Identification of Staphylococcus aureus Cellular Pathways Affected by the Stilbenoid Lead Drug SK-03-92 Using a Microarray

Abstract

The mechanism of action for a new lead stilbene compound coded SK-03-92 with bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) is unknown. To gain insight into the killing process, transcriptional profiling was performed on SK-03-92 treated vs. untreated S. aureus. Fourteen genes were upregulated and 38 genes downregulated by SK-03-92 treatment. Genes involved in sortase A production, protein metabolism, and transcriptional regulation were upregulated, whereas genes encoding transporters, purine synthesis proteins, and a putative two-component system (SACOL2360 (MW2284) and SACOL2361 (MW2285)) were downregulated by SK-03-92 treatment. Quantitative real-time polymerase chain reaction analyses validated upregulation of srtA and tdk as well as downregulation of the MW2284/MW2285 and purine biosynthesis genes in the drug-treated population. A quantitative real-time polymerase chain reaction analysis of MW2284 and MW2285 mutants compared to wild-type cells demonstrated that the srtA gene was upregulated by both putative two-component regulatory gene mutants compared to the wild-type strain. Using a transcription profiling technique, we have identified several cellular pathways regulated by SK-03-92 treatment, including a putative two-component system that may regulate srtA and other genes that could be tied to the SK-03-92 mechanism of action, biofilm formation, and drug persisters.

SK-03-92 Treatment Causes Release of a Lethal Factor Protein That Kills Staphylococcus aureus Cells

“…Based on prior work, we hypothesized that the SK-03-92 treatment affected a putative regulatory system labeled BrpR/S, which led to the release of a lethal factor such as an autolysin, mutacin, holin, or bacteriocin that rapidly killed the S. aureus cells. A previous microarray study of SK-03-92 treated versus untreated S. aureus cells demonstrated that three genes could be tied to a potential mechanism of action of the SK-03-92 drug: brpR, brpS, and lrgA [10]. The brpR and brpS genes had the highest differences in transcript abundance when comparing treated versus untreated cells.…”

“…Transcript abundance differences were the greatest for the MW2284 and MW2285 genes (14.1-and 26.3-fold less, respectively) following treatment with SK-03-92 compared to the untreated cells [10]. Transcription of the lrgA gene also fell 3.2-fold in the drug treated cells versus untreated cells.…”

“…To try to uncover the mechanism of action of the lead compound SK-03-92, an mRNA microarray was conducted comparing the untreated S. aureus cells to those treated with the SK-03-92 drug [10]. A total of 52 genes displayed significant differences in transcription when comparing the untreated versus SK-03-92 treated cells.…”

“…Preliminary bioinformatic analyses suggested that MW2284 and MW2285 comprised a two-component system with homologies to LytTR superfamily proteins [10]. Several Gram-positive bacterial species express LytTR superfamily proteins that regulate biofilm formation, bacteriocin production, competence, or autolysis of the bacteria [15][16][17][18][19][20][21][22][23].…”

Regulation of the Staphylococcal Superantigen-Like Protein 1 Gene of Community-Associated Methicillin-Resistant Staphylococcus aureus in Murine Abscesses

“…All of the RT-qPCRs were performed using the iTaq Universal SYBR Supermix kit according to manufacturer’s instructions (BioRad, Hercules, CA, USA). Primers used in this study have been used in other studies: ftsZ [58], ssl5 [33], and ssl8 [33] and were synthesized by Integrated DNA Technologies (Coralville, IA, USA). A CFX96 machine (BioRad, Hercules, CA, USA) was used throughout the study.…”