Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

Abstract

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...

Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family

“…2 is a two-protein system that consists of a dimeric ATP-binding protein (FakA) and a monomeric fatty acid-binding protein (FakB) (1). Fak expression is widespread in Gram-positive bacteria with most strains possessing a single FakA along with multiple FakBs.…”

“…Staphylococcus aureus expresses two FakBs that both function with FakA. FakB1 binds a saturated fatty acid, although FakB2 prefers an unsaturated fatty acid (1). The FakBs possess a bound fatty acid, but they function as exchange proteins that swap a bound fatty acid for a fatty acid in a detergent micelle or membrane bilayer (Fig.…”

“…1). Acyl-acyl carrier protein is used by either PlsC in the second acyltransferase step of phospholipid synthesis or by FabF, the elongation condensing enzyme of bacterial type II fatty acid synthesis (1). In addition to the role of FakA and FakB in the activation of exogenous fatty acids, Fak inactivation abrogates the expression of a constellation of virulence factor genes ( Fig.…”

“…1) (1, 3). The genes altered by Fak inactivation are the same genes that are regulated by the SaeRS two-component virulence regulatory system suggesting SaeRS is the focal point for Fak regulation of virulence factor transcription (1). Also, the inactivation of fakA increases biofilm formation in S. aureus, although the biochemical role of Fak in biofilm formation is unknown (4).…”

“…All crystallized FakB proteins have a fatty acid or fatty acid-like ligand bound (7,8). The discovery of their role in Fak provided a function for these ubiquitous FakB proteins in bacterial physiology (1), but the role(s) of the highly conserved FakB residues that define the protein family in Fak biochemistry are unknown. In this study, a combination of x-ray crystallography, site-directed mutagenesis, and in vitro and in vivo functional analyses are used to determine the structural and functional roles of the five conserved residues that define the bacterial fatty acid-binding protein family.…”