Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering

Lower ototoxicity and absence of hidden hearing loss point to gentamicin C1a and apramycin as promising antibiotics for clinical use

Trabajo presentado en el 42nd Annual MidWinter Meeting of the Association of Otorhinolaryngology, celebrado en Baltimore (Estados Unidos) del 9 al 13 de febrero de 2019.[Background]: Spread of antimicrobial resistance and shortage of novel antibiotics have led to an urgent need for new antibacterials (Maura et al. 2016, Curr Opin Microbiol 33: 41-46; Tacconelli et al. 2018, Lancet Infect Dis 18: 318-327). Although aminoglycoside antibiotics (AGs) exhibit potent antimicrobial activity, their use has been largely restricted due to serious sideeffects, mainly nephrotoxicity and ototoxicity (Forge and Schacht 2000, Audiol Neurootol 5: 3-22; Huth et al. 2011, Int J Otolaryngol 2011: 937861). It is therefore of great importance to identify AGs of strong antibacterial activity that lack their most harmful side effects.[Methods]: A large number of AGs were tested against a series of multidrug-resistant clinical isolates of the ESKAPE panel; of these, five AGs showing strong antibacterial activity were selected to evaluate their ototoxicity. A stepwise approach was followed, aiming at setting up a protocol that could be used in future high-throughput screenings. In vitro tests were initially conducted by assessing the viability of two established otic cell lines following AG treatment, and subsequently on murine cochlear organotypic cultures, by analysing hair cell survival. In vivo work was then carried out on a guinea pig model, following local round window application of the AGs.[Results]: Commercial gentamicin mixture (GM), the GM congener gentamicin C1a (GM C1a), apramycin (Apra), paromomycin (Paro) and neomycin (Neo) were selected for ototoxicity testing. In vitro analyses confirmed GM and Neo as the most toxic of the tested AGs, and Apra and Paro as those with the lowest toxicity; interestingly, GM C1a appeared to be less toxic than GM. Regarding the in vivo work, a dose-dependent effect of AGs on outer hair cell (OHC) survival and compound action potentials (CAPs) showed that GM C1a and Apra were the least toxic. Strikingly, although no changes were observed in CAP thresholds and OHC survival following treatment with low concentrations of Neo, GM and Paro, the number of inner hair cell (IHC) synaptic ribbons and the CAP amplitudes were reduced. This indication of hidden hearing loss was not observed with GM C1a or Apra at such concentrations.[Conclusion]: These findings have: (a) validated our screening approach, approach that will now be used for high-throughput testing of newly isolated AG congeners, (b) revealed the IHCs as the inner ear’;s most vulnerable element to AG treatment, and (c) identified GM C1a and Apra as promising bases for the development of clinically useful antibiotics

Identification of Lipstatin-Producing Ability in Streptomyces virginiae CBS 314.55 Using Dereplication Approach

Streptomicete su poznate kao bogat izvor bioaktivnih metabolita, poput sekundarnog metabolita lipstatina, koji ima vrlo karakterističnu strukturu β-laktona, a upotrebljava se kao intermedijar u proizvodnji lijeka za mršavljenje, komercijalnog naziva orlistat. Dublje razumijevanje distribucije identičnih ili strukturno sličnih spojeva, izoliranih iz taksonomski sličnih skupina mikroorganizama, bitno je za otkrivanje novih biološki aktivnih spojeva ili kvalitetnijih sojeva što proizvode poznate metabolite od velikog značaja za medicinu. Samo su dva soja, neovisni izolati vrste S. toxytricini, poznati kao proizvođači lipstatina. Prema današnjim taksonomskim spoznajama, vrsta S. toxytricini pripada fenotipskoj skupini S. lavendulae. Taksonomskom dereplikacijom i ispitivanjem in vitro inhibicije lipaze pomoću p-nitrofenolnih derivata C4 i C16 masnih kiselina istraženi su odabrani sojevi fenotipske skupine S. lavendulae iz javno dostupnih kolekcija mikroorganizama. Utvrđeno je da bakterijska kultura soja Streptomyces virginiae CBS 314.55 izrazito inhibira lipazu. Pomoću metoda HPLC i LC-MS/MS po prvi je put otkriveno da ovaj novi soj proizvodi metabolit strukture identične lipstatinu. Dokazano je da se novi soj S. virginiae morfološki i fiziološki bitno razlikuje od sojeva S. toxytricini, ali da im je proizvodni potencijal sličan. Time je potvrđena mogućnost učinkovite primjene metode dereplikacije za brzu identifikaciju novih industrijskih sojeva iz javno dostupnih kolekcija mikroorganizama.Streptomyces species are prolific producers of bioactive metabolites, such as β-lactone-containing lipstatin produced by Streptomyces toxytricini, an intermediate used in semi-synthetic process for production of anti-obesity drug orlistat. Understanding the distribution of identical or structurally similar molecules produced by a taxonomic group is of particular importance when trying to isolate novel biologically active compounds or strains producing known metabolites of medical importance with potentially improved properties. Until now, only two independent isolates of S. toxytricini species have been known to be producers of lipstatin. According to the current taxonomic criteria, S. toxytricini belongs to Streptomyces lavendulae phenotypic cluster. Taxonomy-based dereplication approach coupled with in vitro assay was applied to screen the S. lavendulae phenotypic cluster for production of lipstatin-like lipase inhibitors using synthetic p-nitrophenol derivatives of C4 and C16 lipids. Screening the available strains from public collections belonging to S. lavendulae phenotypic cluster, high lipase inhibitory activity was identified in the Streptomyces virginiae CBS 314.55 culture supernatants. HPLC and LC-MS/MS confirmed lipstatin production by a new Streptomyces species for the first time. We have demonstrated that the new lipstatin-producing strain S. virginiae morphologically and physiologically differs from S. toxytricini substantially; however, the production capacity of the newly identified lipstatin-producing species S. virginiae is comparable to S. toxytricini. We have thus demonstrated the effectiveness of a simple and affordable dereplication approach for identification of potentially novel and useful industrial strains available in public culture collections

