A.J O’Neil, J. A. Lindsay, K. Gould, J. Hinds, and I. Chopra

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

6. Analyzing Microarray Data • Quantitate the fluorescence signal • Calculate the ratio of red/green fluorescence • Log(base 2) transform the ratios • Normalize the log ratios on each microarray slide • Normalize the log ratios for a set of slides in an experiment • Perform statistical analysis on the log ratios • Compare individual genes with known data • Look for patterns (expression profiles) in the data (many programs are available to do this) • Perform Gene Ontology term enrichment analysis (we will use MAPPFinder for this) • 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 MurEunder 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. • Websites: • 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/