The Phospholipase A1 Activity of Glycerol Ester Hydrolase (Geh) Is Responsible for Extracellular 2-12(S)-Methyltetradecanoyl-Lysophosphatidylglycerol Production in Staphylococcus aureus

ABSTRACT Phosphatidylglycerol (PG) is the major membrane phospholipid of Staphylococcus aureus and predominately consists of molecular species with ≥16-carbon acyl chains in the 1-position and anteiso 12(S)-methyltetradecaonate (a15) esterified at the 2-position. The analysis of the growth media for PG-derived products shows S. aureus releases essentially pure 2-12(S)-methyltetradecanoyl-sn-glycero-3-phospho-1′-sn-glycerol (a15:0-LPG) derived from the hydrolysis of the 1-position of PG into the environment. The cellular lysophosphatidylglycerol (LPG) pool is dominated by a15-LPG but also consists of ≥16-LPG species arising from the removal of the 2-position. Mass tracing experiments confirmed a15-LPG was derived from isoleucine metabolism. A screen of candidate secreted lipase knockout strains pinpointed glycerol ester hydrolase (geh) as the gene required for generating extracellular a15-LPG, and complementation of a Δgeh strain with a Geh expression plasmid restored extracellular a15-LPG formation. Orlistat, a covalent inhibitor of Geh, also attenuated extracellular a15-LPG accumulation. Purified Geh hydrolyzed the 1-position acyl chain of PG and generated only a15-LPG from a S. aureus lipid mixture. The Geh product was 2-a15-LPG, which spontaneously isomerizes with time to a mixture of 1- and 2-a15-LPG. Docking PG in the Geh active site provides a structural rationale for the positional specificity of Geh. These data demonstrate a physiological role for Geh phospholipase A1 activity in S. aureus membrane phospholipid turnover. IMPORTANCE Glycerol ester hydrolase, Geh, is an abundant secreted lipase whose expression is controlled by the accessory gene regulator (Agr) quorum-sensing signal transduction pathway. Geh is thought to have a role in virulence based on its ability to hydrolyze host lipids at the infection site to provide fatty acids for membrane biogenesis and substrates for oleate hydratase, and Geh inhibits immune cell activation by hydrolyzing lipoprotein glycerol esters. The discovery that Geh is the major contributor to the formation and release of a15-LPG reveals an unappreciated physiological role for Geh acting as a phospholipase A1 in the degradation of S. aureus membrane phosphatidylglycerol. The role(s) for extracellular a15-LPG in S. aureus biology remain to be elucidated.

donors in biosynthetic pathways to other molecules (4). A well-studied example is the use of phosphatidylethanolamine (PE), the major membrane phospholipid of Escherichia coli, as a substrate for phospholipid:apolipoprotein transacylase (Lnt) (5,6). Lnt catalyzes the transfer of the acyl chain at the 1-position of PE to the lipoprotein amino terminus. The use of PE as an acyl donor, rather than acyl-acyl carrier protein (ACP), is required because this terminal reaction in lipoprotein maturation occurs outside the cell. The resulting 2-acyl-sn-glycero-3-phosphoethanolamine (2-acyl-LPE) is transported into the cell interior by LplT (7) and the 1-position is acylated by the acyl-ACP dependent 2-acyl-LPE acyltransferase (8,9). Staphylococcus aureus also uses membrane phospholipid as the substrate in the N-terminal acylation of lipoproteins by LnsAB, a heterodimeric phospholipid:apolipoprotein transacylase with the subunits derived from separate genes (10). S. aureus LnsAB would use phosphatidylglycerol (PG), the most abundant membrane phospholipid in this organism. The composition of lysophosphatidylglycerols (LPG) and how they are metabolized or reintroduced into the biosynthetic pathway has not been studied in S. aureus.
