Circumventing the Effect of Product Toxicity: Development of a Novel Two-Stage Production Process for the Lantibiotic Gallidermin

ABSTRACT Lantibiotics such as gallidermin are lanthionine-containing polypeptide antibiotics produced by gram-positive bacteria that might become relevant for the treatment of various infectious diseases. So far, self-toxicity has prevented the isolation of efficient overproducing strains, thus hampering their thorough investigation and preventing their exploitation in fields other than the food area. We wanted to investigate the effect of lantibiotic precursor peptides on the producing strains in order to evaluate novel strategies for the overproduction of these promising peptides. In this study, gallidermin was chosen as a representative example of the type A lantibiotics. A Staphylococcus gallinarum Tü3928 mutant, whose gene for the extracellular pregallidermin protease GdmP was replaced by a kanamycin-resistance gene, was constructed. Mass spectrometry (MS) analysis indicated that this mutant produced fully posttranslationally modified gallidermin precursors with truncated versions of the leader peptide, but not the entire leader as predicted from the gdmA sequence. In filter-on-plate assays, these truncated pregallidermins showed no toxicity against Staphylococcus gallinarum Tü3928 up to a concentration of 8 g/liter (corresponding to approximately 2.35 mM), while gallidermin produced clear inhibitory zones at concentrations as low as 0.25 g/liter (0.12 mM). We showed that the lack of toxicity is due entirely to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptides resulting from tryptic cleavage of pregallidermins and gallidermin produced by S. gallinarum Tü3928 had identical masses and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature gallidermin. In nonoptimized fermentations, the gdmP mutant produced pregallidermin to a 50%-higher molar titer, suggesting that the absence of self-toxicity has a beneficial effect on gallidermin production and giving a first confirmation of the suitability of the overproduction strategy.

Colistin: The phoenix Arises

The polymyxins were discovered in the 1940s and represent a group of closely related polypeptide antibiotics obtained fromBacillus polymyxa, which was originally isolated from soil (1,2). Although they have been used extensively worldwide in topical otic and ophthalmic solutions for decades, the intravenous formulations were gradually abandoned in most parts of the world in the early 1980s because of the reported high incidence of nephrotoxicity (3-5). As a result, the use of polymyxin preparations has been mainly restricted to the treatment of lung infections due to multidrug-resistant (MDR) gram-negative bacteria in patients with cystic fibrosis (6,7). The emergence of bacteria resistant to most classes of commercially available antibiotics and the shortage of novel antimicrobial agents with activity against gram-negative microorganisms have led to the reemergence of polymyxins as a valuable addition to the therapeutic armamentarium. It was thus considered timely to review colistin and its emerging role in managing infections due to MDR gram-negative bacteria.