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FRIMEDBIO-Fri prosj.st. med.,helse,biol

Purification, characterization, and structure determination of a novel antibiotic highly active against Streptococcus pneumoniae

Alternative title: Rensing, karakterisering og strukturbestemmelse av et nytt antibiotikum som er svært aktivt mot Streptococcus pneumoniae

Awarded: NOK 6.9 mill.

Rapid emergence of antibiotic resistant bacteria is occurring worldwide. This endangers the efficacy of antibiotics, a group of substances that have allowed modern medicine to advance rapidly and saved millions of lives. To stem the current crisis new antibacterial compounds are urgently needed. The goal of the current project is to characterize two narrow-spectrum antimicrobial agents, streptovoracin I and II, discovered in a screening of marine bacteria from the Oslo fjord. The identified compounds are highly active against Streptococcus pneumoniae, a major human pathogen causing about 2 million deaths annually. An important goal of the project was to solve the molecular structure of streptovoracin I and II. The chemist Jens M. J. Nolsøe has been working on these structures from July 1th 2019 to the 30th of June 2021. Despite considerable problems caused by the Covid-19 pandemic, he and his collaborators have now succeeded in solving the structure of streptovoracin I. It turned out to be identical to the structure of viscosin, a cyclic lipopeptide that has been characterized and published previously. In the project proposal we wrote that streptovoracin I and II probably represent novel antimicrobial compounds. This assumption was based on the fact that their masses were different from all cyclic lipopeptides registered in the NORINE database. We determined the mass of streptovoracin I to be 1148.7 dalton, while the mass of viscosine was reported to be 1126.4 dalton. After we discovered that the structure of streptovoracin I is identical to that of viscosin, we understood that the mass difference of about 22 dalton corresponds to a sodium ion. Hence, our results show that streptovoracin/viscosin has a strong affinity for sodium ions. Consequently, it is very likely that the binding of a sodium ion is a prerequisite for biological activity. In sum, the reason we believed that we had discovered a new antibiotic was that the mass of viscosin in the NORINE database was given without the sodium ion. Streptovoracin II proved to be a variant of streptovoracin I that contains two additional carbon atoms and a carbon-carbon double bond in the lipid moiety attached to the peptide ring structure. Since the structure of viscosin has been published previously, it will be difficult publish the structural work that has been carried out by the chemists involved in this project. Streptovoracin, hereafter called viscosin, is a very effective antimicrobial agent with an unknown mode of action. The mechanism by which it kills susceptible bacteria has not been studied before. Our results strongly indicate that viscosin kills S. pneumoniae by disrupting the integrity of its cell membrane. However, considering the high specificity of this drug it cannot just act as a surfactant or detergent. Hence, it must recognize a target, i.e. a docking molecule, in the membrane of sensitive streptococci. In order to understand the mode of action of viscosin it is very important to identify this docking molecule. Understanding the mode of action will make it possible for chemists to synthesize derivatives of the antibiotic that have improved properties, e.g. better antimicrobial effect, lower toxicity and better pharmaceutical properties. In addition, since viscosin targets the membrane of sensitive organisms it might be possible to synthesize variants that are designed to kill specific enveloped viruses and/or cancer cells. For this reason, Daniel Straume has focused on elucidating the mechanism by which viscosin kills streptococci. He subjects cultures of S. pneumoniae to sublethal concentrations of the drug to select for mutants that can tolerate higher concentrations than wild type. So far, we have not succeeded in identifying mutants with high resistance against viscosin, but we have isolated several mutants with higher resistance than the wild-type strain. Furthermore, the genomes of these mutants have been sequenced and mutations have been found in genes encoding proteins involved in transcription, protein synthesis and phospholipid synthesis. Results describing our pipeline of producing pure viscosin and the mode of action study will be published during the first halve of 2024. In this project we have discovered another antimicrobial compound that kills S. pneumoniae and other streptococci. This compound is produced by a new bacterium in the genus Lysinibacillus (we have sequenced and annotated the complete genome, gene bank accession nr. CP102798), which we have isolated from the Oslo fjord. This bacterium is called Lysinibacillus sp. OF-1 (OF = Oslo fjord), and the antimicrobial compound it produces is a novel antimicrobial peptide or glycopeptide. We have named the compound lysinicin OF, and it uses an oligo peptide uptake system in S. pneumoniae to kill the bacterium. Results about our discovery and characterisation of lysinicin OF have been published in the international research journal Microbiology.

- Forsterket tverrfaglig samarbeid mellom forskningsmiljø innen mikrobiologi og kjemi ved NMBU. - Oppdagelsen av nytt antimikrobielt stoff (lysinicin OF) har initiert et forskningssamarbeid mellom forskere ved NMBU og Universitetet i Bern (Prof. Lucy Hathaway). - Både lysinicin OF (nytt antimikrobielt stoff oppdaget i prosjektet) og fremtidige derivater av viscosin har potensial til å fungere som antibiotika mot S. pneumoniae og andre streptokokker. Hvis noen av stoffene passerer kliniske tester i fremtiden, vil det sannsynligvis styrke muligheten helsevesenet har for å behandle og evt. forhindre streptokokkinfeksjoner.

The continuous emergence and worldwide spread of antibiotic resistant bacteria endangers the efficacy of antibiotics. Adoption of antibiotic stewardship programs, better diagnosis and improved therapeutic regimens are important measures that contribute to control antibiotic resistance, but to stem the current crisis new antibacterial compounds are required. The discovery and development of new agents are therefore of the utmost importance. The emergence of drug-resistance in the important human pathogen S. pneumoniae represents a considerable problem in many European countries as well as in other parts of the world. The rise in resistance has been driven by widespread use and misuse of broad-spectrum antibiotics such as beta-lactams and macrolides in the treatment of pneumococcal infections. A growing body of research has begun to elucidate the harmful effects of broad-spectrum antibiotic therapy on the beneficial host microbiota. To combat these threats, increasing efforts are being directed toward the development of precision antimicrobial therapeutics that target key virulence determinants of specific pathogens while leaving the remainder of the host microbiota undisturbed. Moreover, as narrow-spectrum antibiotics impose a selection pressure only on a minor part of the microbiota, the risk of resistance development is reduced. The goal of the proposed project is to characterize an antimicrobial agent discovered in a screening of marine bacteria from the Oslo fjord. The purpose of our screening was to identify new drugs that inhibit the growth of S. pneumoniae. Preliminary data show that the identified compound is highly active against pneumococci, and that it has a narrow inhibition spectrum. In sum, the data indicate that the new compound has very interesting properties that should be further explored in order to determine whether the new compound has a therapeutic potential in the treatment of pneumococcal infections.

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FRIMEDBIO-Fri prosj.st. med.,helse,biol

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