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.

to infection followed by another cyclophosphamide (100 mg/kg) injection 2 days prior to infection.
Bacteria were grown in Luria broth overnight at 37C and back diluted in fresh medium and allowed to grow to an OD600 = 0.4. Bacteria were washed twice with sterile phosphate buffered saline (PBS) and suspended into fresh PBS to obtain an inoculum of 2 x 10 6

Skin-soft tissue (SSTI) infection model. The mouse SSTI model was developed to investigate
factors related to staphylococcal pathogenesis of skin and soft tissue infections, including the ability to adapt to environmental changes and the production of specific bacterial molecules that contribute to virulence (10). Bacteria were enumerated to determine the exact number of bacteria in each infection dose. The SSTI model used 8-week-old female Balb/C mice that were injected subcutaneously with 5 x 10 7 CFUs and the infection allowed to proceed for 24 hours. Mock infections were carried out by injecting sterile PBS. The infected tissues were excised and homogenized in 1 ml of ice-cold PBS for cytokine analysis and determination of the bacterial burden by serial dilution and plating.
Lipid extractions. Two individual thighs from neutropenic mice were pooled together, homogenized, and the lipids from 100 l of the homogenate supernatant were extracted using the Bligh and Dyer method (11). The lipid extracts were dried under N2, resuspended and analyzed by LC-MS/MS as described (12). The ion current in the free fatty acid peak was measured and reported in Fig. 1E. To quantify the hFA, the tissue homogenates were spiked with [U-13 C]18:1 as the internal standard, and the lipids were extracted (11). The fatty acid fraction was isolated using a solid phase column (Discovery DSC-NH2, 500 mg) conditioned with 8 ml of hexane (12)(13)(14). Non-polar lipids were eluted with 6 ml of 2:1 (v/v) chloroform:isopropanol; fatty acids were eluted with 6 ml of ether + 2% acetic acid; PC and PE were eluted with 6 ml of methanol; and PG and PI were eluted with 6 ml of chloroform:methanol:0.8 M sodium acetate 60:30:4.5 (v/v/v). The fatty acid fraction was dried under N2 and the free fatty acids were converted to their 3picolylamide derivatives (15) and analyzed by LC-MS/MS as described below. Alternately, the hFA were analyzed without derivatization by LC-MS/MS in the negative mode (16) as described below.
Lipid mass spectrometry. Lipid extracts were resuspended in chloroform/methanol (1:1). FA and hFA were analyzed using a Shimadzu Prominence UFLC attached to a QTrap 4500 equipped with a Turbo V ion source (Sciex). Samples (5 l) were injected onto an Acquity UPLC BEH HILIC, 1.7 m, 2.1 x 150 mm column (Waters) at 45C with a flow rate of 0.2 ml/min. Solvent A was acetonitrile, and solvent B was 15 mM ammonium formate, pH 3. The HPLC program was the following: starting solvent mixture of 96% A/4% B; 0 to 2 min, isocratic with 4% B; 2 to 20 min, linear gradient to 80% B; 20 to 23 min, isocratic with 80% B; 23 to 25 min, linear gradient to 4% B; 25 to 30 min, isocratic with 4% B. The QTrap 4500 was operated in the Q1 negative mode.
The ion source parameters for Q1 were as follows: ion spray voltage, -4,500 V; curtain gas, 25 psi; temperature, 350C; ion source gas 1, 40 psi; ion source gas 2, 60 psi; and declustering potential, -40 V. The system was controlled and analyzed by the Analyst software (Sciex).
Picolylamide-hFA were analyzed using a Shimadzu Prominence UFLC attached to a QTrap 4500 equipped with a Turbo V ion source (Sciex). Samples (5 l

FIG S2
The impact of Geh lipase on hFA formation in human serum. Quantification of hFA recovered from the media following growth of S. aureus strains PDJ171 (geh) and PDJ171(geh)/pGeh in 50% human serum as compared to AH1263 (WT) (Fig. 1C). ND means < 0.01 pmoles/l.