Transcript Microassay Journal Club

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
Journal Club Presentation
BIOL398/S10: Bioinformatics Lab
J’aime Moehlman & Amanda Wavrin
April 13th, 2010
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.
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.
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 1:10, it was
corrected to a 1:1 output ratio.
• Bacteria was recovered from the small intestine and
was then plated on a medium.
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.
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 77.5.
• They were then mixed with the in vitro grown
competitor strain.
• When this mixture was infected into mice, the
hyperinfectious state remained.
The competitive indices for human-shed V.
cholerae shows an increase in infectivity
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.
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.
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.
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
Transcriptional profile of human-shed V.
cholerae
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.
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.
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 nonchemotactic during dissemination, which could:
– Increase the shedding from the GI tract
– Increase infectivity
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 humanshed 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.
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.