Identification of four genes responsible for antimicrobial resistance of MEL-B against S. aureus.

There is increasing interest in the antimicrobial activity of mannosylerythritol lipids-B (MEL-B) against Gram-positive bacteria such as Staphylococcus aureus (S. aureus). However, the specific molecules involved in MEL-B's antimicrobial action against S. aureus have not been identified. This study utilized the Nebraska transposon mutant library (NTML), which contains 1920 mutants, each lacking three-quarters of the genes found in S. aureus. The NTML was screened to identify mutants resistant to MEL-B. Four mutants (Accession Number: SAUSA300_0904, SAUSA300_0752, SAUSA300_0387, and SAUSA300_2311) largely unaffected by incubation with MEL-B, indicating MEL-B resistance. Despite the strong binding of MEL-B to these mutants, the four molecules encoded by the deleted genes (yjbI, clpP, pbuX, or brpS) in each mutant were not directly recognized by MEL-B. Given that these molecules are not localized on the outer surface of S. aureus and that the antibacterial activity of MEL-B against S. aureus is facilitated by the effective transfer of two antibacterial fatty acids (caprylic acid and myristoleic acid) to S. aureus via ME, the deletion of each of the four molecules may alter the peptidoglycan structure, potentially inhibiting the effective transfer of these antimicrobial fatty acids into S. aureus.

Improvement of Oil Degradation and MEL Production in a Yeast Strain, Pseudozyma tsukubaensis, by Translation Elongation Factor 1 Promoter-driven Expression of a Lipase.

The basidiomycetous yeast Pseudozyma tsukubaensis produces a mannosylerythritol lipid (MEL) homologue, a diastereomer type of MEL-B, from olive oil. In a previous study, MEL-B production was increased by the overexpression of lipase PaLIPAp in P. tsukubaensis 1E5, through the enhancement of oil consumption. In the present study, RNA sequence analysis was used to identify a promoter able to induce high-level PaLIPA expression. The recombinant strain, expressing PaLIPA via the translation elongation factor 1 alpha/Tu promoter, showed higher lipase activity, rates of oil degradation, and MEL-B production than the strain which generated in our previous study.

Roles of mannosylerythritol lipid-B components in antimicrobial activity against bovine mastitis-causing Staphylococcus aureus.

Mannosylerythritol lipid-B (MEL-B), which comprises ester-bonded hydrophilic ME and hydrophobic fatty acids, is a bio-surfactant with various unique properties, including antimicrobial activity against most gram-positive bacteria. The gram-positive Staphylococcus aureus is a causative pathogen of dairy cattle mastitis, which results in considerable economic loss in the dairy industry. Here, we demonstrate the efficacy of MEL-B as a disinfectant against bovine-derived S. aureus and elucidate a mechanism of action of MEL-B in the inhibition of bacterial growth. The growth of bovine mastitis causative S. aureus BM1006 was inhibited when cultured with MEL-B above 10 ppm. The activity of MEL-B required fatty acids (i.e., caprylic and myristoleic acids) as ME, the component of MEL-B lacking fatty acids, did not inhibit the growth of S. aureus even at high concentrations. Importantly, ME-bound fatty acids effectively inhibited the growth of S. aureus when compared with free fatty acids. Specifically, the concentrations of ME-bound fatty acids and free caprylic and myristoleic acids required to inhibit the growth of S. aureus were 10, 1442, and 226 ppm, respectively. The involvement of ME in the antimicrobial activity of MEL-B was confirmed by digestion of MEL-B with alkali, which dissociated ME and fatty acids. These results indicated that a mechanism of action of MEL-B in inhibiting the growth of S. aureus could be explained by the effective transporting of antimicrobial fatty acids to the bacterial surface via hydrophilic ME.