Transcript V. cholerae - OpenWetWare

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
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Introduction to Vibrio cholerae
Results
Discussion
Methods
Microarray analysis
Further research
References
Vibrio cholerae is a waterborne
disease, that is infectious humans.
• It produces a cholera toxin that acts on the
mucosal epithelium and is responsible for the
characteristic diarrhea.
• Cholera is one of the most rapidly fatal illnesses
known.
• A healthy person who is infected may die within
2-3 hours if no treatment is provided.
• Usually, the disease progresses to shock in 4-12
hours, with death following in 18 hours to several
days after the onset of symptoms.
Is stomach acid a factor that contributes
to the epidemic spread of cholera?
• The study took place in Dhaka, Bangladesh
because of the common outbreaks of cholera in
its natural setting.
• They collected stool samples at the ICDDR and
identified those positive with the O1 Inaba El Tor
strain (marked by the deletion of the lacZ gene).
• This strain was mixed with a strain grown in vitro;
and then was used to inoculate infant mice.
• Bacteria was recovered from the small intestine
and was then plated on a medium.
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.
• These results suggest that passage through
the human GI tract will increase the infectivity
of the cholera.
Testing the hyperinfectious phenotype
after incubation in pond water.
• The samples were diluted in pond water that
was free of V. cholerae.
• They were then mixed with the in vitro grown
competitor strain.
• When this mixture was infected into mice, the
hyperinfectious state remained.
• From this they proposed that passage of V.
cholerae enhances infectivity by lowering the
infectious dose in secondary hosts.
Representation of the competitive
indices for human-shed V. cholerae.
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 and were then filtered and frozen.
• The stool RNA was analyzed by agarose gel
electrophoresis to ensure its integrity.
RNA from each sample was used for
DNA synthesis.
• 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.
The Statisical Analysis for Microarrays
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.
The transcriptomes of the V. cholerae
were similar to that of the cultured
DSM-V99 strain.
• The transcriptome is consistent with bacterial
growth conditions that was also found in ricewater stools.
• With these observations they propsed that V.
cholerae moves from a nutrient rich
environment in the small intestine to a
nutrient poor lumenal fluid that 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 in infectivity is
unknown.
• There are some genes that are needed for
chemotaxis that are also known to be required
for expression of the cholera toxin.
• Many of the genes required for chemotaxis were
repressed when being shed.
• This suggests that the motile bacteria are nonchemotactic during dissemination into the
environment, which could:
– Increase the shedding from the GI tract.
– Increase infectivity.
Strains, sample collection and
competetion assays
• Fresh stool samples were collected in beakers, filtered
through cheese cloth, and frozen at 80 degrees Celsius
• Protocols were reviewed and approved by the Research
Reviem Committee, the Ethical Review Committee, and the
Institutional Review Board
• Competition assays were done by mixing DSM-V984 grown
overnight with stool bacteria in a ratio of 1:10
• It was given to 3-5 day old mice by gavage
• The mice were then euthanized and the small intestines
removed
• Output ratios were corrected
• The PH of the two pond water samples used were 7-7.5
Microarray analysis
• ORFs were found using and ORF-finding program and portions of
the ORFs were amplified by polymerase chain reaction and
spotted onto slides.
• V. cholerae RNA was collected from stool samples and DSMV999 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.
• Labelling reactions were done on two separate days which
resulted in quadruplicate arrays for each strain.
• Control arrays were also hybridized to identify potential affects
of freezing the stools.
Opportunities for Further Research
• Induction of the acid tolerance response (ATR) could
potentially be involved in the increased infectivity of
human-shed V. cholerae.
• If this is true, the mechanism of action is unknown.
• Transcriptional profiling only provides a small look at
the gene expression right at the time of dissemination,
the next step would be making sense of the proteome
of human-shed V. cholerae.
• By discovering how the human host preps the bacteria
for infection of additional humans, it can aid it 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.