Oleate Hydratase (OhyA) Is a Virulence Determinant in Staphylococcus aureus

Abstract

Staphylococcus aureus is an important pathogen that relies on a variety of mechanisms to evade and counteract the immune system. We show that S. aureus uses oleate hydratase (OhyA) to convert host cis-9 unsaturated fatty acids to their 10-hydroxy derivatives in human serum and at the infection site in a mouse neutropenic thigh model. Wild-type and ΔohyA strains were equally infective in the neutropenic thigh model, but recovery of the ΔohyA strain was 2 orders of magnitude lower in the immunocompetent skin infection model. Despite the lower bacterial abundance at the infection site, the levels of interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), IL-1β, and tumor necrosis factor alpha (TNF-α) elicited by the ΔohyA strain were as robust as those of either the wild-type or the complemented strain, indicating that the immune system was more highly activated by the ΔohyA strain. Thus, OhyA functions to promote S. aureus virulence. IMPORTANCE The oleate hydratase protein family was discovered in commensal bacteria that utilize host unsaturated fatty acids as the substrates to produce a spectrum of hydroxylated products. These hydroxy fatty acids are thought to act as signaling molecules that suppress the inflammatory response to create a more tolerant environment for the microbiome. S. aureus is a significant human pathogen, and defining the mechanisms used to evade the immune response is critical to understanding pathogenesis. S. aureus expresses an OhyA that produces at least three 10-hydroxy fatty acids from host unsaturated fatty acids at the infection site, and an S. aureus strain lacking the ohyA gene has compromised virulence in an immunocompetent infection model. These data suggest that OhyA plays a role in immune modulation in S. aureus pathogenesis similar to that in commensal bacteria.

Keywords: Staphylococcus aureus; hydroxy fatty acids; oleate hydratase; soft tissue infection; unsaturated fatty acids; virulence; virulence determinants.

FIG 1

OhyA-dependent hFA formation in human serum and the neutropenic thigh infection model. (A) A representative gas chromatogram illustrating the fatty acid composition of the human serum lot with the average fatty acid composition from three replicates (inset). (B) Representative LC-MS/MS chromatograms of picolylamide derivatized hFA recovered from the medium following growth of S. aureus strains PDJ68 (ΔohyA) and PDJ68 (ΔohyA)/pOhyA in 50% human serum. (C) Quantification of hFA recovered from the medium following growth of S. aureus strains AH1263 (WT), PDJ68 (ΔohyA), and PDJ68 (ΔohyA)/pOhyA in 50% human serum. (D) Enumeration of the bacteria recovered from infected neutropenic thighs. The gray shaded bar represents the range of initial inoculum. Numbers of animals are in parentheses. (E) Representative total ion chromatograms of the fatty acid fraction in the LC-MS/MS analysis of mock-infected (black) and infected (red) neutropenic thighs. (F) Quantification of hFA recovered from neutropenic thighs infected with the strain set. ND means <5 pmol. Mean ± standard error of the mean (SEM).

FIG 2

OhyA is a virulence determinant in an SSTI infection model. (A) Enumeration of the bacteria recovered from the infection site. S. aureus strains AH1263 (WT), PDJ68 (ΔohyA), and PDJ68 (ΔohyA)/pOhyA were used to infect mice by subcutaneous infection. Mock-infected mice were given an injection of sterile phosphate-buffered saline (PBS). Kruskal-Wallis test determined whether overall differences between groups have statistical significance, and P values were calculated using Mann-Whitney test. The gray shaded bar represents the range of initial inoculum determined by serial dilution. (B to E) Measurements of cytokine analytes that were recovered from the infection sites in the SSTI model. Data are normalized to the number of bacteria recovered. The cytokine levels per infection site are shown in Figure S3. (F) Model for OhyA-dependent hFA production at the infection site. Unsaturated cis-9 fatty acids (UFA) (16:1, 18:1, 18:2) are converted to hydroxy fatty acids (hFA) (h16:0, h18:0, h18:1) by OhyA. hFA are released into the extracellular environment. This process inactivates the antimicrobial fatty acids (16:1 and 18:2) and generates mediators that inhibit cytokine production by mechanisms that remain to be delineated. Numbers of animals are in parentheses.