Transcript microarray - OpenWetWare

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
Outline
• Purpose of Microarray
• Microarray Information
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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
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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
healthy
Experiment
Infected
Red- genes
of interest
(infected/
increased)
mRNA
cDNA
Microarray/
hybridizatio
n
http://www.cs.wustl.edu/~jbuhler/research/array/arra
y.png
Greenhealthy
(turned
down)
Yellowhybridized/
expressed in
both
Analyzing Microarray Data
1.
2.
3.
4.
5.
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
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/BIOL39801/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
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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
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Dereg in stage II/III
_________________
Transcriptional signature
For 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/BIOL39801/S10:DNA_Microarrays#Get_Acquainted_with_Your_Microarray
_Dataset
– DNA Microarray Virtual Lab
• learn.genetics.utah.edu/content/labs/microarray/