J'aime C. Moehlman's Week 11

Microarray Journal Club Assignment

Vibrio cholerae Journal Article: 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. doi:10.1038/nature00778

Terms & Definitions

1. Chemotaxis: A response of motile cells or organisms in which the direction of movement is affected by the gradient of a diffusible substance, the gradient on which diffusion occurs is in one direction
2. Murine: Of, or relating to, a member of the rodent family muridae, including rats and mice.
3. Gavage: Therapeutic use of a high-potency diet administered by stomach tube.
4. lacZ gene: A gene that encodes for beta-galactosidase that is a lactase which cleaves lactose.
5. Homogenization:The process by which a material is made homogeneous, which means; consisting of or composed of similar elements or ingredients, of a uniform quality throughout.
6. In- vitro: Processes or reactions that would normally occur within an organism but are made to occur in an artificial environment; such as a test tube, beaker, etc.
7. Dissemination: The act/ property of dispersing or diffusing something.
8. Hypervirulent: A high quantity, above normal or a high state or degree of something that is extremely toxic, denoting a markedly pathogenic microorganism.
9. 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.
10. Regulon: A collection of genes or operons under regulation by the same regulatory protein.
11. Transcriptome: The set of all RNA molecules, including mRNA, rRNA, tRNA, and non-coding RNA produced in one or a population of cells and the transcriptome can vary with external environmental conditions.

Definitions from: biology-online.org dictionary

Article Outline

• The main result presented in the paper is to show that when cholera is passed through humans it significantly increases the transmission of the disease to other people leading to the chances of an epidemic.
• By testing naturally occuring strains of cholera and strains grown in vitro; they discovered that the cholera from human stools created a hyperinfectious bacterial state that may lead to epidemic spread.
• There are many cholera outbreaks in Bangladesh, making it easy to study the bacterium in its naturally setting.
• The strain they wanted to identify in the stool samples was the O1 Inaba El Tor.
• Then they mixed the isolated El Tor samples with a strain that was grown in vitro; which they injected into mice.
• The isolated strain is missing a lacZ gene.
• The competetive indices of the stool sample bacterium showed that the human shed strain enhanced infectivity 700-fold to that of the in vitro strain.
• The competitive advantage of stool-derived bacteria has to persist after dissemination in order for it to play a large role in the epidemic spread.
• The next step was to test whether or not the human shed strand would maintain its hyperinfectious state after incubation in a pond; after waiting for 5 hours the hyperinfectious state of the bacteria remained.
• They used a spotted microarray that contained representations of the identified open reading frames of the El Tor strain.
• Stool RNA preparations were analysed by agarose gel electrophoresis to make sure the RNA was pure.
• Cy3 was the common reference- exponentially growing El Tor strain and the samples were marked as Cy5.
• 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
• 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
• 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

• 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

Figures

• Figure 1:This figure shows the CI's for the human-shed V. cholera. This comes from the GI tract in 1a and b shows the mix between the human shed strain and the in vitro strain.
• Figure 2: This figure shows the log expression ratio for the three patients (A,B,C)and their 4 samples(replicate arrays). On the left it shows the technical replications and the right shows bilogical replications. The color representations: red shows a minimum 2 fold increase and green shows a minimum 2 fold decrease in expression.

Merrell Journal Club

Microassay Journal Club