S. aureus is an important human pathogen that secretes a wide spectrum of protein factors that promote virulence (11,12). Glycerol ester hydrolase (Geh, SAUSA3300_0320) is a well-known lipase that is one of the most abundant exoproteins (13) that is produced in nearly all S. aureus isolates (14). The transcription of the geh gene depends on the activity of the Agr quorum-sensing signal transduction system that acts as a master regulator of S. aureus virulence factor transcription (12). Geh has a role in virulence based on its hydrolysis of host lipids at the infection site to provide fatty acids for membrane biogenesis (15 to 18), substrates for oleate hydratase (19,20), and by hydrolyzing lipoprotein glycerol esters to attenuate immune cell activation (21). The primary geh translation product is preproGeh, and the 37-residue signal sequence is removed concomitant with its transport out of the cell (22). The extracellular Geh (proGeh; 75 kDa) is cleaved by the aureolysin metalloprotease to mature Geh (mGeh; 44 kDa) that contains the catalytic triad (22,23). The function of the pro-peptide is unclear because both forms appear equally active in vitro (23,24) and both proGeh and mGeh are abundant exoproteins (23,25). Because Geh is a secreted lipase that hydrolyzes host lipids (15)(16)(17)(18), most research has focused on its potential role in promoting tissue invasion and pathogenesis (26). Here, we show that Geh has a physiological role in S. aureus membrane phospholipid homeostasis. Geh acts as a 1-position specific phospholipase A1 responsible for the formation of 2-12(S)-methyltetradecanoyl-sn-glycero-3-phospho-sn-19-glycerol (a15-LPG) that is released into the extracellular environment.

RESULTS
Composition of the S. aureus LPG pool. S. aureus PG has a strict positional distribution of fatty acids when grown in rich broth (27)(28)(29). The sn-2-position is dominated by 12(S)-methyltetradecanoic acid (a15), and the sn-1-position contains $16-carbon acyl chains ($16) giving rise to the prototypical PG molecular species distribution annotated in Fig. S1. The cellular LPG pool composition was determined by LC/MS/MS (Fig. 1). The most abundant LPG was a15-LPG (Fig. 1A), which implies that it arises from the removal of the 1-position acyl chain of PG. The other LPGs contain acyl chains normally found in the 1-position of PG and would arise from the removal of the a15 from the 2-position. The composition of the $16-LPGs (Fig. 1A, Inset) reflects their relative abundance in the PG molecular species (Fig. S1). We detected substantial quantities of a15-LPG in S. aureus conditioned media, whereas the extracellular concentrations of the $16-LPGs were almost below detection (Fig. 1B). The growth media alone had undetectable a15-LPG (,0.6 pmol/mL). These data suggest that the bulk of the a15-LPG produced by S. aureus was released into the environment.
The abundance of the LPG molecular species in different fractions from 1 mL of cultured wild-type strain AH1263 cells were quantified using the [d5]17-LPG internal standard (Fig. 2). The cellular LPG pool was a mixture of a15-LPG and $16-LPG ( Fig. 2A). The media samples were filtered through a 0.2 mm filter to eliminate intact cells, and a15-LPG was the dominate component of the media fraction (Fig. 2B). However, exosomes or other membrane fragments may pass through the filter. The filtered media sample was centrifuged a 100,000 Â g for 60 min to pellet exosomes, and the pellet and supernatant fractions analyzed for LPG (Fig. 2). The $16-LPGs molecular species in the pellet fraction reflected the composition of the cells; however, a15-LPG was enriched relative to $16-LPGs in the media vesicle fraction (Fig. 2C) compared to the cells ( Fig. 2A). Only a small fraction of extracellular a15-LPG was associated with the vesicle fraction ( Fig. 2C and 2D). The bulk of the a15-LPG was recovered in the supernatant from the high-speed centrifugation, and this fraction contained only a minor contamination with 14-LPG (Fig. 2D). Lysophospholipids can partition into phospholipid bilayers but are also water soluble. For example, 14-LPG solubility is ;300 mM (30) so finding a15-LPG in the medium is consistent with the biophysical properties of the molecule.
