A Novel Microbisporicin Producer Identified by Early Dereplication during Lantibiotic Screening

Abstract

With the increasing need of effective antibiotics against multi-drug resistant pathogens, lantibiotics are an attractive option of a new class of molecules. They are ribosomally synthetized and posttranslationally modified peptides possessing potent antimicrobial activity against aerobic and anaerobic Gram-positive pathogens, including those increasingly resistant to β-lactams and glycopeptides. Some of them (actagardine, mersacidin, planosporicin, and microbisporicin) inhibit cell wall biosynthesis in pathogens and their effect is not antagonized by vancomycin. Hereby, we apply an efficient strategy for lantibiotic screening to 240 members of a newly described genus of filamentous actinomycetes, named Actinoallomurus, that is considered a yet-poorly-exploited promising source for novel bioactive metabolites. By combining antimicrobial differential assay against Staphylococcus aureus and its L-form (also in the presence of a β-lactamase cocktail or Ac-Lys-D-alanyl-D-alanine tripeptide), with LC-UV-MS dereplication coupled with bioautography, a novel producer of the potent microbisporicin complex was rapidly identified. Under the commercial name of NAI-107, it is currently in late preclinical phase for the treatment of multi-drug resistant Gram-positive pathogens. To our knowledge, this is the first report on a lantibiotic produced by an Actinoallomurus sp. and on a microbisporicin producer not belonging to the Microbispora genus.

Figure 1

MS-HPLC profiles of the F31/11 broth screening extract: (a) MS trace in negative and positive mode; (b) bioautography: each HPLC fraction was tested versus S. aureus MRSA L1400, MSSA L100, and L-form L3751 in dose dilution; (c) MS spectrum of the peak eluting at 11.7 min in negative and positive mode; (d) UV spectrum of the peak eluting at 11.7 min; (e) MS spectrum of the peak eluting at 12.2 min in negative and positive mode; (f) UV spectrum of the peak eluting at 12.2 min. In UV spectra, the λ values of the maximum and of the shoulder are indicated.

Figure 2

Morphology of F31/11 observed at the light microscope (model ULWD-CDPlan; Olympus, with 40x magnification).

Figure 3

Phylogenetic tree derived from the 16S rRNA gene sequences of Actinoallomurus species and related actinomycetes belonging to the Thermomonosporaceae family. Sequences from actagardine, planosporicin, and microbisporicin actinomycete producers were also included. For the construction of the phylogenetic tree, selected sequences were aligned with Clustal-Omega (from the EMBL-EBI site) and analyzed with BioEdit [30]. Distance matrices were calculated with MEGA5.2, using the Maximum Likelihood method implemented in the program and the method of Jukes and Cantor. Trees were inferred using the Nearest-Neighbor-Interchange (NNI) heuristic method and making the initial tree with both Neighbour Joining and BioNJ, and selecting the superior tree (all methods are included in the MEGA package). All analyses were performed on a bootstrapped data set containing 500 replicates.