Investigation of NAI-107 immunity in the lantibiotic producer Microbispora ATCC PTA-5024

Abstract

The actinomycete Microbispora ATCC PTA-5024 produces the lantibiotic NAI-107, currently in pre-clinical development for the treatment of multi-drug resistant Gram-positive bacteria. NAI-107 displays its antibacterial activity by binding bactoprenol-pyrophosphate-coupled peptidoglycan precursors, thereby interfering with peptidoglycan biosynthesis. In the present study, a comprehensive analysis of the immunity of this lantibiotic-producing actinomycete was carried out. In order to understand how Microbispora counteracts the action of its own antibiotic, its peptidoglycan composition was analysed in detail. Microbispora peptidoglycan consists of muropeptides with Ala, Gly and Ser in the fourth or fifth position of peptide stems. Muropeptides with alternative 3-3 cross-links besides the classical 4-3 cross-links were detected. The non-producing Microbispora RP0 and Microbispora spp. JCM 10266 and JCM 10267 possess the same muropeptides found in Microbispora ATCC PTA-5024, thus the diversity of the muropeptides is not correlated with NAI-107 production. In addition, the NAI-107 biosynthetic gene cluster (mlb) was analysed for the expression of immunity proteins. Distinct immunity determinants are encoded in the mlb cluster: the ABC transporter MlbYZ acting cooperatively with the transmembrane protein MlbJ and the lipoprotein MlbQ. MlbJ was hypothesized to act as substrate-binding protein for MlbYZ, while MlbQ to have an independent mode of action. MlbQ was further investigated by genetic and biochemical analyses. Deletion analysis showed that MlbQ is required for normal growth under NAI-107 production. NMR structural analysis of MlbQ revealed a hydrophobic surface patch, which was proposed to bind the cognate lantibiotic. Moreover, localization studies by fluorescence microscopy showed a spore localization of the chimeric protein MlbQ-mCherry. This study demonstrates that immunity in Microbispora is not only based on one determinant but on the action of the distinct immunity proteins MlbYZ, MlbJ and MlbQ.