Mass spectrometry alone cannot directly determine whether a 15-carbon LPG has a straight chain or has an iso or anteiso branch. We performed a mass tracing experiment using strain AH1263 grown in defined media where the normal concentrations of branched-chain amino acids were replaced with [ 13 C 6 ]isoleucine, [d 3 ]leucine, and [ 13 C 5 , 15 N] valine to impart specific mass tags into the derivative branched-chain fatty acids allowing the identification of their origin and structure (29). The anteiso odd-carbon acyl chains derived from Ile have a 15-mass tag, the iso odd-carbon acyl chains have a 13-mass tag derived from Leu and iso even carbon acyl chains have a 14-mass tag derived from Val (29). The 15-LPG species in both the cells (Fig. S2A) and the media (Fig. S2B) predominantly contained the 15-mass tag derived from Ile, meaning that it is a15. There was also a normal mass (unlabeled) 15-LPG peak that arises from 2-methylbutyryl-CoA derived from the de novo Ile biosynthetic pathway, and a smaller proportion of Leu-derived (13) iso 15-carbon acyl chains. These data show that a15:0 is the predominant acyl chain in extracellular LPG.
Identification of Geh as the source of extracellular a15-LPG. The appearance of extracellular a15-LPG suggested that an extracellular lipase/phospholipase may be involved. There are three major secreted lipases in S. aureus: a short-chain esterase/lipase (Lip or SAL1), glycerol ester hydrolase (Geh or SAL2), and (SAL3) (11,31). The concentrations of extracellular a15-LPG produced by strains from the Nebraska transposon library (32) containing inactivating insertions in each of these lipase genes (Table S1) were compared to the parental strain JE2. There was no impact on the size or composition of the cellular $16-LPG pools in the three knockout strains compared to strain JE2 (Fig. 3A). The cellular a15-LPG concentration was reduced by half in strain NE338 (geh::f NR) but was not reduced in the other knockouts (Fig. 3A). Strain NE338 (geh::f NR) was significantly (P , 0.001) deficient in the production of extracellular a15-LPG (Fig. 3B). The fact that a pool of cellular a15-LPG remained present in the geh::f NR strain means that there is also a Geh-independent source of cellular a15-LPG.
These results were validated by constructing strain PDJ171 (Dgeh), a derivative of wildtype strain AH1263 that was constructed to contain an unmarked deletion of the geh gene coding sequence (20). The production of extracellular a15-LPG was measured in strains AH1263/pPJ480, PDJ171 (Dgeh)/pPJ480 containing the empty vector, and strain PDJ171 (Dgeh)/pGeh programed to express geh driven by the sarA promoter (Table S1). This Dgeh strain was also severely deficient in the production of extracellular a15-LPG, which was restored by the plasmid-driven expression of the geh gene (Fig. 3B, Inset). Orlistat, a covalent inhibitor of pancreatic lipase, also blocks Geh activity (33). The treatment of strain AH1263 with 20 mg/mL Orlistat significantly reduced the formation of extracellular a15-LPG (Fig. 3C). Orlistat treatment also reduced the cellular concentration of a15-LPG ( Fig. 3C) similar to the reductions in cellular a15-LPG noted in the Dgeh strain (Fig. 3A). These experiments show Geh is the most important source of extracellular a15-LPG.
Geh biochemistry. The geh gene encodes preproGeh (34) (Fig. S3A). The signal sequence (amino acids 1 to 37) is cleaved coincident with the export of the protein  through the plasma membrane to form proGeh. Then, a proportion of proGeh is cleaved by aureolysin, a secreted metalloprotease, to produce mature Geh (mGeh) (23, 35) (Fig. S3A). Both proGeh and mGeh are abundant extracellular proteins (23), and in the prior experiments with cells, Geh refers to the mixture of proGeh and mGeh that normally arise in S. aureus from expression of the geh gene. Prior biochemical work with the two isoforms has not revealed a difference in their activities toward the substrates employed in the studies (23,24). Geh is considered a nonspecific lipase (36) that at sufficient concentrations of enzyme and substrate is capable of hydrolyzing a wide array of long-chain esters, including p-nitrophenyl esters of butyrate (33) or palmitate (21), triacylglycerols (36), and diacyl-glycerol-modified lipoproteins (21). However, for Geh to be responsible for a15-LPG formation, its physiological function would be to act as a 1-position-specific phospholipase A1, an activity that has not been studied in Geh.
