The Bactericidal Fatty Acid Mimetic 2CCA-1 Selectively Targets Pneumococcal Extracellular Polyunsaturated Fatty Acid Metabolism

Abstract

Streptococcus pneumoniae, a major cause of pneumonia, sepsis, and meningitis worldwide, has the nasopharynges of small children as its main ecological niche. Depletion of pneumococci from this niche would reduce the disease burden and could be achieved using small molecules with narrow-spectrum antibacterial activity. We identified the alkylated dicyclohexyl carboxylic acid 2CCA-1 as a potent inducer of autolysin-mediated lysis of S. pneumoniae, while having low activity against Staphylococcus aureus. 2CCA-1-resistant strains were found to have inactivating mutations in fakB3, known to be required for uptake of host polyunsaturated fatty acids, as well as through inactivation of the transcriptional regulator gene fabT, vital for endogenous, de novo fatty acid synthesis regulation. Structure activity relationship exploration revealed that, besides the central dicyclohexyl group, the fatty acid-like structural features of 2CCA-1 were essential for its activity. The lysis-inducing activity of 2CCA-1 was considerably more potent than that of free fatty acids and required growing bacteria, suggesting that 2CCA-1 needs to be metabolized to exert its antimicrobial activity. Total lipid analysis of 2CCA-1 treated bacteria identified unique masses that were modeled to 2CCA-1 containing lysophosphatidic and phosphatidic acid in wild-type but not in fakB3 mutant bacteria. This suggests that 2CCA-1 is metabolized as a fatty acid via FakB3 and utilized as a phospholipid building block, leading to accumulation of toxic phospholipid species. Analysis of FabT-mediated fakB3 expression elucidates how the pneumococcus could ensure membrane homeostasis and concurrent economic use of host-derived fatty acids.IMPORTANCE Fatty acid biosynthesis is an attractive antibiotic target, as it affects the supply of membrane phospholipid building blocks. In Streptococcus pneumoniae, it is not sufficient to target only the endogenous fatty acid synthesis machinery, as uptake of host fatty acids may bypass this inhibition. Here, we describe a small-molecule compound, 2CCA-1, with potent bactericidal activity that upon interactions with the fatty acid binding protein FakB3, which is present in a limited number of Gram-positive species, becomes metabolized and incorporated as a toxic phospholipid species. Resistance to 2CCA-1 developed specifically in fakB3 and the regulatory gene fabT. These mutants reveal a regulatory connection between the extracellular polyunsaturated fatty acid metabolism and endogenous fatty acid synthesis in S. pneumoniae, which could ensure balance between efficient scavenging of host polyunsaturated fatty acids and membrane homeostasis. The data might be useful in the identification of narrow-spectrum treatment strategies to selectively target members of the Lactobacillales such as S. pneumoniae.

FabT, a Bacterial Transcriptional Repressor That Limits Futile Fatty Acid Biosynthesis

“…Interestingly, FabT represses the expression of two non-FASII genes involved in FA and phospholipid metabolism. i-fakB3 encodes FakB3, which is involved in the incorporation of polyunsaturated FAs, which are not synthesized by Firmicutes (22,23); and ii-plsC encodes PlsC, which catalyzes joining of a second FA to the 1-acyl-sn-glycerol 3-phosphate (bearing a single FA) to complete phospholipid acylation. S. pneumoniae FabT thus couples regulation of FA and phospholipid synthesis, similar to FapR regulation in B. subtilis and Staphylococcus aureus, where the regulatory mechanisms are different (24,25).…”

“…Investigation of its bactericidal effect led to the isolation of resistant mutants (23). One mutation mapped to fakB3, encoding a FA binding protein involved in the uptake of polyunsaturated FAs.…”