Angela A. Garibaldi Week 11

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

Introduction

• Studying the Mode Of Action of how antibacterial agents, such as antibiotics, work is important in coming up with new antibacterials against Staph aureus in light of all of the resistance issues.
• Analyzing structure-activity relationships are key in developing new antibiotics to attack staph in a novel way to avoid resistance
• DNA microarrays is a good way to do transcriptional profiling to see the cellular response to a novel antibacterial across the entire genome.
  • The MOA of a new antibacterial can be predicted by comparing the profile with that of a profile from an antibiotic with a previously well characterized MOA.

Review of Common Antibacterial Inhibitor Mechanism

• Targets Cell wall biosynthesis (CWB) to kill Staph aureus
• Researchers are trying to come up with an antibacterial inhibitor that targets the early stage of CWB via the Mur enzymes
  • This stage I or Cytoplasmic stage of peptidoglycan synthesis has not been widely targeted in recent studies, providing a great potential avenue to find an antibacterial with a different system of effectiveness.
• Previous studies have characterized transcriptional responses of genes being downregulated or upregulated in response to antibiotics that target the later stages of CWB such as vanomycin and oxacillin
  • So far only fosfomycin is the only Mur enzyme inhibitor that inhibits the earlier stage of CWB by affecting the function of MurA and MurZ.

Methods

1. Create universal transcriptional profile of S. aureus after the earlier stage (Stage I) is inhibited in CWB
2. Use fosfomycin as test inhibitor (control)
3. utilized genetic and postranslational challenge to inhibit/deplete the cell of other enzymes that are active in the early stage I of CWB
4. S. aureus RN4220 and derivatives used
5. Strain TS2557 has a mutation in MurB making it temperature sensitive
6. CYL368 has been modified so that MurE is controlled by the Pspac promoter, meaning that its expression depends on the presence of IPTG

• CLY368 requires tetracycline in the growth medium to maintain the integrity of the lacI repressor plasmid (pMJ8246).
  • pMJ8246 also put into RN4220 so that both conditional and control strains were subject to identical conditions (ie with tetracycline)

1. Conditional mutants resulted in 80% inhibition of growth in mutant vs wild-type
2. Drug-treated cultures exposed to a concentration of antibiotic causing 25% reduction in growth relative to untreated cultures after 40 minutes.
3. Cyl368 and RN4220(pMJ8246) were grown overnight in presence of 3ug tetracycline and .3mM IPTG.
4. Cells harvested, washed to remove IPTG
5. Resuspended in fresh broth lacking IPTG at OD600 of .05
6. Grow at 37C, harvest at OD600 of .25
7. TS2557 and RN4220 grown overnight at 30C, resuspended to OD600 at .075 in fresh broth
8. TS2557 and RN4220 grown at 42C and harvested at OD600 of .25

Fosfomycin treatment

1. O/N RN4220 at 37C used to inoculate prewarmed TSB to OD600 of .02 and grown at 37C to OD600 of .1
2. Culture split into 2 prewarmed flasks (one of these has fosfomycin), harvest cells after 40 min incubation

RNA preparation

1. RNA midi kit from cells treated with lysostaphin.
2. Control RNA and sample RNA used to make cDNA by reverse transcription that will be differentially labeled in presence of Cy3 and Cy5 fluorescent dyes.
3. Both control and sample RNA are cohybridized, scanned, analyzed.

Chips

1. Cultures grown in triplicate
2. Hybridized in duplicate for a total of 6 arrays per condition.
3. Feature extraction via ImageGeneSoftware
4. Fluorescent intensities processed via MAVI Pro software
5. Normalization via GeneSpring v7.3.1 software
6. Differentially expressed genes for each condition identified

Figure 1

• shows the pathway of stage I CWB, involving the biosynthesis of UDP-MurNAc-pentapeptide from UDP-GlcNAc, mediated by the Mur enzymes
• Inhibition in the pathway was achieved at

1. MurA/Z
2. MurB (thermosensitive protein)
3. MurE (conditional gene expression)

Definitions

1. cytoplasmic stage -
2. peptidoglycan
3. repressor plasmid -
4. lysostaphin -
5. cohybridize -
6. feature extraction -
7. chaperone -

1. • What is the main result presented in this paper? (Hint: look at the last sentence of the introduction and restate it in plain English.)
   • What is the importance or significance of this work?
   • What were the limitations in previous studies that led them to perform this work?
   • What were the methods used in the study?
     • What samples did they collect and use for the microarray experiment?
     • How many microarray chips did they hybridize in the experiment?
     • Which samples were paired to hybridize on the chip?
     • Which was labeled red (Cy5)? Which was labeled green (Cy3)?
     • How many replicates did they perform of each type?
       • Biological replicates are made from entirely different biological samples.
       • Technical replicates are made when one biological sample is split at a particular stage in the procedure and then carried through to the end of the procedure.
     • What do they say about how they performed each of the steps listed in the Overview of Microarray Data Analysis section above?
   • Briefly state the result shown in each of the figures and tables.
   • How do the results of this study compare to the results of previous studies (See Discussion).