Both proGeh with an amino-terminal His tag (Fig. S3B) and mGeh with a carboxyterminal His tag (Fig. S3C) were expressed in E. coli and purified by Ni 21 affinity and gel filtration chromatography. The proGeh was primarily a dimer, whereas mGeh was a mixture of monomers and dimers based on gel filtration chromatography ( Fig. S3B and S3C). Lipid metabolic enzymes exhibit stereo-selectivity for utilizing either the 1-or 2positions of the sn-glycerol-3-phosphate backbone. Bee venom phospholipase A2 (PLA2) was used as a control for a phospholipase that selectively hydrolyzes acyl chains at the 2-position of phospholipids. S. aureus was labeled with [1-14 C]oleate and the PG fraction was isolated by solid-phase chromatography. Oleate is selectively incorporated into the 1-position of PG (27,37), therefore [ 14 C]fatty acid is formed by a phospholipase A1 reaction and [ 14 C]LPG is formed by a phospholipase A2 reaction. The [ 14 C]PG was digested with either proGeh or PLA2 and the products separated by thin-layer chromatography (Fig. 4A). The product of the Geh digest was [ 14 C]fatty acid indicating hydrolysis of the 1-position acyl chain, and [ 14 C]LPG was the PLA2 product indicating hydrolysis at the 2-position acyl chain.
Product formation by proGeh and PLA2 was examined further using 1-palmitoyl-2oleoyl-PG (1-16:0-2-18:1-PG) as the substrate at pH 6.0 followed by immediate characterization of the products by LC-MS/MS. The 1-and 2-position LPGs are separated from each other in the liquid chromatography fractionation step in the LC-MS/MS workflow ( Fig. 1) (38). The product of the proGeh reaction was 2-18:1-LPG and the PLA2 product was 1-16:0-LPG (Fig. 4B) showing that proGeh is a 1-position-specific phospholipase A1 and that PLA2 selectively hydrolyzes the 2-position. The acyl chains of lysophospholipids undergo spontaneous migration between the 1-and 2-positions of the glycerol backbone, and at equilibrium they reach about 90% 1-position (39)(40)(41). Suppressing this acyl migration during the analytical workflow was important to obtaining the data in Fig. 4B. Acyl chain migration is base catalyzed (39)(40)(41). Performing Geh assays at pH 8.5 followed by the immediate injection and analysis of the products resulted in detecting a mixture of 1-and 2-18:1-LPGs (Fig. S5A). If the sample was analyzed 12 h later, the 1-18:1-LPG isomer was the predominant form detected (Fig. S5B). When the Geh reaction was performed at pH 6.0, 2-18:1-LPG was the major product with only a minor amount 1-acyl-LPG detected (Fig. S5C). At this pH, only about 10% of the sample isomerized to the 1-position after 12 h (Fig. S5D). Thus, performing Geh assays at pH 6.0 greatly minimizes acyl chain migration, and coupled with an immediate analysis of the products by LC-MS/MS, provides a clear indication of the enzyme's positional specificity. In practice, samples are usually extracted, dried, and resuspended in solvent. It is common for sample preparation to take a day and for the samples to sit in the autosampler for 12-24 h before injection when a series of experiments with multiple samples are being analyzed. It is not possible to prevent acyl chain migration in these circumstances resulting in the detection of a mixture of isomers in most experiments. We also tested if both proGeh and mGeh were active in the formation of 2-acyl-LPG and found that both enzyme forms had robust phospholipase A1 activity, although mGeh was slightly less active under these conditions (Fig. 4C). The hydrolysis of a S. aureus lipid extract with proGeh at pH 6.0 with immediate injection showed that 2-a15-LPG was the only LPG detected (Fig. 4D). S. aureus plasma membranes were isolated by lysostaphin treatment, sonication and ultracentrifugation (42). The isolated membranes incubated with proGeh generated large quantities of a15-LPG (Fig. S4) illustrating that the membrane PG is a substrate for Geh. These data show that both proGeh and mGeh are phospholipase A1 enzymes and that the biologically relevant Geh product is 2-a15-LPG. This biologically relevant product undergoes acyl chain migration during sample workup and analysis to give rise to the two peaks of a15-LPG observed in the chromatograms (Fig. 1).

