Ryan N. Willhite Week 11

Journal Club

Transcriptional Signature following Inhibition of Early-Stage Cell Wall Biosynthesis in Staphylococcus aureus

File:StaphAureusMicroArray.ppt

Journal Club Week 11

Transcriptional Signature following Inhibition of Early-Stage Cell Wall Biosynthesis in Staphylococcus aureus

Biological Terms

1. DNA microarrays- method used to measure the expression of thousands of genes simultaneously. It allows one to visualize through color the changes in gene expression.
2. MOA studies-mode of action studies.
3. Mur enzymes
4. isoenzymes-are enzymes that differ in amino acid sequence but catalyze the same chemical reaction.
5. tetracycline- are a group of broad-spectrum antibiotics
6. aeration- process in which air is circulated
7. optical density- index of refraction
8. triplicate-one set of three objects or copies
9. phosphoenolpyruvate-is an enzyme used in the metabolic pathway of gluconeogenesis. It converts oxaloacetate into phosphoenolpyruvate and carbon dioxide.

(wikipedia.org)

Outline

• Introduction
  • Purpose: Defining the mode of action of a anitbacterial agent for further knowledge into the development process such as optimization of structure-activity relationships. Along with microarray analysis providing an overview of the genome and cellular responses to anitbacterial inhibitors at the transcriptional level. The main focus is cell wall biosynthesis for antibiotic action in Staphylococcus aureus and overall establishing a transcriptional signature of S. aureus following inhibition of stage I CWB.

• Methods

1. Use fosfomycin as a test inhibitor like previous studies
2. S. aureus RN4220 and derivatives were used
3. Strain TS2557 engineered to place murE under the control of the Pspac promoter and therefore rendering expression in the presence of IPTG
4. pMJ8246 introduced into S. aureus RN4220 to enable both conditional and control strains cultured under the same conditions int he presence of tetracycline.
5. Strains cultured in TSB
6. Conditional mutants cultured under conditions inhibiting 80 percent in mutant vs wt
7. drug-treated cultures exposed to antibiotic causing 25 percent growth reduction relative to untreated cultures after 40 min
8. Cells were harvested with 2 volumes of RNAprotect solution to culture
9. S. aureus CYL368 and RN4220 (pMJ8246) grown at 37 degree C with tetracycline and IPTG
10. grown and harvested
11. TS2557 and RN4220 were grown overnight at 30 degrees C then grown at 42 degree C and harvested
12. Fosfomycin treatment
13. Culture split into prewarmed flasks and one contained fosfomycin and incubation for 40 min before harvesting
14. RNA was prepared using RNA midi kit from cells with lysostaphin
15. 2 RNA's, one a control and the other a sample, used to make differentially labeled cDNA by RT with dyes Cy3 and Cy5
16. RNAs were cohybridized, scanned and analyzed
17. Cultures were then grown in triplicate and hyrbridized in duplicate for 6 arrays
18. Mircroarray feature extraction- "BioDiscovery"
19. Further processing with MAVI Pro software
20. Normalization and statistical analysis performed

• Results

1. Little deregulation was found in genes encoding enzymes invovled with CWB with some exceptions
2. Deregulation in expression of Mur enzymes were not consistent
3. Obtained a universal transcriptional signature for inhibition

• Conclusion

1. Members of the transcriptional signature for inhibition of CWB

• Inhibition/depletion of MurA or MurZ, MurB, and MurE

1. 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