Download Host-induced epidemic spread of the cholera bacterium

Host-induced epidemic spread of
the
cholera bacterium
Theresa Graebener, Salomon Garcia, Claudia Campos
Journal Club Presentation
Biological Databases 367-01
October 19, 2010
Outline
Background on Vibrio cholerae
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It’s a gram negative bacterium
Has polar flagellum
Comma shaped
Main cause for cholera
V. cholerae has 2 circular chromosomes of
unequal size that encode for an approximate
of 3885 genes.
The experiment itself…
• For this study human subjects were gathered
from the International Centre for Diarrhoeal
Disease Research (ICDDR)
• Subjects were from Dhaka, Bangladesh mainly
due to bacterium being in it’s natural habitat
• Serological analysis was done in order to
identify if stool samples that were positive for
V.cholerae 01 Inaba El Tor
Bacterial Strain responsible for the
spread of V.cholerae
• Strain (DSM-V984) was strain that had been
previously isolated and is marked by the deletion
of the lacZ gene. It allows for the enumeration of
in vitro and the stool samples
• Strain (DSM-V984) was mixed with V.cholerae in
stool
• Mixture was injected to 3-5 day old Swiss
Webster mice.
• Mice were euthanized after 20-24hrs and
bacteria was recovered
Testing of the human shed V.cholerae occurred
in order to test if the hyperinfectious phenotype
was maintatined.
• V.cholerae samples that were freshly shed were diluted
in pond water that was free from contaminants of
V.cholerae
• Incubation at room temperature for 5 hrs, then diluted
samples were mixed with in vitro grown competitor
strains
• This mixture was injected to mice
• Hyperinfectious state remained the same the only
difference being human passage of the bacteria
enhances water bourne spread which lowers the dose to
secondary individuals
Transcriptional profiling of human
shed V.cholerae
• Spotted DNA microarray with 87% of known
open reading frames
• Samples of the 3 patients were taken then
later frozen in order to prepare for total RNA.
• RNA analyzed using agarose gel
electrophoresis
• Strain DSM-V999 from one of the 3 samples,
RNA was isolated after in vitro growth
Statistical Analysis reveals significant genes
are responsible for gene expressions
• 237 genes were differentially regulated
• 44 genes were induced
• 193 genes were repressed in human stool
samples
• Transcriptomes were similar to strain DSMV999, 3120 out of 3357 open reading frames
that were examined were expressed
Competition assays take advantage of
concentration differences
• On A samples mixed directly with in-vitro grown DSMV984
• On B samples incubated in local pond water for 5 hours
• The Data points represent the output ratio of stoolderived V. cholerae to in-vitro grown competitor strain
after correction for deviations in input rations.
• Each data point represents output from a single
animal
• The geometric mean is indicated by horizontal
bar
• (n) is the total number of animals per
experiment
The transcription profiles were used to
compare genetic similarities and
differences
The cluster
diagram shows
the wholegenome
expression
profile of V.
cholerae
recovered
from the stools
of three ICDDR
patients
• Patients labeled A B or C (in blue)
• Replicate arrays are numbered 1-4 (in orange)
• Right panel is cluster
diagram showing
consistent differential
regulation from patients
A B and C
• Fold changed relative to
V. cholerae grown in
vitro calculated using the
avg values from the
quadruplicate arrays on
left
• Red indicated a
minimum twofold
increase in
expression
• Green represents
a minimum
twofold reduction
in expression
• Representative
genes listed and
their relative
location indicated
by arrow
Differences found between human
shed V.cholerae and strain DSM-V999
• Increased expression of genes required for
biosynthesis of amino acids, iron uptake
systems, ribosomal proteins, and formation of
periplasmic nitrate reductase complex
• V. cholerae moves from rich nutrient
environment to poor environment which is
purged.
Several genes were found to be
differentially expressed in stool samples
• These genes were found to be factors for
infection of mice and humans
• VC0468 strain was found to play a significant
role in the ability of V.cholerae to tolerate acid
References
Merrell D. Scott, Butler Susan M., QadriFirdausi,
Dolganov Nadia A., Alam Ahsfaqui, Cohen
Mitchell B., Calderwood Stephen B., Schoonik
Gary K., & Camilli Andrew. Host-induced
epidemic spread of the cholera bacterium.
Nature. 2002. Vol, 417,