DISCUSSION
The discovery that Geh is the major contributor to the formation and release of a15-LPG in S. aureus reveals an unappreciated physiological role for Geh acting as a phospholipase A1 in the degradation of S. aureus membrane PG (Fig. 5A). It is known that the acyl chains of S. aureus PG turnover and that the resulting fatty acids are metabolized by the fatty acid kinase system (Fig. 5A). Fatty acid kinase consists of a kinase domain protein (FakA) that phosphorylates a fatty acid bound to a fatty acid binding protein (FakB) (37,(43)(44)(45). Fatty acid kinase knockout strains have an elevated cellular fatty acid pool that consists of $16 fatty acids derived from the 1-position of PG (46). These data suggested that the a15 acyl chain in the 2-position of PG is used for another purpose and is not reincorporated into the membrane. This work identifies the Geh-dependent release of a15-LPG into the environment, leaving the fatty acid product associated with the cells, as one source of cellular $16 fatty acid arising from PG turnover. The fatty acid kinase system activates these fatty acids to acyl-phosphates FIG 5 Role of Geh as a phospholipase A1 in membrane PG turnover. (A) Geh cleaves the one position fatty acid of PG, the major membrane phospholipid of S. aureus. The bulk of the a15-LPG product is released from the cell. The fatty acid product ($16) is activated by the fatty acid kinase system that consists of the kinase domain protein (FakA) that phosphorylates the fatty acid bound to the fatty acid binding protein (FakB). The resulting acyl-phosphate ($16;P) is a substrate for PlsY, the glycerol-phosphate acyltransferase that initiates PG synthesis. PlsC specifically utilizes a15-ACP arising from the fatty acid elongation cycle (FASII). Phospatidic acid (PA) is converted to PG in 3 steps. The cycle regenerates the same spectrum of PG molecular species that Geh hydrolyzed, thus maintaining membrane compositional homeostasis. (B) 1-Palmitoyl-2-oleoyl-PG docked into the active site cavity of Geh (PDB 6KSM) with the carbonyl of the sn-1 acyl chain positioned in the oxyanion hole created by Ser116 and Phe17. An initial docking solution was obtained using the SwissDock server, and energy minimization of the ligand was performed using Molecular Operating Environment (MOE) (2018.01; Chemical Computing Group). The sn-1 and sn-2 position fatty acids extend into two hydrophobic substrate channels in Geh, and the PG headgroup (GroP) lies in the more polar cavity. (C) Surface representation of the Geh substrate cavity with PG docked in the Geh active site. Electrostatic potential surface of Geh was drawn using the Adaptive Poisson-Boltzmann Solver (APBS) package within PyMOL. The electrostatic potential ranges from 25 to 5 kT/e. Red is negative, blue is positive and white is neutral/hydrophobic potential. All structures were rendered with PyMOL (version 2.5.1, Schrödinger, LLC). and they are reintroduced into the phospholipid biosynthetic pathway at the PlsY step (Fig. 5A). Geh is not the only source for a15-LPG, which may arise from N-terminal acylation of lipoproteins by LnsAB (10), other cellular biosynthetic processes or other a/b-hydrolases. PlsC is highly specific for a15-ACP arising from the fatty acid elongation cycle. Therefore, this PG turnover cycle maintains membrane compositional homeostasis by regenerating the same spectrum of PG molecular species that were degraded by Geh to initiate the turnover cycle (Fig. 5A).
