g., CFTR) for ion secretion.
d. The cAMP increase leads to massive fluid/electrolyte loss, which is clinically manifested as moderate to severe diarrhea.

Answer 18

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Question 19

Vibrio cholerae:
Pathogenesis:

e. This diarrhea typically develops after an incubation period of several hours to several days.
f. In severe cholera (known as cholera gravis), the diarrheal fluid loss is ~1 L/hour. This diarrheal fluid has a pale yellow/brown color with flecks of small white mucous material and thus is known as “rice water stool”.
g. Fluid and electrolyte loss from the diarrhea, can lead to poor skin turgor, and sunken eyes. Patients may exhibit vomiting. In mild-moderate cases the patient remains mentally alert but can become comatose in severe cases due to severe dehydration.
h. There is no invasion and, thus, an absence of fecal leucocytes.
i. The severe fluid/electrolyte loss of cholera gravis can lead to cardiac problems and shock, often resulting in death.

Answer 19

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Question 20

Vibrio cholerae:
Prevention:

i) Good hygiene, good water, and cooked food.
ii) An ineffective parental vaccine of whole killed V. cholerae cells was licensed for many years.
iii) Three oral vaccines are available, but not licensed in the USA.

A. Dukoral: Formalin killed V. cholerae O1 and recombinant B-subunit of cholera toxin (www.crucell.com). The recombinant B-subunit makes this vaccine more costly to produce that the others.

B. Shanchol: Heat and formalin killed whole cell V. cholerae O1 and O139 (www.shanthabiotech.com).

C. mORCVAX is produced in Vietnam and has a formulation that is similar to Shanchol

iv) Work is ongoing for the development of a live attenuated vaccine strain

Answer 20

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Question 21

Vibrio cholerae:

Treatment:

i) Must rapidly restore fluid/electrolyte balance.
ii) In severe cases, must give i.v. fluids, but in most cases can use oral rehydration solution.
iii) Antimicrobial therapy (e.g. azithromycin) helps lessen disease severity and duration. Also, limits spread of the organism.

Diagnosis:

a. Initial diagnosis made on clinical grounds.
b. Lab can confirm diagnosis by stool culture and biochemical/serologic tests.

Answer 21

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