Transcriptomic Responses and Survival Mechanisms of Staphylococci to the Antimicrobial Skin Lipid Sphingosine

Abstract

Sphingosines are antimicrobial lipids that form part of the innate barrier to skin colonization by microbes. Sphingosine deficiencies can result in increased epithelial infections by bacteria including Staphylococcus aureus. Recent studies have focused on the potential use of sphingosine resistance or its potential mechanisms. We used RNA-Seq to identify the common d-sphingosine transcriptomic response of the transient skin colonizer S. aureus and the dominant skin coloniser S. epidermidis. A common d-sphingosine stimulon was identified that included downregulation of the SaeSR two-component system (TCS) regulon and upregulation of both the VraSR TCS and CtsR stress regulons. We show that the PstSCAB phosphate transporter, and VraSR offer intrinsic resistance to d-sphingosine. Further, we demonstrate increased sphingosine resistance in these staphylococci evolves readily through mutations in genes encoding the FarE-FarR efflux/regulator proteins. The ease of selecting mutants with resistance to sphingosine may impact upon staphylococcal colonization of skin where the lipid is present and have implications with topical therapeutic applications.

Keywords: Staphylococcus; drug resistance evolution; resistance; skin.

FIG 1

Comparison of up- and downregulated genes of S. aureus and S. epidermidis after d-sphingosine challenge. Genes with homologs in both species are indicated in the central ovals, whereas genes without homologues are shown in the peripheral ovals. Common differentially expressed genes are indicated in overlapping portions of the colored shapes, comprising the d-sphingosine challenge stimulon.

FIG 2

Transcription of key genes postchallenge with sphingosine and their role in resistance. (a) Transcription of pstSCABphoU for control untreated (U, blue) and treated cells (T, purple) of S. aureus and S. epidermidis. Read counts were normalized by total reads per library. Representative data of 3 replicates is shown. (b) qPCR of pstB and vraX across different S. aureus and S. epidermidis strains showing fold change after challenge with d-sphingosine, average fold change and standard error for 3 replicates is shown. (c) d-sphingosine MICs of S. aureus wild type and mutant strains with standard error for 3 replicates.

FIG 3

d-sphingosine MIC of experimentally evolved isolates. d-sphingosine MICs S. aureus (a) and S. epidermidis (b) from passage days 0–9 and 0–23 respectively. For each day of sampling, individual isolates of separate growth passages are labeled A, B, C. Data represent a minimum of 3 independent assays. White bars indicate the representative isolates selected for whole genome sequencing.

FIG 4

Variants and MICs of S. aureus and S. epidermidis experimental evolution isolates. SNPs that occurred in more than one isolate, within a gene and caused non-synonymous changes are shown. Dotted line indicates d-sphingosine MIC of WT.

FIG 5

Role of FarE/FarR in d-sphingosine resistance. (a) S. aureus SH1000 farR::tet and farE::tet mutant MICs compared to WT. The mean and standard error of 3 replicates is shown. (b) Survival of SH1000 WT, farR::tet and farE::tet in 15 μM d-sphingosine after overnight growth in control conditions (circles) or with 5 μM d-sphingosine (crosses). Representative data from 2 replicates is shown.