The role(s) for extracellular a15-LPG in S. aureus biology remain to be elucidated. Bacillus subtilis also releases a15-LPG into the environment (47). These investigators named the molecule bacilysocin and proposed it may function as an antibiotic against certain fungi. The fact a15-LPG release is common to these related bacteria that inhabit different environments suggests there may be a shared physiological function for the process. PG hydrolysis by extracellular Geh would create a15-LPG on the outer leaflet of the PG bilayer with the potential to introduce positive curvature to the membrane. The role of bilayer curvature introduced by asymmetric lysophospholipid generation in the formation of budding vesicles in other systems (48,49) suggests that Geh hydrolysis of the outer leaflet of the PG bilayer may function similarly to facilitate formation and budding of exosomes (50)(51)(52). The a15-LPG content of the media vesicle fraction shows it is enriched in a15-LPG compared to the cellular membrane; however, more research is needed to determine if this correlation is functionally relevant. The bulk of the extracellular a15-LPG is soluble, and a signaling role for a15-LPG in S. aureus is possible. Lysophospholipids are established second messengers in mammalian biology (1) suggesting that LPG release may have a role in either S. aureus signaling and/or virulence by altering host responses. Although the role of LPG in signaling is less studied than other lysophospholipids (1), it is known that LPG specifically triggers calcium mobilization and phospholipase C activation in cell lines (53), is a competitive inhibitor of the LPA receptor in Jurkat T cells (54), and stimulates chemotactic migration of natural killer cells (55,56). Validating the potential targets for a15-LPG action in modulating host responses will be important to understanding the role of this molecule in pathogenesis.
PG modeled into the active site of Geh using the GehOrlistat complex structure provides insight into Geh phospholipase A1 activity (Fig. 5B). The sn-2 chain of PG lies in the hydrophobic cavity occupied by the Orlistat b-side chain and the sn-1 acyl chain sits in the space occupied by the a-side chain of Orlistat (Fig. S6A). The glycerol head group sits in the cavity occupied by the shorter and more polar amino ester side chain of Orlistat ( Fig. 5B and S6A). The sn-1 ester is positioned adjacent to the active site serine with the carboxyl group sitting in the oxyanion hole formed by the backbone amide protons of Ser116 and Phe17 (Fig. 5B). The fixed orientation of the two acyl chains in the active site cavity (Fig. 5C) explain the selectivity of Geh for the sn-1 position of PG. The Geh homolog from S. hyicus (Shl) has robust lipase and phospholipase A1 activity (22,57). The similarities between the crystal structures of S. hyicus Shl and Geh are striking (Fig. S6B). The only region where the two proteins diverge is in the relative orientation of the lid, which is a dynamic region consisting of two a-helices that move to allow substrate entry to and exit from the active site (58). Modeling dioctanoyl-phosphatidylcholine into the Shl active site yields similar conclusions concerning the positioning of the sn-1 ester for hydrolysis (59). The Lip lipase of S. aureus, which does not have a homolog in S. hyicus, is most active on short-chain triacylglycerols and lacks phospholipase A1 activity (57). Understanding the structural differences between Geh and Lip that confer the substrate selectivity of the two proteins awaits crystal structures of Lip.

MATERIALS AND METHODS
Materials. All chemicals and reagents were reagent grade or better. Bacterial strains and media. S. aureus strains are listed in Table S1. Defined media components and other reagents were purchased from Millipore Sigma or Thermo Fisher Scientific. All mass spectrometry reagents are HPLC grade or better. The defined media used in this work consists of M9 salts supplemented with glucose, amino acids and other nutrients exactly as described (29). Defined media consisted of M9 salts, 1 mM MgSO 4 , 0.1 mM CaCl 2 , 15 mM vitamin B 1 , 32 mM vitamin B3, 0.4% glucose, 0.1 mg/L biotin, 2 mg/L pantothenic acid, 10 mM FeCl 2 , 6 mg/L citrate, 10 mg/L MnCl 2 , 4 mg/L L-tryptophan, 0.1 mg/L L-lipoic acid; and the amino acid concentrations were the same as in RPMI 1640 media (Sigma-Aldrich). S. aureus strains were routinely grown in rich broth (10 g tryptone, 5 g yeast extract, 5 g NaCl per L). Bacterial strains were grown in 15 mL culture tubes containing 3 mL rich broth overnight at 37°C with shaking at 200 rpm. The bacteria were subcultured in 30 mL culture flasks containing 10 mL fresh rich broth at OD 600 0.05 and grown to OD 600 4.0 at 37°C with shaking at 200 rpm.
