Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus.T he pathway for the uptake and incorporation of host fatty acids (FA) by bacterial pathogens is important to understanding their physiology and determining if type II fatty acid synthesis (FASII) inhibitors will be useful antibacterial therapeutics. FASII is an energy-intensive process, and the advantage of being able to use host FA for membrane assembly obviously allows ATP to be diverted to the synthesis of other macromolecules. Gram-positive pathogens are capable of incorporating extracellular FA into their phospholipids. In species related to Streptococcus agalactiae, extracellular FA can completely replace endogenously synthesized FA, rendering the FASII inhibitors ineffective growth inhibitors if sufficient FA are present (1, 2). In contrast, FASII inhibitors exemplified by the enoyl-acyl carrier protein (ACP) reductase therapeutic AFN-1252 are effective against Staphylococcus aureus, even when extracellular FA are abundant (2, 3). A major impediment to our understanding of this diversity is that the pathway and enzymes responsible for the incorporation of extracellular FA is not established in the Firmicutes. A recent analysis of a ΔplsX knockout strain of S. aureus ruled out a role for either acyl-CoAs or acyl-ACP as intermediates in host FA metabolism and instead provided evidence for the existence of a new enzyme that phosphorylates FA, called FA kinase (Fak) (4) (Fig. 1A). Host FA are phosphorylated by Fak, and the acyl-PO 4 formed is either used by the PlsY glycerol-3-phosphate acyltransferase or converted to acyl-ACP by PlsX. The acyl-ACP may be either elongated by FASII or used by PlsC. Despite the strong evidence for their existence, the genes/proteins that encode this novel enzyme in FA metabolism were previously unidentif...

The skin represents an important barrier for pathogens and is known to produce fatty acids that are toxic toward Gram-positive bacteria. A screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity. Fatty acids like oleate (18:1⌬9) were nontoxic, whereas palmitoleate (16:1⌬9) was a potent growth inhibitor. Cells treated with 16:1⌬9 exhibited rapid membrane depolarization, the disruption of all major branches of macromolecular synthesis, and the release of solutes and low-molecular-weight proteins into the medium. Other cytotoxic lipids, such as glycerol ethers, sphingosine, and acyl-amines blocked growth by the same mechanisms. Nontoxic 18:1⌬9 was used for phospholipid synthesis, whereas toxic 16:1⌬9 was not and required elongation to 18:1⌬11 prior to incorporation. However, blocking fatty acid metabolism using inhibitors to prevent acyl-acyl carrier protein formation or glycerol-phosphate acyltransferase activity did not increase the toxicity of 18:1⌬9, indicating that inefficient metabolism did not play a determinant role in fatty acid toxicity. Nontoxic 18:1⌬9 was as toxic as 16:1⌬9 in a strain lacking wall teichoic acids and led to growth arrest and enhanced release of intracellular contents. Thus, wall teichoic acids contribute to the structure-specific antimicrobial effects of unsaturated fatty acids. The ability of poorly metabolized 16:1 isomers to penetrate the cell wall defenses is a weakness that has been exploited by the innate immune system to combat S. aureus. Staphylococcus aureus is a common cutaneous pathogen responsible for serious infections that are becoming increasingly dangerous due to the prevalence of antibiotic-resistant organisms (8). Lipids have an important role in innate immunity. Human skin deploys a variety of innate defenses against S. aureus colonization that include antimicrobial peptides and fatty acids (9,18,47,49,51). In mice, 16:1⌬9 is the most potent antibacterial fatty acid, whereas humans synthesize a different isomer, 16:1⌬6 (49). It has been known for decades that these skin fatty acids block the growth of S. aureus (21,27,44). Humans (49) and mice (18) deficient in the production of these 16-carbon monounsaturated fatty acids are more susceptible to S. aureus skin infections. However, it is much less clear how these specific fatty acids produce their antibacterial effect. Ideas include the destabilization of the bacterial membrane due to their surfactant properties (19), uncoupling of ATP synthesis (17), the formation of fatty acid hydroperoxides that elicit oxidative stress (29), increased membrane fluidity due to the incorporation in unsaturated fatty acids in phospholipid (5, 7), and the inhibition of de novo fatty acid synthesis at the FabI step (44, 54). These toxic properties of fatty acids stand in contrast to the observations that S. aureus readily incorporates exogenous fatty acids into membrane phospholipids (1, 3, 4, 41) and that acetyl-coenzyme A (CoA) carboxylase knockout mutants can be isolated a...

