Kzebrows Week 11

Preparation for Journal Club on Your Species

Researching Unknown Terms

First I made a list of 10 biological terms from my team's microarray paper (full PDF text here) for which I did not know the definitions. I then ran a search through Google for the biological definition of each of these terms. No one online biology dictionary had definitions for all of these so I often had to click on several links before finding an appropriate definition. I used a combination of online biological or medical dictionaries and journal articles with the definitions in their abstracts or early in the background information. I avoided using sites like Wikipedia in my search.

regulon: a group of genes regulated by the same regulatory molecule with a common binding site or promoter (definition here)
pouchitis: after a proctocolectomy an IPAA is performed (ileal pouch-anal anastomosis) in which the lower part of the small intestine is connected to the anus, creating a pouch. Pouchitis is the inflammation of the pouch that can occur following the surgery. (definition here)
chaperonin: a protein that aids in assembly and folding of protein molecules in living cells (definition here)
QRT-PCR: Real TIme Quantitative Reverse Transcription PCR, or a procedure to detect products generated during each cycle of PCR by cleaving the probe during PCR. Nuclease activity of Taq polymerase can then be used to detect amplification of a certain product. (definition here)
beta-subunit: a subdivision or distinct component of a larger protein unit (definition here)
island: a cluster of genes acquired by horizontal gene transfer that are frequently associated with bacterial adaptation (definition here)
serotype: an immunology defined variant of a micro-organism that bears a unique epitope on its surface (definition here)
type III secretion system (TTS): a syringelike apparatus used by some bacteria to penetrate the plasma membrane of a host cell and inject something, such as a toxin (definition here)
isoniazid: first-line and most commonly used anti-tuberculosis drug. Organisms are able to develop resistance very rapidly if it is used alone. Hepatic toxicity is the major side effect. (definition here)
cold shock protein: a protein expressed at low temperatures that protects cell components from changes caused by suboptimal temperatures, e.g. CspA in E. coli which binds to RNA and prevents 2-degree structure formation. (definition here)

