Proteomics Analysis of Tea Tree Oil‐Selected Staphylococcus aureus Small Colony Variant

Abstract

Staphylococcus aureus small colony variants (SCV) are associated with chronic and recurring infections that are recalcitrant in antimicrobial therapy. SCVs demonstrate: slower growth rates; defective metabolism and electron transport; and reduced antimicrobial susceptibility. Tea tree oil (TTO) kills bacteria by denaturing proteins and disrupting membrane structure and TTO reduced‐susceptibility (TTORS) S. aureus mutants exhibit an “unique” SCV phenotype. Similar to previously described SCVs, all TTORS SCVs investigated were less susceptible to both the cell wall antibiotics vancomycin and oxacillin. A TTORS SCV mutant (TTORS‐1) harbored numerous mutations, including a mutation within acpP which encodes the acyl carrier protein (ACP) essential for fatty acid biosynthesis. Comparative proteomics revealed that TTORS‐1 demonstrated increases in 39 proteins and decreases in 74 proteins compared to parent strain SH1000. In TTORS‐1, the fatty acid biosynthesis proteins FabF (3‐oxoacyl‐synthase) and FabD (malonyl CoA‐acyl carrier protein transacylase) and the bifunctional phosphopantothenoylcysteine decarboxylase/phosphopantothenate‐cysteine ligase were found in greater abundance. This latter enzyme is required for the synthesis of 4′‐phosphopantetheine, which when linked to ACP acts as the anchor on which fatty acid biosynthesis takes place. Furthermore, RT‐PCR analysis revealed that the 5 gene fapR operon gene member plsX (encodes glycerol‐3‐phosphate acyltransferase), was up‐regulated in TTORS‐1. These findings indicate an alteration in fatty acid biosynthesis in TTORS‐1. S. aureus SCVs can result from the deletion of menB or cold shock protein cspB, and menB SCVs demonstrate a decrease in citric acid cycle activity. TTORS‐1 harbored less menaquinone biosynthetic protein MenB (1,4‐dihydroxy‐2‐naphthoyl‐CoA synthase), cold shock proteins CspB and CspC, ATP synthase subunit gamma, and proteins involved with the citric acid cycle. Collectively our data indicates that fatty acid biosynthesis is altered in TTORS‐1, as would be expected in an acpP mutant. We also demonstrate the reduced synthesis of certain proteins in TTORS‐1 mirroring what has been observed in previously described SCV phenotypes.Support or Funding InformationOklahoma Agricultural Experimental Station