Summary Acyl-CoA and acyl-acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram-negative bacteria. However, Gram-positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn-glycerol-3-phosphate by PlsY using an acyl-phosphate (acyl-PO4) intermediate. PlsX generates acyl-PO4 from the acyl-ACP end-products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1-position and endogenous acyl groups were channeled into the 2-position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl-ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP-dependent fatty acid kinase activity, and the acyl-PO4 was converted to acyl-ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids.

The regulation of phosphatidylinositol synthesis was examined by cloning and expressing in COS-7 cells the human cDNAs encoding the two enzymes in the biosynthetic pathway. Human CDP-diacylglycerol synthetase (cds1) and phosphatidylinositol synthase (pis1) clones were identified in the human expressed sequencetagged (EST) data base, and full-length cDNAs were obtained by library screening. The cds1 cDNA did not possess a recognizable mitochondrial import signal, and the activity of the expressed Cds1 protein was stimulated by nucleoside triphosphates in vitro, indicating that cds1 did not encode the mitochondrial-specific isozyme. There were two mRNA species (3.9 and 5.6 kilobases) detected on Northern blots hybridized with the cds1 probe that were expressed at distinctly different levels in various human tissues. Consistent with the presence of the two mRNAs, a cDNA predicted to encode a second human CDP-diacylglycerol synthetase (cds2) was also uncovered in the EST data base. In contrast to the two cds mRNAs, a single, 2.1-kilobase pis1 mRNA was uniformly expressed in all human tissues examined. Expression of the pis1 gene led to the overproduction of both phosphatidylinositol synthase and phosphatidylinositol:inositol exchange reactions, indicating that the Pis1 polypeptide catalyzed both of these activities. Phosphatase treatment of cell extracts abolished the CMP-independent phosphatidylinositol:inositol exchange reaction, and exchange activity was completely restored by the addition of CMP. Overexpression of cds1 or pis1 alone or in combination did not enhance the rate of phosphatidylinositol biosynthesis. Also, overexpression did not result in a significant proportional increase in the cellular levels of CDP-diacylglycerol or phosphatidylinositol. These data illustrate that the levels of Cds1 and Pis1 protein expression are not critical determinants of cellular PtdIns content and argue against a determining role for the activity of either of these enzymes in the regulation of PtdIns biosynthesis.

Phosphoethanolamine cytidylyltransferase (ECT) catalyzes the rate-controlling step in a major pathway for the synthesis of phosphatidylethanolamine (PtdEtn). Hepatocyte-specific deletion of the ECT gene in mice resulted in normal appearing animals without overt signs of liver injury or inflammation. The molecular species of PtdEtn in the ECT-deficient livers were significantly altered compared with controls and matched the composition of the phosphatidylserine (PtdSer) pool, illustrating the complete reliance on the PtdSer decarboxylase pathway for PtdEtn synthesis. PtdSer structure was controlled by the substrate specificity of PtdSer synthase that selectively converted phosphatidylcholine molecular species containing stearate paired with a polyunsaturated fatty acid to PtdSer. There was no evidence for fatty acid remodeling of PtdEtn. The elimination of diacylglycerol utilization by the CDP-ethanolamine pathway led to a 10-fold increase in triacylglycerols in the ECTdeficient hepatocytes that became engorged with lipid droplets. Triacylglycerol accumulation was associated with a significant elevation in the expression of the transcription factors and target genes that drive de novo lipogenesis. The absence of the ECT pathway for diacylglycerol utilization at the endoplasmic reticulum triggers increased fatty acid synthesis to support the formation of triacylglycerols leading to liver steatosis. PtdEtn2 is the second most abundant phospholipid in mammals and has many important functions in cell physiology (for reviews see Refs.