Molecular biology. The geh gene was amplified by PCR using primers designed for Gibson Assembly cloning into NdeI and HindIII digested pET28a to obtain pPJ628 with a carboxy-terminal His tag used for E. coli expression and purification of proGeh and mGeh. These His-tagged constructs were then cloned into pPJ480 at the NcoI and HindIII sites to derive plasmid pPJ630 for expression in S. aureus driven by the sarA promoter (20). Plasmids were rendered transformable into normal S. aureus strains by passing them through strain RN4220.
LPG extraction and isoleucine labeling. Strains AH1263, PDJ171 (Dgeh) (20), JE2, NE338 (SAUSA300_ 2603::f NR), NE1175 (SAUSA300_0320::f NR) or NE104 (SAUSA300_0641::f NR) were grown in rich broth to a A 600 of 4.0. LPG was extracted from 5 mL of cells or 1 mL of supernatant from 0.2 mm filtered media. The cells were resuspended in 0.5 mL water and 0.5 mL of cold methanol containing 1% acetic acid was added. To the 1 mL of filtered media, 1 mL of cold methanol containing 1% acetic acid was added. Samples were incubated on ice for 10 min and centrifuged at 20,000 Â g for 20 min. Supernatants were dried in a speed vac concentrator, resuspended in 80% methanol and 500 picograms of [d5]17-LPG was added. For complementation experiments, strain AH1263 containing the vector pPJ480, strain PDJ171 (Dgeh) containing either the pPJ480 or pPJ630 vectors were grown in rich broth plus 10 mg/mL chloramphenicol to an A 600 of 4.0. LPG was extracted from cells and media as described above. For heavy labeling of LPG, AH1263 cells were grown on defined media plates and grown overnight in defined media. The next morning, cells were washed with media containing no isoleucine, leucine or valine and then inoculated into defined media containing 0.5 mg/mL [ 13 C 6 ] isoleucine, 0.2 mg/mL [ 13 C 5 , 15 N]valine and 0.5 mg/mL of [d3]leucine at an OD 600 of 0.05, and then grown to an A 600 of 1.0. LPG was extracted from cells and media as described above. AH1263 and PDJ171 cells were treated with 20 mg/mL of Orlistat and grown to a final A 600 of 4.0. LPG was extracted from cells and media as described above.
LPG mass spectrometry. LPG was analyzed using a Shimadzu Prominence UFLC attached to a QTrap 4500 equipped with a Turbo V ion source (Sciex). Samples were injected onto an Acquity UPLC HSS C18, 2.5 mm, 3.0 Â 150 mm column at 30°C (Waters) using a flow rate of 0.2 mL/min. Solvent A was 5 mM ammonium acetate 1 0.1% formic acid, and Solvent B was 95% methanol 1 5 mM ammonium acetate 1 0.1% formic acid. The HPLC program was the following: starting solvent mixture of 35% A/ 65% B, 0 to 1 min isocratic with 65% B; 1 to 3 min linear gradient to 100% B; 3 to 30 min isocratic with 100% B; 30 to 32 min linear gradient to 65% B; 32 to 35 min isocratic with 65% B. The QTrap 4500 was operated in the negative mode, and the ion source parameters were: ion spray voltage, -4500 V; curtain gas, 30 lb/in 2 ; temperature, 500°C; collision gas, medium; ion source gas 1, 20 lb/in 2 ; ion source gas 2, 35 lb/in 2 ; decluttering potential, -80 V; and collision energy, -30 V. The multiple reaction monitoring transitions for LPG species are listed in Table S2. [d5]17-LPG was used as the internal standard. The system was controlled by the Analyst software (Sciex) and analyzed with MultiQuan 3.0.2 software (Sciex). Data used in the figures was output using the Gaussian smoothing routine in the Analyst software (Sciex). Peaks corresponding to individual LPG species were quantified relative to the internal standard. The lower limit of LPG detection based on a standard curve using 16-LPG was 5 femtomoles.
