Oleate hydratase from Staphylococcus aureus protects against palmitoleic acid, the major antimicrobial fatty acid produced by mammalian skin

Abstract

Edited by Dennis R. Voelker Oleate hydratases (OhyAs) belong to a large family of bacterial proteins catalyzing the hydration or isomerization of double bonds in unsaturated fatty acids. A Staphylococcus aureus gene (Sa0102) is predicted to encode an OhyA. Here, we recombinantly expressed and purified SaOhyA and found that it forms a homodimer that requires FAD for activity. SaOhyA hydrates only unsaturated fatty acids containing cis-9 double bonds, but not fatty acids with trans-9 double bonds or cis double bonds at other positions. SaOhyA products were not detected in S. aureus phospholipids and were released into the growth medium. S. aureus does not synthesize unsaturated fatty acids, and the SaOhyA substrates are derived from infection sites. Palmitoleate (16:1(9Z)) is a major mammalian skin-produced antimicrobial fatty acid that protects against S. aureus infection, and we observed that it is an SaOhyA substrate and that its hydroxylated derivative is not antimicrobial. Treatment of S. aureus with 24 M 16:1(9Z) immediately arrested growth, followed by growth resumption after a lag period of 2 h. The ⌬ohyA mutant strain did not recover from the 16:1(9Z) challenge, and increasing SaOhyA expression using a plasmid system prevented the initial growth arrest. Challenging S. aureus with sapienic acid (16:1(6Z)), an antimicrobial fatty acid produced only by human skin, arrested growth without recovery in WT, ⌬ohyA, and SaOhyA-overexpressing strains. We conclude that SaOhyA protects S. aureus from palmitoleic acid, the antimicrobial unsaturated fatty acid produced by most mammals, and that sapienic acid, uniquely produced by humans, counters the OhyA-dependent bacterial defense mechanism. Bacterial oleate hydratase (OhyA) 2 activity (EC 4.2.1.53) was first detected in 1962 (1), and the product was characterized as 10(R)-hydroxy-18:0 using soluble enzyme preparations from

Exploring the abundance of oleate hydratases in the genus Rhodococcus—discovery of novel enzymes with complementary substrate scope

“…The kinetics of RpOhy showed a significant cooperative effect, which has rarely been reported for Ohys. Only recently for Staphylococcus aureus Ohy, similar cooperative kinetics was reported with a similar Hill coefficient of 2.2 ± 0.3 (Subramanian et al 2019). Most Ohys have been reported to display regular Michaelis-Menten kinetics with K M in the range from 0.1 to 0.6 mM and V max from 0.6 to 4 U/mg (Bevers et al 2009;Volkov et al 2010;Rosberg-Cody et al 2011;Joo et al 2012a;Kim et al 2012;Joo et al 2012b).…”

A fatty acid-binding protein of Streptococcus pneumoniae facilitates the acquisition of host polyunsaturated fatty acids

“…Each S. aureus fakB sequence was cloned into plasmid pCS119, which has a pCM28 backbone (31), no ribosomal binding site, and uses a sarA P1 promoter to drive expression. DNA sequences for S. pneumoniae strain TIGR4 fakB1 (SP_1557), fakB2 (SP_1112), and fakB3 (SP_0742) genes did not contain a ribosomal binding site or a His 6 tag and were cloned into pET28a (Novagen), to add an N-terminal His 6 tag, and ligated into pPJ480 (32), which is a pCS119 plasmid derivative containing the sarA P1 promoter as well as a ribosomal binding site. Restriction followed by ligation reactions were used to insert the fakB gene of interest into either pCS119 (S. aureus expression) or pPJ480 (S. pneumoniae expression).…”

Staphylococcus aureus Releases Proinflammatory Membrane Vesicles To Resist Antimicrobial Fatty Acids

“…However, efflux pumps FarE ( 20 ) and Tet38 ( 21 ) prevent the cellular accumulation of AFAs, which are still able to bind to S. aureus . Additionally, this bacterium possesses a functional oleate hydratase, which hydrates and thereby detoxifies AFAs containing cis -9 double bonds ( 22 ).…”

“…However, efflux pumps FarE (20) and Tet38 (21) prevent the cellular accumulation of AFAs, which are still able to bind to S. aureus. Additionally, this bacterium possesses a functional oleate hydratase, which hydrates and thereby detoxifies AFAs containing cis-9 double bonds (22). Strikingly, S. aureus grown for a few hours in the presence of subinhibitory amounts of AFAs survives subsequent exposures to otherwise bactericidal AFA concentrations (20,23), suggesting that the bacteria activate an AFA stress response program.…”