Outline

Background:
- traits of Shigella flexneri: one of 4 known Shigella species
  - gram negative
  - facultative anaerobes
  - intracellular pathogen
  - genes required for Shigella to invade and spread within the cell are encoded by a virulence plasmid
- Infection controlled by antibiotics
- common antibiotic is rifamycin bind to RNA polymerase beta subunit and inhibit synthesis of RNA
  - rifamycin has low absorption, low systemic toxicity, and low interaction with other drugs, making it ideal
  - two compounds: rifampin (RA) and rifaximin (RX)
  - problem: drug resistance is on the rise
- expression profiling using microarrays allows researchers to see which genes the drug is targeting and can help find out if a better target exists
  - can also help identify genes involved in different effects
Methods of the study and experimental design:
- used S. flexneri 2a strain (Sf301) grown at 37C in Mueller-Hinton broth
  - determined the minimal inhibitory concentrations (MIC) of the two drugs
  - Sf301 inoculated to optimum density and added drugs dissolved in methanol to the concentrations of 0xMIC, 0.25xMIC, 0.5xMIC, 1xMIC, 2xMIC, and 4xMIC
  - As cultures reached early exponential growth phase, drugs were added to make concentrations of 0.5xMIC and 1xMIC and samples collected after predetermined amounts of time had passed and washed 2X with PBS
  - Methanol solution without drug was added to control cultures at same concentration
  - bacterial densities were measured in terms of absorbance/optical density (OD600)
- total RNA extracted and reverse transcribed to prepare cDNA and labeled with Cy3-dCTP (control) and Cy5-dCTP (drug-treated samples)
- Microarray construction: PCR products spotted onto microarrayer and hybridized using protocol found here
- Microarray analysis using GenePix Pro 6.0 software
- Real-time PCR assay was performed
- 72 total samples (each of the samples listed below, equaling 36 samples, had a corresponding control with no drug treatment bringing the total number of samples to 72). You can see the sample data here.
  - shigella flexneri strain 301 plus 0.5 x MIC rifamaxin for 10 minutes (3)
  - shigella flexneri strain 301 plus 0.5 x MIC rifamaxin for 30 minutes (3)
  - shigella flexneri strain 301 plus 0.5 x MIC rifamaxin for 60 minutes (3)
  - shigella flexneri strain 301 plus 1 x MIC rifamaxin for 10 minutes (3)
  - shigella flexneri strain 301 plus 1 x MIC rifamaxin for 30 minutes (3)
  - shigella flexneri strain 301 plus 1 x MIC rifamaxin for 60 minutes (3)
  - shigella flexneri strain 301 plus 0.5 x MIC rifampin for 10 minutes (3)
  - shigella flexneri strain 301 plus 0.5 x MIC rifampin for 30 minutes (3)
  - shigella flexneri strain 301 plus 0.5 x MIC rifampin for 60 minutes (3)
  - shigella flexneri strain 301 plus 1 x MIC rifampin for 10 minutes (3)
  - shigella flexneri strain 301 plus 1 x MIC rifampin for 30 minutes (3)
  - shigella flexneri strain 301 plus 1 x MIC rifampin for 60 minutes (3)
- type of replicates
  - the first of each type was a technical replicate because the samples are all of Shigella flexneri strain 301 but treated differently
  - the second and third repetitions of the experiment were biological replicates because each experiment was independently performed three times
- no dye swaps occurred
- Significant change identified if ratio of gene is higher than twofold (up or down-regulated)
Overview of figures:
- Figure 1. Growth curve for S. flexneri in the presence or absence of RP and RX
  - This graph exhibits how growth is both concentration- and time-dependent for the bacteria
  - different concentrations: MHB and 0.25, 0.5, 1, 2, and 4 times the minimum inhibitory concentration (MIC)
  - After 90 minutes RX is more inhibitive than RP
  - Growth rate was not affected by 0.25 MIC of drugs, was inhibited more at 0.5 MIC, and severely inhibited at 1 x MIC
- Figure 2. Percentage of genes induced (open bars) and repressed (black bars) for each functional class
  - RX: 535 genes altered: 236 displayed increased expression and 299 displayed reduced expression
  - RP: 367 genes altered: 158 up-regulated and 209 down-regulated
- Figure 3. Relative fold change for genes in Table 1 determined by QRT-PCR
  - QRT-PCR was used to validate microarray data for 14 selected genes with the same procedure as used for CDNA preparations. There was a strong positive correlation between the microarray and QRT-PCR data (Table 1), that indicated that the data was reliable.
Overview of tables:
- Table 1: Gene-specific primers for the 14 genes of interest were designed. This table shows the sense and antisense primer sequences used.
- Table S1. Shows differentially expressed genes of Shigella flexneri exposed to RX in different concentrations.
- Table S2: Shows differentially expressed genes of Shigella flexneri exposed to RP in different concentrations.
- Table S3. Shows the ratios for genes and target genes induced by treatment with RX and treatment with RP (overlap).
- Table S4. Shows the ratios for genes and target genes repressed by treatment with RX and treatment with RP (overlap).
Discussion: overall, the two drugs showed similar trends
- 42% of shared genes were encoded by virulence plasmid
  - includes type III secretion system genes and proteins necessary to assemble it, virulence factor regulators, and heat shock genes (early time point only)
- commonly repressed genes were involved in cold shock proteins, chaperonin, ribosomal proteins, glycolysis, metabolism, and other normal cell growth processes
- Antibiotics did not influence expression of entry genes or their activators so invasion ability was not substantially enhanced
- Intracellular movement intercellular dissemination genes were enhanced, showing that RNA polymerase contributes to pathogenesis AFTER invasion
- Rho gene inhibited, possibly the reason why virulence genes were up-regulated
Comparing results of this study to previous studies
- Previous study showed that RP moderately up-regulated RNA synthesis genes, consistent with this study
  - Found that high concentration elicits strong induction but prolonged incubation results in rapid decrease in expression
- Found more virulence genes affected by RX and RP than in previous studies and also some association genes
- Studies have shown that changing ribosome synthesis can control expression of thermal stress, which was supported in this paper, but it is unclear why (further study

Flow Chart

The following flow chart outlines the experimental methods of the research.

Power Point

File:KZShigellamicroarray.pdf

Guild Meeting Notes 11/10: GenMAPP Users

Experimental design (for sure do by next week)
- Treatment vs. control
- Biological vs. technical replicates: which one and how many
  - Biological replicates: independent samples
  - Technical replicates: if at any point sample is split into two and treated differently
  - NOTE: should be able to find # chips or microarrays (a combo of the above should add up to this)
- dye swaps: green and red dye behave differently in experiments
  - Figure out how many dye swaps occurred (best is even, but they could have done half and half and swapped it or used technical replicates)
- Flow chart (will go into final paper and presentation!)
Sample and data relationships: Match each chip/filename with a sample. One file should correspond to one chip. (look at by next week)
- raw.zip should have the files in it
- sdrf.txt (Sample Data Relationship File) spreadsheet
- NOTE: Double-check with Dr. Dahlquist
Create compiled raw data file with ID, sample log 2 fold, sample log size, etc.
Normalization and stats (scaling and centering data, calculating T tests and B-H/Bonferroni corrections). Can consult with Dr. Dahlquist for customized procedure.
- Goal: do a sanity check and compare to what was actually written in the paper to see if it matches (probably not identical but same magnitude and direction)
GenMAPP: format data and run through GenMAPP and MAPPFinder (converge here with Coder and QA)
- Do biological interpretation of the data, analyze, and find out something new because the paper did not have this kind of analysis previously
- Identify exceptions and feed info back to QA person (a few hundred is ok, just hopefully not more than 10% of genome)

Deliverables: responsible for describing methods, how raw data file was found, etc., data tables, GenMAPP and MAPPFinder analysis and interpretation. Compare or contrast in discussion what was found vs. what coder and QA found.