Staphylococcus aureusUtilizes Host-Derived Lipoprotein Particles as Sources of Fatty Acids

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) is a threat to global health. Consequently, much effort has focused on the development of new antimicrobials that target novel aspects ofS. aureusphysiology. Fatty acids are required to maintain cell viability, and bacteria synthesize fatty acids using the type II fatty acid synthesis (FASII) pathway. FASII is significantly different from human fatty acid synthesis, underscoring the therapeutic potential of inhibiting this pathway. However, many Gram-positive pathogens incorporate exogenous fatty acids, bypassing FASII inhibition and leaving the clinical potential of FASII inhibitors uncertain. Importantly, the source(s) of fatty acids available to pathogens within the host environment remains unclear. Fatty acids are transported throughout the body by lipoprotein particles in the form of triglycerides and esterified cholesterol. Thus, lipoproteins, such as low-density lipoprotein (LDL), represent a potentially rich source of exogenous fatty acids forS. aureusduring infection. We sought to test the ability of LDLs to serve as a fatty acid source forS. aureusand show that cells cultured in the presence of human LDLs demonstrate increased tolerance to the FASII inhibitor triclosan. Using mass spectrometry, we observed that host-derived fatty acids present in the LDLs are incorporated into the staphylococcal membrane and that tolerance to triclosan is facilitated by the fatty acid kinase A, FakA, and Geh, a triacylglycerol lipase. Finally, we demonstrate that human LDLs support the growth ofS. aureusfatty acid auxotrophs. Together, these results suggest that human lipoprotein particles are a viable source of exogenous fatty acids forS. aureusduring infection.IMPORTANCEInhibition of bacterial fatty acid synthesis is a promising approach to combating infections caused byS. aureusand other human pathogens. However,S. aureusincorporates exogenous fatty acids into its phospholipid bilayer. Therefore, the clinical utility of targeting bacterial fatty acid synthesis is debated. Moreover, the fatty acid reservoir(s) exploited byS. aureusis not well understood. Human low-density lipoprotein particles represent a particularly abundantin vivosource of fatty acids and are present in tissues thatS. aureuscolonizes. Herein, we establish thatS. aureusis capable of utilizing the fatty acids present in low-density lipoproteins to bypass both chemical and genetic inhibition of fatty acid synthesis. These findings imply thatS. aureustargets LDLs as a source of fatty acids during pathogenesis.

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogenListeria monocytogenesencode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype ofEscherichia colistrain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate ofL. monocytogenesin laboratory medium. Robust exogenous fatty acid incorporation was not detected inL. monocytogenesunless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth ofL. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth ofL. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth ofL. monocytogenes.

Triclosan Resistance in Francisella tularensis: A Site-Directed Mutagenesis Study of the FabI Enzyme

An NADH-dependent enoyl-acyl carrier protein reductase, FabI, catalyzes the final step of bacterial fatty acid biosynthesis, reducing the double bond of trans-2-enoyl-ACP to a single bond forming acyl-ACP. Given its importance in bacterial fatty acid synthesis, FabI has become a recognized drug target. Triclosan, a diphenyl ether, targets the FabI, inhibits its activity, and stops bacterial growth. However, as a consequence of triclosan's popularity, and thus its overuse, bacterial resistance to triclosan has been reported. The mutation G93V in Escherichia coli (E. coli) FabI allows E. coli to resist the action of triclosan. We have identified the equivalent residue of G93 in Francisella tularensis FabI (ftFabI) as A92, and prepared a mutant A92V. E. coli cells, transformed with a plasmid containing the ftFabI-A92V gene, were grown, and the gene was overexpressed. From two growths (6 G of cells), 62 mG of protein, with a histidine tag, at a purity of 85% were obtained. Enzymatic activity was assayed by monitoring the absorbance of NADH at 340 nm. In the presence of triclosan, the wild-type protein was almost completely inhibited after NADH was converted to NAD$^+$ in the enzymatic reaction; however the A92V mutant exhibited similar activity with and without triclosan, demonstrating that triclosan resistance may also develop in Francisella tularensis.