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A family of class I lantibiotics from actinomycetes and improvement of their antibacterial activities Sonia I. Maffioli, Paolo Monciardini, Bruno Catacchio, Carlo Mazzetti, Daniela Münch, Cristina Brunati, Hans-Georg Sahl, and Stefano Donadio ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb500878h • Publication Date (Web): 09 Jan 2015 Downloaded from http://pubs.acs.org on January 12, 2015
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A family of class I lantibiotics from actinomycetes and improvement of their antibacterial activities Sonia I. Maffioli, *,†,‡ Paolo Monciardini†,‡, Bruno Catacchio†,§, Carlo Mazzetti†,§, Daniela Münch┴, Cristina Brunatiǁ, Hans-Georg Sahl┴, Stefano Donadio†,‡
†
Naicons srl, Milano, Italy
‡
KtedoGen srl, Milano, Italy
§
ITB-CNR Segrate, Milano, Italy
┴
Institute of Medical Microbiology, Immunology and Parasitology, Pharmaceutical Microbiology
Section, University of Bonn, Bonn, Germany ǁ
Need Pharma, Milano, Italy
SUMMARY Lantibiotics, abbreviation for “lanthionine containing antibiotics", interfere with bacterial metabolism by a mechanism not exploited by the antibiotics currently in clinical use. Thus, they have aroused interest as source for new therapeutic agents because they can overcome existing resistance mechanisms. Starting from fermentation broth extracts preselected from a high throughput screening program for discovering cell-wall inhibitors, we isolated a series of related, class I lantibiotics produced by different genera of actinomycetes. Analytical techniques together with explorative chemistry have been used to establish their structures: the newly described compounds share a common 24–aa sequence with the previously reported lantibiotic planosporicin (aka 97518), differing at positions 4, 6, and 14. All these compounds maintain an overall –1 charge at physiological pH. While all these lantibiotics display modest antibacterial activity, their potency can be substantially modulated by progressively eliminating the negative charges, with the most active compounds carrying basic amide derivatives of the two carboxylates originally present in the 1 ACS Paragon Plus Environment
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natural compounds. Interestingly, both natural and chemically modified lantibiotics target the key biosynthetic intermediate lipid II, but the former compounds do not bind as effectively as the latter in vivo. Remarkably, the basic derivatives display an antibacterial potency and a killing effect similar to those of NAI–107, a distantly related, actinomycete-produced class I lantibiotic which lacks altogether carboxyl groups and which is a promising clinical candidate for treating Grampositive infections caused by multidrug-resistant pathogens. Keywords: lantibiotics, Streptomyces, Streptosporangiaceae, lipid II, semi-synthesis INTRODUCTION Lantibiotics, abbreviation for “lanthionine containing antibiotics”, represent a subgroup of the larger family of lanthipeptides1. They are ribosomally and post–translationally modified peptides of which nisin is the best-known example. As these compounds interfere with bacterial cell wall metabolism by a mechanism not exploited by the antibiotics currently in clinical use, they usually overcome existing resistance mechanisms and thus represent a promising alternative to existing antibiotics. In recent years site-directed and random mutagenesis, mutasynthesis, semi- and total synthesis have been applied to a relevant number of class II lantibiotics like mersacidin2, nukacin ISK–13, actagardine4 providing some structure-activity relationships for these lantibiotics5. Among the class I compounds, nisin is by far the most studied example, thanks to the numerous natural variants and the analogs obtained by engineering.6 While most lantibiotics have been isolated and characterized from different genera of the Firmicutes, one of the most active compounds is actually produced by the actinomycete Microbispora: the lantibiotic NAI–107, also known as 107891 or microbisporicin.7,8 This lantibiotic has shown to be highly effective in sophisticated experimental models of infection.9 NAI–107 binds to the key peptidoglycan intermediate lipid II, as well as other bactoprenol-bound precursors, and impairs membrane function in Gram-positive bacteria.10 Some insights on the structural features modulating NAI–107 potency have been recently provided by the characterization of naturally 2 ACS Paragon Plus Environment
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occurring congeners.11 NAI–107 was isolated during a screening program12 aimed at identifying new bacterial cell wall inhibitors, which also afforded the related but less active 97518,13 as well as the recently described lanthipeptides NAI–80214 and NAI–112.15 In this paper we report the isolation and characterization of four additional class I, 97518–related lantibiotics produced by different genera of actinomycetes. We also show that the antibacterial potency of these compounds can be substantially improved by simple chemical manipulations.
RESULTS AND DISCUSSION Screening and taxonomy of the producing strains The high throughput screening program has been previously described12 and found to be unexpectedly selective in identifying lanthipeptides from actinomycetes.16 We thus reasoned that there was a relatively high probability of discovering novel lanthipeptides by evaluating further hits emerging from that screening. Six actinomycete strains were thus further characterized. All producing strains were independent isolates deriving from soil samples collected in Europe (ID114623), Africa (ID105857), SouthEast Asia (ID106130) or Central America (Table 1). The strains, classified at genus level by partial sequencing of the 16S rRNA gene, were classified as members of the genera Streptomyces (four strains), Planomonospora (two instances) and Streptosporangium (one instance). Among the four Streptomyces strains, pairwise 16S rRNA gene sequence identity values were 98.1% or lower, indicating that they most likely belong to different species. The highest level of sequence identity with described Streptomyces species was found for strains ID116506 and ID106130, showing 99.5% identity with S. heteromorphus JCM 5031 (Acc. no. AB045865) and 99.4% identity with S. fumigatiscleroticus NRRL 12999 (NR_112337) respectively. The two other Streptomyces strains have lower 16S identity (
