Amanda N. Wavrin Week 11

Vibrio cholerae Journal Club Article

Host-induced Epidemic Spread of the Choler Bacterium

Vocabulary

1. Electrophoresis-separation of ionic molecules, (principally proteins) by the differential migration through a gel according to the size and ionic charge of the molecules in an electrical field. High resolution techniques normally use a gel support for the fluid phase
2. Gavage-Forced feeding by stomach tube
3. Enumerate-To count; to reckon; to ascertain the units of
4. Chemotaxis-a response of motile cells or organisms in which the direction of movement is affected by the gradient of a diffusible substance. Differs from chemokinesis in that the gradient alters probability of motion in one direction only, rather than rate or frequency of random motion
5. Inoculation-The process of introducing an antigenic substance or vaccine into the body to trigger immune response against a specific disease.
6. Dissemination-The act of dispersing or diffusing something
7. Homogenization-The process by which a material is made homogeneous
8. Chromogenic-Producing colour, a chromogenic colony is a pigmented colony
9. Murine-Of, relating to, a member of the rodent family muridae, including rats and mice
10. Shine-Dalgarno sequence-A short stretch of nucleotides on a prokaryotic mRNA molecule upstream of the translational start site, that serves to bind to ribosomal RNA and thereby bring the ribosome to the initiation codon on the mRNA

Dictionary used: biology-online.org dictionary

Outline

• It is unclear as to what factors enhance the transission of pathogens during epidemic spread
• Vibrio Cholera is a water-borne diarrhoel disease
• Cholera was studied in its natural habitat in Dhaka, Bangladesh
• Stool samples were collected and tested for V. cholerae O1 Inaba El Tor from patients at the International Centre for Diarrhoeal Disease Research
• These strains were used in murine infection studies by mixing an in vitro strain with the stool V. cholerae and using the mixture to inoculate mice
• The mice were euthanized after around 24 hours and the bacteria was collected from the mice and plated.
• out put ratios of stool-sample V. cholerae to the in vitro strain were corrected and represent the competitive indices
• A CI above one indicates enhanced infectivity
• A CI below one indicates decreased infectivity
• Human shed V. cholerae consistently showed enhances infectivity
• However, when the V. cholerae strain was purified and cultured in vitro this competitive advantage was lost
• These results suggest that passage through the human gastointestinal tract increases infectivity of the cholera bacterium
• They tested to see whether or not the human shed V. cholerae maintained its hyperinfectious state after being dispersed back into the environment
• After being infected into mice, they found that the hyperinfectious state remained
• They proposed that human passage enhances the infectivity of V. cholerae by lowering the infectious dose in secondary individuals
• They then conducted transcriptional profiling of human shed V. cholerae by using DNA microarray
• The DNA microarray contained around 87 percent of the ORFs of the Bangladeshi O1 El Tor reference strain
• stool samples were collected from three patients in the ICDDR
• The samples were filtered and frozen
• The samples showed the 'rice water' appearance typically found with V. cholerae
• The contained around 100 million V. cholerae per millilitre and showed few contaminating bacteria
• The stools were analyzed using gel electrophoresis
• One microgram of RNA from each sample was used for DNA synthesis, labelled with Cy5 and hybridized to the microarray with a Cy3 labelled reference strain- an expotentially growing O1 Inaba El Tor strain
• The relative flourescent intensities were determined using an Axon scanner and the data was quantified, normalized, and corrected
• Using the Statistical Analysis for Microarrays program, they were able to identify significant differences in the intensity ratios
• An in vitro strain was used as class I and each individual stool sample as class II
• They obtained the following results: 237 genes were differentially regulated, 44 genes were induced, and 193 genes were repressed in human shed V. cholerae
• The transcriptomes of the stool derived V. cholerae and the strain of DSM-V999 were similar
• The transcriptome in consistent with bacterial growth in conditions similar to those found in rice water stools
• Hierarchical clustering of stationary phase, expotential phase, and human shed V. cholerae shows that the transcriptome bears traits of both growth phases
• Results suggest that before it is shed, V. cholerae turns off the expression of particular genes, genes that are needed for successful infection of humans and mice
• This indicates that increased expression of these genes is not necessary for increased infectivity
• In vitro induction of the acid toerlance response enhances the infectivity of V. cholerae
• The role of chemotaxis in the infectivity of V. cholerae in uncertain
• Most of the genes for chemotaxis were repressed durinf infection
• The results suggest that motile bacteria are non-chemotactic when being shed
• These results shed some light on the infectivity of the cholera bacterium
• Passage through the human gastrointestinal tract increase the infectivity of V. cholerae
• Humans help prepare the cholera bacterium for infection of other hosts
• These findings may help with the understand of the epiemic spread of other microorganisms
• This work could also aid in the development of a vaccine to prevent infection

Methods

• patients at the ICDDR had cholera and rice water stools
• 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 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
• 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