Host-induced epidemic spread of the cholera bacterium Merrell DS, Butler SM, Qadri F, Dolganov NA, Alam A, Cohen MB, Calderwood SB, Schoolnik GK, and Camilli A. Nature 2002 Jun 6; 417(6889) 642-5. doi:10.1038/nature00778

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Host-induced epidemic spread of the cholera
bacterium Merrell DS, Butler SM, Qadri F,
Dolganov NA, Alam A, Cohen MB, Calderwood SB,
Schoolnik GK, and Camilli A. Nature 2002 Jun 6
417(6889) 642-5. doi10.1038/nature00778

• Journal Club Presentation
• BIOL398/S10 Bioinformatics Lab
• Jaime Moehlman Amanda Wavrin
• April 13th, 2010

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Outline

• Vibrio cholerae is a highly infectious waterborne
  disease.
• The human-shed form of V. cholerae proves to be
  more pathogenic than a strain grown in vitro.
• Strains, sample collection and Microarray
  analysis.
• Further research can be done on the proteome.

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Vibrio cholerae is a waterborne disease that is
infectious to humans

• It produces a cholera toxin that acts on the
  mucosal epithelium and is responsible for
  diarrhea.
• Cholera is one of the most rapidly fatal
  illnesses.
• The disease can progress to shock in as little as
  4-12 hours.
• Death can follow 18 hours to several days after
  the onset of symptoms.
• A healthy person who is infected may die within
  2-3 hours if no treatment is provided.

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Samples of the O1 Inaba El Tor strain were
collected and another strain was grown in vitro

• The study took place in Dhaka, Bangladesh due to
  the commonality of outbreaks in a natural
  setting.
• The O1 Inaba El Tor strain is distinguished by
  its deletion of the lacZ gene.
• The two strains were combined and used to
  inoculate infant mice.
• The ratio of the mixed assays was 110, it was
  corrected to a 11 output ratio.
• Bacteria was recovered from the small intestine
  and was then plated on a medium.

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Passage through the human GI tract increases the
infectivity of cholera

• The output ratios were corrected to represent the
  competitive indices (CI) of the V. cholerae.
• A CI above 1 indicates increased infectivity.
• A CI below 1 indicates decreased infectivity.
• The human-shed V. cholerae had a CI above 1
  indicating an enhanced infectivity.
• V. cholerae that was cultured and purified in
  vitro did not show enhanced infectivity.

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Passage of V. cholerae enhances infectivity in
secondary hosts

• The samples were diluted in pond water that was
  free of V. cholerae.
• The pH of the two pond water samples used were
  7-7.5.
• They were then mixed with the in vitro grown
  competitor strain.
• When this mixture was infected into mice, the
  hyperinfectious state remained.

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The competitive indices for human-shed V.
cholerae shows an increase in infectivity
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Microarray analysis

• ORFs were found and portions were amplified by
  polymerase chain reaction and spotted onto
  slides.
• V. cholerae RNA was collected from stool samples
  and DSM-V999 strain was grown overnight in vitro.
  
• DNAse treatment to remove DNA contamination was
  carried out.
• Equal concentrations of each test RNA and common
  reference RNA were used for reverse transcription
  reactions.
• Control arrays were also hybridized to identify
  potential affects of freezing the stools.

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Transcriptional profiling using DNA microarray

• A spotted DNA microarray containing about 87 of
  the identified ORFs of the El Tor strain was
  used.
• Positive samples were attained from 3 patients.
• The samples were collected in beakers, filtered
  through cheese cloth, and frozen at 80 (C)
• Protocols were reviewed and approved by three
  different review committees.

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The stool RNA was analyzed by agarose gel
electrophoresis to ensure its integrity

• RNA from each sample was used for DNA synthesis
• The stool RNA was analyzed by agarose gel
  electrophoresis to ensure its integrity.
• This was labeled with Cy5 and hybridized to the
  microarray with a Cy3- labeled common reference
  strain (exponential growing).
• The samples were hybridized in quadruplicate and
  relative fluorescent intensities were determined.
  
• The data was quantified, normalized and corrected
  to yield intensity ratios.

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SAM program was used to determine significant
differences in the intensity ratios

• The in vitro strain was used as class I, and each
  individual sample as class II.
• They obtained these results
• 237 genes were differentially regulated, of
  these
• 44 were induced
• 193 were repressed

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Transcriptional profile of human-shed V. cholerae
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Transcriptomes of the V. cholerae were similar to
that of the cultured DSM-V99 strain

• It was consistent with bacterial growth
  conditions that were also found in rice-water
  stools.
• They proposed that V. cholerae moves from a
  nutrient rich environment in the small intestine
  to a nutrient poor lumenal fluid.
• This fluid is quickly removed.

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Before being shed, V. cholerae turns off
expression of specific genes

• This has the potential to be for dissemination to
  the environment or transmission to a new host.
• These genes are necessary for infection of humans
  and mice.
• These genes include those for the cholera toxin,
  and the Vibrio pathogenecity island.
• These results also suggest that increased
  expression of these genes is not necessary for
  the increased infectivity of cholera.

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The role of chemotaxis during infectivity is
unknown

• Some genes that are needed for chemotaxis are
  also required for expression of the cholera
  toxin.
• Within both cholera strains the genes required
  for chemotaxis were repressed while being shed.
• This suggests that the motile bacteria are
  non-chemotactic during dissemination, which
  could
• Increase the shedding from the GI tract
• Increase infectivity

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Opportunities for Further Research

• The next step would be making sense of the
  proteome of human-shed V. cholerae.
• Induction of the acid tolerance response (ATR)
  could be involved in the increased infectivity of
  human-shed V. cholerae.
• If this is true, the mechanism of action is
  unknown.
• Discovering how the human host preps the bacteria
  for infection of additional humans can aid in the
  further study of human to human transmission of
  other microorganisms.
• The work done in this study could also aid in the
  development of a vaccine.

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References

• Merrell DS, Butler SM, Qadri F, Dolganov NA, Alam
  A, Cohen MB, Calderwood SB, Schoolnik GK, and
  Camilli A. Host-induced epidemic spread of the
  cholera bacterium. Nature 2002 Jun 6 417(6889)
  642-5.
• Todar, Kenneth. Online Textbook of Bacteriology
  Vibrio cholerae http//www.textbookof
  bacteriology.net/cholera.html. 11 April 2010.