Genetic and chemical control of tuberculostearic acid production in Mycobacterium avium subspecies paratuberculosis

Authors: Bannantine, John; DUFFY, SHANNON - McGill University - Canada; COLOMBATTI OLIVIERI, MARIA - US Department Of Agriculture (USDA); BEHR, MARCEL - McGill University - Canada; BIET, FRANCK - Universite De Tours; Price, Neil

Submitted to: American Society for Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/28/2024
Publication Date: 3/19/2024
Citation: Bannantine, J.P., Duffy, S.C., Colombatti Olivieri, M.A., Behr, M.A., Biet, F., Price, N.P. 2024. Genetic and chemical control of tuberculostearic acid production in Mycobacterium avium subspecies paratuberculosis. American Society for Microbiology. https://doi.org/10.1128/spectrum.00508-24.
DOI: https://doi.org/10.1128/spectrum.00508-24

Interpretive Summary: We discovered a unique way to control production of a specific fatty acid, important in biofuels, using either genetic disruption (gene knockout) or a compound called pivalic acid. The fatty acid is called tuberculostearic acid and its production control is exhibited by Mycobacterium species. Analysis of additional strains of the bacterial agent that causes Johne's disease (a.k.a. Mycobacterium avium subspecies paratuberculosis, abbreviated as Map) showed that a genetic lesion (termed a SNP) prevents tuberculostearic acid production in all sheep strains of Map. We constructed a mutant in a bovine strain of Map to show that it also stops tuberculostearic acid production in that condition. Collectively, these results document yet another of the many genetic and notable differences between sheep and cattle strains of Map. These results also help us understand disease host range and disease pathology results.

Technical Abstract: Branch-chain fatty acids are a predominant cell-wall component among species belonging to the Mycobacterium genus. One of these is tuberculostearic acid (TBSA), which is a long-chain middle branched fatty acid used as a diagnostic marker for M. tuberculosis. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a single-gene knockout of bfaA which encodes an FAD-binding oxidoreductase. Mycobacterium avium subspecies paratuberculosis (Map) growth and TBSA production was inhibited in pivalic acid supplemented cultures (0.5 mg/ml), but higher concentrations were needed to have a similar effect in other mycobacteria tested including Mycobacterium smegmatis, Mycobacterium avium subspecies hominissuis (Mah) and M. avium subspecies silvaticum (Mas). Gas chromatography/mass spectrometry (GC/MS) of fatty acid methyl esters (FAMEs) revealed a build-up of the C18:0 fatty acid (stearic acid) with no conversion to TBSA in Map cultures containing 0.5 mg /ml pivalic acid. While C-type strains of Map will produce TBSA when pivalic acid is below 0.5 mg/ml, the S-type strains of Map do not produce TBSA regardless of the presence of pivalic acid. The SAM-dependent methyltransferase, encoded by bfaB, and FAD-dependent oxidoreductase, encoded by bfaA, are both required in the two-step biosynthesis of TBSA; however, S-type strains of Map contain a SNP in the bfaA gene, rendering the enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected by GC/MS of FAMEs in S-type strains. A bfaA knockout was constructed in C-type Map to determine if the intermediate could be detected. FAMEs analysis of the bfaA knockout reveals loss of TBSA production, but the intermediate (10-methylene stearate) was present similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between C- and S-type strains of Map and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA not be used as a diagnostic marker for Map.