Generation of Branched-Chain Fatty Acids through Lipoate-Dependent Metabolism Facilitates Intracellular Growth of Listeria monocytogenes

The gram-positive bacterial pathogen Listeria monocytogenes has evolved mechanisms to rapidly replicate in the host cytosol, implying efficient utilization of host-derived nutrients. However, the contribution of host nutrient scavenging versus that of bacterial biosynthesis toward rapid intracellular growth remains unclear. Nutrients that contribute to growth of L. monocytogenes include branched-chain fatty acids (BCFAs), amino acids, and other metabolic intermediates generated from acyl-coenzyme A, which is synthesized using lipoylated metabolic enzyme complexes. To characterize which biosynthetic pathways support replication of L. monocytogenes inside the host cytosol, we impaired lipoate-dependent metabolism by disrupting two lipoate ligase genes that are responsible for bacterial protein lipoylation. Interrupting lipoate-dependent metabolism modestly impaired replication in rich broth medium but strongly inhibited growth in defined medium and host cells and impaired the generation of BCFAs. Addition of short BCFAs and amino acids restored growth of the A1A2-deficient (A1A2−) mutant in minimal medium, implying that lipoate-dependent metabolism generates amino acids and BCFAs. BCFAs alone rescued intracellular growth and spread in L2 fibroblasts of the A1A2− mutant. Lipoate-dependent metabolism was also required in vivo, as a wild-type strain robustly outcompeted the lipoylation-deficient mutant in a murine model of listeriosis. The results of this study suggest that lipoate-dependent metabolism contributes to both amino acid and BCFA biosynthesis and that BCFA biosynthesis is preferentially required for intracellular growth of L. monocytogenes.