1. Transcriptional Signature following Inhibition of Early-Stage Cell Wall Biosynthesis in Staphylococcus aureus A.J O’Neil, J. A. Lindsay, K. Gould, J. Hinds, and I. Chopra ( 2009)Antimicrobial Agents and Chemotherapy 01309-08: 1701-1704 Angela Garibaldi & Ryan Willhite BIOL398-01/S10: Bioinformatics Laboratory April 13, 2010

2. Outline Purpose of Microarray Microarray Information – Microarray Steps – Experiment in detail – Analyzing Microarray Data – Limitations to Microarray Methods Goals of the Experiment The Experiment Design Tables/Results Conclusion

3. Purpose of Microarray To determine which genes are repressed/activated Visualize differences in gene expression To measure the expression of many genes simultaneously

4. Microarray Steps Collect Tissue Isolate RNA and later mRNA Make cDNA using Reverse Transcription Apply to the chip Scan Microarray Analyze Data http://www.youtube.com/watch?v=VNsThMNjKhM

5. Experiment http://www.cs.wustl.edu/~jbuhler/research/array/arra y.png Infected healthy mRNA cDNA Microarray/ hybridizatio n Red- genes of interest (infected/ increased) Green- healthy (turned down) Yellow- hybridized/ expressed in both

6. Analyzing Microarray Data 1.Quantitate the fluorescence signal 2.Calculate the ratio of red/green fluorescence 3.Log(base 2) transform the ratios 4.Normalize the log ratios on each microarray slide 5.Normalize the log ratios for a set of slides in an experiment 6.Perform statistical analysis on the log ratios 7.Compare individual genes with known data 8.Look for patterns (expression profiles) in the data (many programs are available to do this) 9.Perform Gene Ontology term enrichment analysis (we will use MAPPFinder for this) 10.Map onto biological pathways (we will use GenMAPP for this) (Dahlquist steps of analyzing off wiki) http://www.openwetware.org/wiki/BIOL398- 01/S10:DNA_Microarrays#Get_Acquainted_with_Your_Microarray_Dataset

7. Limitations to Microarray Cannot see if genes are defective in protein production Can only be seen through protein analysis AFTER microarray analysis – Time consuming

8. Methods in Relation to Microarray Info. Previous described The comparison is between – Those treated and untreated with fosfomycin This includes derivatives of S. aureus RNA extraction step performed by Qiagen – Rna midi kit, RNA protectant solution cDNA made with RT using dyes – Cy3 and Cy5 RNA’s then cohybridized, scanned, and analyzed – Image Gene Software used: Biodiscovery – Mavi Pro software: MWG Biotech

9. Goals of the Experiment Post-inhibitor exposure Transcriptional signature profile for late-stage CWB already exists Create a post-inhibitor exposure transcriptional profile of the early-stage of Cell Wall Biosynthesis (CWB) MOA can be predicted by comparing the gene deregulation following exposure to a new antimicrobial with profiles created from established antibiotics with known MOAs.

10. Stage I Biosynthesis pathway

11. Experimental Design Inhibit the Mur enzymes (A/Z, B, and E) 3 Biological Replicates 2 Technical Replicates 18 hybridizations (6 per condition) Dye swap design – label orientations are reversed

12. S. aureus strains utilized RN4220 serves as wild-type strain (control) T52557 contains temperature sensitive mutation in MurB Cyl368 puts MurE under control of the Pspac hybrid promoter that contains the lac operator region with lacI gene that encodes lac repressor – inhibits the transcription/expression of downstream genes in the presence of IPTG.

13. Creating the MurA/Z inhibition

14. Creating the MurB inhibition

15. Creating the MurE inhibition

16. Table 1 Deregulated following inhibition Shows genes deregulated after inhibition, but not after exposure to inhibitors of stages II and III of CWB Only genes showing more than 2 fold deregulation in the same direction under all 3 experimental conditions Provide Precursors Environmental Stress Encode enzymes directly involved in CWB/cell wall turnover

17. Table 1 continued Genes with: Similar dereg to MurF _ Dereg in stage II/III _________________ Transcriptional signature For Inhibition of Stage I !

18. Results of Mur enzyme inhibition Upregulation of genes involved in providing precursors that are essential for CWB Upregulation of genes involved in the environmental stress response No pattern of deregulation, meaning expression of genes involved in Stage 1 peptidoglycan synthesis is essential Little deregulation in genes encoding enzymes directly involved in CWB/cell wall turnover – EXCEPT for: Upregulation of dal, sgtB AND Downregulation of atl.

19. Conclusions Members of the transcriptional signature for inhibition of CWB –Inhibition/depletion of MurA or MurZ, MurB, and MurE Suggest that transcriptional profiling can be employed –Not only to identify inhibitors of CWB –Also to establish whether they act on early or late stages in the biosynthetic pathway

20. References O'Neill AJ, Lindsay JA, Gould K, Hinds J, and Chopra I. Transcriptional signature following inhibition of early-stage cell wall biosynthesis in Staphylococcus aureus. Antimicrob Agents Chemother 2009 Apr; 53(4) 1701-4. doi:10.1128/AAC.01309-08 pmid:19164146.O'Neill AJ, Lindsay JA, Gould K, Hinds J, and Chopra I. Transcriptional signature following inhibition of early-stage cell wall biosynthesis in Staphylococcus aureus. Antimicrob Agents Chemother 2009 Apr; 53(4) 1701-4. Websites: – http://www.openwetware.org/wiki/BIOL398- 01/S10:DNA_Microarrays#Get_Acquainted_with_Your_Microarray _Dataset http://www.openwetware.org/wiki/BIOL398- 01/S10:DNA_Microarrays#Get_Acquainted_with_Your_Microarray _Dataset – DNA Microarray Virtual Lab learn.genetics.utah.edu/content/labs/microarray/