Staphylococcus aureus produces several virulence factors that allow it to cause a variety of infections. One of the major virulence factors is the capsule, which contributes to the survival of the pathogen within the host as a way to escape phagocytosis. The production of the capsular polysaccharide is encoded in a 16 gene operon, which is regulated in response to several environmental stimuli including nutrient availability. For instance, the capsule is produced in the late-and post-exponential growth phases, but not in the early-or mid-exponential growth phase. Several regulators are involved in capsule production, but the regulation of the cap operon is still poorly understood. In this study, we show that MsaB activates the cap operon by binding directly to a 10 bp repeat in the promoter region. We show that despite the fact that MsaB is expressed throughout four growth phases, it only activates capsule production in the late-and post-exponential growth phases. Furthermore, we find that MsaB does not bind to its target site in the early and mid-exponential growth phases. This correlates with decreased nutrient availability and capsule production. These data suggest either that MsaB binding ability changes in response to nutrients or that other cap operon regulators interfere with the binding of MsaB to its target site. This study increases our understanding of the regulation of capsule production and the mechanism of action of MsaB.

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

Staphylococcus aureus utilizes the fatty acid (FA) kinase system to activate exogenous FAs for membrane synthesis. We developed a lipidomics workflow to determine the membrane phosphatidylglycerol (PG) molecular species synthesized by S. aureus at the thigh infection site. Wild-type S. aureus utilizes both host palmitate and oleate to acylate the 1 position of PG, and the 2 position is occupied by pentadecanoic acid arising from de novo biosynthesis. Inactivation of FakB2 eliminates the ability to assimilate oleate and inactivation of FakB1 reduces the content of saturated FAs and enhances oleate utilization. Elimination of FA activation in either ΔfakA or ΔfakB1 ΔfakB2 mutants does not impact growth. All S. aureus strains recovered from the thigh have significantly reduced branched-chain FAs and increased even-chain FAs compared to that with growth in rich laboratory medium. The molecular species pattern observed in the thigh was reproduced in the laboratory by growth in isoleucine-deficient medium containing exogenous FAs. S. aureus utilizes specific host FAs for membrane biosynthesis but also requires de novo FA biosynthesis initiated by isoleucine (or leucine) to produce pentadecanoic acid. IMPORTANCE The shortage of antibiotics against drug-resistant Staphylococcus aureus has led to the development of new drugs targeting the elongation cycle of fatty acid (FA) synthesis that are progressing toward the clinic. An objection to the use of FA synthesis inhibitors is that S. aureus can utilize exogenous FAs to construct its membrane, suggesting that the bacterium would bypass these therapeutics by utilizing host FAs instead. We developed a mass spectrometry workflow to determine the composition of the S. aureus membrane at the infection site to directly address how S. aureus uses host FAs. S. aureus strains that cannot acquire host FAs are as effective in establishing an infection as the wild type, but strains that require the utilization of host FAs for growth were attenuated in the mouse thigh infection model. We find that S. aureus does utilize host FAs to construct its membrane, but host FAs do not replace the requirement for pentadecanoic acid, a branched-chain FA derived from isoleucine (or leucine) that predominantly occupies the 2 position of S. aureus phospholipids. The membrane phospholipid structure of S. aureus mutants that cannot utilize host FAs indicates the isoleucine is a scarce resource at the infection site. This reliance on the de novo synthesis of predominantly pentadecanoic acid that cannot be obtained from the host is one reason why drugs that target fatty acid synthesis are effective in treating S. aureus infections.

Flavin adenine dinucleotide (FAD)-dependent bacterial oleate hydratases (OhyAs) catalyze the addition of water to isolated fatty acid carbon–carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regiospecific and stereospecific hydration is required to understand their biological functions and the potential for engineering new products. In this study, we deduced the catalytic mechanism of OhyA from multiple structures of S. aureus OhyA in binary and ternary complexes with combinations of ligands along with biochemical analyses of relevant mutants. The substrate-free state shows Arg81 is the gatekeeper that controls fatty acid entrance to the active site. FAD binding engages the catalytic loop to simultaneously rotate Glu82 into its active conformation and Arg81 out of the hydrophobic substrate tunnel, allowing the fatty acid to rotate into the active site. FAD binding also dehydrates the active site, leaving a single water molecule connected to Glu82. This active site water is a hydronium ion based on the analysis of its hydrogen bond network in the OhyA•PEG400•FAD complex. We conclude that OhyA accelerates acid-catalyzed alkene hydration by positioning the fatty acid double bond to attack the active site hydronium ion, followed by the addition of water to the transient carbocation intermediate. Structural transitions within S. aureus OhyA channel oleate to the active site, curl oleate around the substrate water, and stabilize the hydroxylated product to inactivate antimicrobial fatty acids.

Staphylococcus aureus is an important human pathogen that causes nosocomial and community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host’s immune response and antimicrobial agents. Biofilm development is very complex and involves several regulators that ensure cell survival on surfaces within the extracellular polymeric matrix. Previously, we identified the msaABCR operon as an additional positive regulator of biofilm formation. In this study, we define the regulatory pathway by which msaABCR controls biofilm formation. We demonstrate that the msaABCR operon is a negative regulator of proteases. The control of protease production mediates the processing of the major autolysin, Atl, and thus regulates the rate of autolysis. In the absence of the msaABCR operon, Atl is processed by proteases at a high rate, leading to increased cell death and a defect in biofilm maturation. We conclude that the msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm.