Corynebacterium accolens Releases Antipneumococcal Free Fatty Acids from Human Nostril and Skin Surface Triacylglycerols

Model human skin surface TAGs triolein (triO) and trilinolein (triL) support the growth of the fatty acid-requiring species C. accolens. Black arrows indicate the oleic acid esters in (A) triolein and the linoleic esters in (B) trilinolein. (C) The mean turbidity (OD₆₀₀) after 48 h of shaken aerobic growth at 37°C demonstrated increased C. accolens growth in BHI broth supplemented with one of the TAGs compared to solvent-only controls (n = 9). Data were analyzed using an ANOVA and Tukey’s multiple-comparisons test. Error bars represent standard errors of the means. ****, P ≤ 0.0001.

C. accolens inhibits S. pneumoniae. C. accolens KPL1818 (Cac) inhibited S. pneumoniae 603 (Spn) growth on BHIC agar spread with 50 µl of a 100-mg/ml solution of (A) triolein solubilized in chloroform (triO + CHCl₃) or (C) trilinolein emulsified in ethanol (triL + EtOH), but not on medium spread with 50 µl of solvent alone: (B) chloroform (CHCl₃) or (D) EtOH. Representative images are shown (n = 3). To permit visualization of partial inhibition of the pneumococcal spot, S. pneumoniae was inoculated at various distances from C. accolens, as shown by the varied sizes of ZOIs, which are likely due to differences in diffusion of the antipneumococcal molecule(s).

Oleic acid is enriched in methanolic extracts of C. accolens CFCM. We used mass spectrometric analysis to identify compounds uniquely enriched in C. accolens CFCM. Methanolic extracts were analyzed by direct line infusion into a triple-quadrupole mass spectrometer operating in negative-ion scanning mode from m/z 50 to m/z 500. (A) To identify novel compounds present in the CFCM, the limited mass spectrum of the methanolic extract from unconditioned medium was subtracted from the spectrum for C. accolens CFCM. (B) Limited mass scanning of the novel m/z 281 peak present in CFCM, determined via high-resolution mass spectrometry, which gave a measured mass of m/z 281.2485, consistent with the calculated mass of oleic acid (m/z 281.2486). (C) An FFA standard mixture containing authentic oleic acid and heptadecanoic acid (HdA), as an internal standard, was analyzed by reversed-phase ultraperformance liquid chromatography coupled to tandem mass spectrometry, operating in multiple reaction mode monitoring transition of m/z 281 to m/z 281 at 10 eV for HdA and m/z 269 to m/z 269 at 10 eV for oleic acid, respectively. (D) CFCM with HdA added as an internal standard, analyzed under the same chromatographic conditions. (E) Unconditioned medium with HdA added as an internal standard, analyzed under the same chromatographic conditions. Representative spectra (A and B) or chromatographs (C, D, and E) are shown.

C. accolens LipS1 is necessary for growth on triolein (triO) as the sole source of fatty acids. (A) A C. accolens mutant strain (LipS1⁻) harboring an in-frame deletion in lipS1 did not grow in BHI supplemented with triolein (0.1 mg/ml), whereas the wild-type (WT) did. Growth was measured as the mean OD₆₀₀ following 48 h of aerobic growth with shaking at 37°C. Data were analyzed using a Welch two-sample t test. n = 3. Error bars represent standard deviations. *, P ≤ 0.05. (B) The lipase-deficient mutant carrying the empty vector (LipS1⁻ pCGL⁺) did not grow in BHI supplemented with triolein; however, the complemented mutant (LipS1⁻ cLipS1⁺) did. For comparison, the empty vector and complemented vector controls for the parental strain (WT pCGL⁺ and WT cLipS1⁺, respectively) are included. Data (n = 3) were analyzed using an ANOVA and Tukey’s multiple-comparisons test. Error bars represent standard deviations. **, P ≤ 0.01; ****, P ≤ 0.0001.