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 WillhiteBIOL398-01/S10: Bioinformatics Laboratory
April 13, 2010
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
Purpose of Microarray
• To determine which genes are repressed/activated
• Visualize differences in gene expression• To measure the expression of many genes
simultaneously
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
Experiment
http://www.cs.wustl.edu/~jbuhler/research/array/array.png
Infectedhealthy
mRNA
cDNA
Microarray/
hybridization
Red- genes of interest (infected/ increased)
Green- healthy (turned down)
Yellow-hybridized/ expressed in both
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
Limitations to Microarray
• Cannot see if genes are defective in protein production
• Can only be seen through protein analysis AFTER microarray analysis – Time consuming
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
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.
Stage I Biosynthesis pathway
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
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.
Creating the MurA/Z inhibition
Creating the MurB inhibition
Creating the MurE inhibition
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
Table 1 continued
Genes with:
Similar dereg to MurF_
Dereg in stage II/III_________________
Transcriptional signatureFor Inhibition of Stage I !
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.
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
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/