Staphylococcus aureus Fatty Acid Auxotrophs Do Not Proliferate in Mice

Abstract

Inactivation of acetyl-coenzyme A (acetyl-CoA) carboxylase confers resistance to fatty acid synthesis inhibitors in Staphylococcus aureus on media supplemented with fatty acids. The addition of anteiso-fatty acids (1 mM) plus lipoic acid supports normal growth of ⌬accD strains, but supplementation with mammalian fatty acids was less efficient. Mice infected with strain RN6930 developed bacteremia, but bacteria were not detected in mice infected with its ⌬accD derivative. S. aureus bacteria lacking acetyl-CoA carboxylase can be propagated in vitro but were unable to proliferate in mice, suggesting that the acquisition of inactivating mutations in this enzyme is not a mechanism for the evasion of fatty acid synthesis inhibitors.T he steady increase in multidrug-resistant Staphylococcus aureus prevalence has spurred the search for novel antibiotics to attack this clinically important organism (1). The bacterial type II fatty acid biosynthesis system (FASII) is a novel target that has engendered considerable attention, and there are multiple natural-product antibiotics that target the pathway (2). Most Gramnegative bacteria are susceptible to FASII inhibitors even when exogenous fatty acids are provided because they lack the ability to activate exogenous fatty acids to produce acyl carrier proteins (ACPs) and synthesize the hydroxyacyl-ACPs to support lipopolysaccharide biosynthesis (2). However, the behavior of Grampositive bacteria is different. These organisms do not produce hydroxy-fatty acids, and they are capable of incorporating exogenous fatty acids by ligating them to ACPs (3). These acyl-ACPs are either elongated by the FASII system or incorporated into phospholipids via the acyl-PO 4 /acyl-ACP-specific PlsX/PlsY/PlsC acyltransferase system (3). Thus, the FASII pathway can be inactivated through genetic deletions or FASII drugs in S. agalactiae, and the organism is able to grow if supplied with an exogenous fatty acid supplement (4). In S. pneumoniae, endogenous fatty acid synthesis is repressed in the presence of exogenous fatty acids, leading to the replacement of endogenous fatty acids with those provided in the medium even if FASII is not chemically or genetically inactivated (3, 4). Although S. aureus also incorporates exogenous fatty acids into membrane phospholipids via acyl-ACP, FASII inhibitors remain effective against this Gram-positive organism even in the presence of exogenous fatty acids (3).AFN-1252 is a compound in clinical development that blocks the enoyl-ACP reductase step of S. aureus FASII (5-7). Two classes of AFN-1252-resistant S. aureus mutants were isolated (3). One class consists of missense mutations in the fabI gene that lead to the production of a mutant FabI protein that is refractory to AFN-1252. When exogenous fatty acids were supplied in the media during selection, AFN-1252-resistant clones appeared 100 times more frequently (3). Genetic analysis showed that these isolates harbored mutations that completely inactivated one of the four genes required for acetyl-coenzyme A (ac...

Clinical Relevance of Type II Fatty Acid Synthesis Bypass in Staphylococcus aureus

“…A previous report suggested that fatty acid auxotrophs would be noninfectious based on the behavior of constructed S. aureus acc mutants in a mouse model (6). In contrast to those conclusions, this study establishes that at the least, fatty acid auxotrophs (i) are maintained in in vivo reservoirs and (ii) define a new category of VBNC pathogens that would escape detection in current routine identification screens.…”

“…In vitro-selected FASII bypass in S. aureus was associated with mutations mapping to the acc and fabD genes (6,19,29). Among 10 analyzed Exo isolates, 6 carried polymorphisms in accC, accD, or accB, including stop codons (Table 1).…”

“…The question of the survival of such mutants in a clinical context remains in debate (6,7,12,19). It is therefore essential to determine whether FASII bypass, as characterized mainly in vitro, is relevant to natural staphylococcal populations.…”

“…Acquisition of antimicrobial resistance, likely due to overuse of antibiotics, is the principal cause of S. aureus drug resistance (1). A class of new-generation antimicrobials in intensive development uses the type II fatty acid synthesis (FASII) pathway as an antibacterial target (2-5) to treat S. aureus infections (6)(7)(8). A widely used biocide, triclosan (5-chloro-2-[2,4-dichlorophenoxy]phenol; commercialized as Irgasan or Microban), is a prototype for further anti-FASII development (4,9,10).…”

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria

“…Lipoic acid was provided because it is an essential cofactor for ketoacid dehydrogenases and is derived from octanoyl-ACP arising from FASII (52)(53)(54). S. aureus lacking FASII require exogenous lipoate in addition to fatty acids to support maximum growth (35,55). These experiments showed that exogenous fatty acids cannot bypass the inhibition of FabI by AFN-1252 in both N. gonorrhoeae and N. meningitidis.…”