Purification of Geh and size exclusion chromatography. pPJ628 (proGeh) and pPJ650 (mGeh) expression vectors were transformed into BL21(DE3) cells and grown in rich broth to an OD 600 of 0.7 at 37°C with shaking at 200 rpm. The culture was cooled to 16°C and the cells were induced with 1 mM IPTG overnight with shaking at 200 rpm. Cells were harvested and resuspended in 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM imidazole. Cells were broken by two passages through a cell disruptor and centrifuged at 20,000 g for 45 min. Geh was purified from the supernatant using a Ni-NTA column by washing with 20 column volumes of each 20 mM Tris (pH 8.0), 200 mM NaCl containing 10 mM imidazole or 20 mM imidazole or 40 mM Imidazole. Geh was eluted with 20 mM Tris (pH 8.0), 200 mM NaCl, 250 mM imidazole. The eluant was further purified by gel filtration chromatography using a HiLoad 16/600 Superdex 200 column (Cytiva Life Sciences) equilibrated with 200 mM NaCl, 20 mM Tris, pH 7.5. The molecular weight was estimated by analyzing the peak size on a HiLoad 13/300 Super 200 analytical column (Cytiva Life Sciences). A standard curve was generated using thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), RNase A (13.7 kDa), and aprotinin (6.5 kDa).
Geh and PLA2 biochemistry. In vitro activity of Geh and PLA2 was analyzed by mass spectrometry. Liposomes for Geh assays were prepared with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(19-rac-glycerol) by drying the lipid under N 2 and hydrating with 50 mM Tris (pH 8.0) for 2 h at 37°C. Hydrated lipids were sonicated for 2 min twice in a water bath sonicator and then passed 30 times through an Avanti Mini Extruder with a 100 nm membrane. A 50 mL assay reaction mixture containing 100 mM bis-Tris (pH 6.0), 150 mM NaCl, 1 mM PG liposomes and 500 nM Geh was incubated at room temperature for 15 min and 50 mL of methanol containing 1% acetic acid was added to stop the reaction. The rection was centrifuged at 20,000 Â g for 20 min and the samples were analyzed by LC-MS/MS. For PLA2 assay, a 50 mL assay reaction mixture containing 100 mM bis-Tris (pH 6.0), 50 mM CaCl 2 , 1 mM PG liposomes, and 0.5 mg PLA2 was incubated at room temperature for 1 h, and 50 mL of methanol containing 1% acetic acid was added to stop the reaction. The reaction was centrifuged at 20,000 Â g for 20 min and the samples were analyzed by LC-MS/MS.
Docking PG in the Geh active site. The 1-palmitoyl-2-oleoyl-PG substrate was docked with Geh by using SwissDock server (61,62). For docking calculation, the ligand was energy minimized with Molecular Operating Environment (MOE) (2018.01; Chemical Computing Group). Geh monomer protein structure was generated from Geh-Orlistat complex structure (PDB 6KSM). All solvent molecules and ligands except calcium and zinc ions were removed from the structure and Ser116 was mutated to Ala. After completion of docking processes, the obtained one of the poses was manually adjusted so that the carbonyl oxygen atom of cleavage site was located in the oxyanion hole. The fitted ligand and Geh (Ser116) structures were imported into MOE and prepared using QuickPrep and the ligand was minimized energy with carbonyl oxygen atom fixed in the oxyanion hole.
Ethics statement. All work with S. aureus described herein were done in accordance with protocols approved by the Institutional Biosafety Committees at St. Jude Children's Research Hospital (SJCRH).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.