シンポジウム

Additional file 14. A table with description of strains and plasmids generated and used in this study and graphical representation of over-expressed operons/genes

Učinak ATP sulfurilaze na prooksidativne značajke selenata u kvascu Schizosaccharomyces pombe

Selenium is an essential microelement in human and animal nutrition, whose intake can be in inorganic (e.g. selenite, selenate) or organic form (e.g. selenomethionine). The prooxidant effect of inorganic selenium sources in the animal nutrition has been found as a great disadvantage. Therefore, in this study the effect of the ATP sulphurylase on the prooxidant properties and toxicity of selenate in the fission yeast model organism Schizosaccharomyces pombe has been studied. Two strains of yeast Schizosaccharomyces pombe were used, selenate resistant (SeR) ZIM 1889 and selenate sensitive (SeS) ZIM 1878 strains, with inactive and active ATP sulphurylase, respectively. During the yeasts’ exposure to selenate growth, intracellular oxidation, cell viability and antioxidative defence systems were determined. Also, activities of antioxidative enzymes (catalase, superoxide dismutases, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase) and intracellular content of reduced glutathione were measured. The results show altered enzymatic activities and decreased intracellular content of reduced form of glutathione in the selenate sensitive strain as a consequence of active ATP sulphurylase, which enables selenate reduction leading to oxidative stress. During the selenate reduction, reactive oxygen species (ROS) are generated and therefore antioxidative defence systems are induced. In contrast, in the selenate resistant strain with inactive ATP sulphurylase, where selenate reduction does not occur, no induction of antioxidative defence systems was found. Consequently, the active ATP sulphurylase is the key enzyme for the prooxidant properties of selenate and it seems to be the main reason for selenate toxicity and ROS formation during the selenate reduction.Selen je esencijalni mikroelement u prehrani ljudi i ishrani životinja, čiji unos može biti u anorganskom (npr. selenit, selenat) ili u organskom obliku (npr. selenometionin). Prooksidativni učinak selena u anorganskom obliku u ishrani životinja ima niz nedostataka. Da bi se oni otklonili, proučavan je učinak ATP sulfurilaze na prooksidativne značajke i toksičnost selenata u modelnom kvascu Schizosaccharomyces pombe. U istraživanju su upotrijebljeni različiti sojevi kvasca Schizosaccharomyces pombe, i to ZIM 1889 otporan na selenat (SeR) s neaktivnom ATP sulfurilazom, te ZIM 1878 osjetljiv na selenat (SeS) s aktivnom ATP sulfurilazom. Kvasci su izloženi selenatu i tijekom rasta određivana je intracelularna oksidacija, preživljavanje stanica i antioksidativni obrambeni sustav. Također su određivani različiti antioksidativni enzimi (katalaza, superoksid dismutaza, glutation peroksidaza i glukoza-6-fosfat dehidrogeneza) i intracelularni udjel reduciranoga glutationa. Rezultati pokazuju promijenjene enzimske aktivnosti i smanjeni intracelularni udjel glutationa u soju osjetljivom na selenat, kao posljedica aktivne ATP sulfurilaze koja omogućava redukciju selenata, što dovodi do oksidativnog stresa. Tijekom redukcije selenata stvaraju se reaktivni kisikovi spojevi (ROS) i zbog toga aktivira antioksidativni obrambeni sustav. Suprotno, u soju otpornom na selenat, s neaktivnom ATP sulfurilazom, gdje nema redukcije selenata, nije ni došlo do aktiviranja antioksidativnog obrambenog sustava. Zbog toga je aktivna ATP sulfurilaza ključni enzim za prooksidativne značajke selenata i čini se da je glavni uzrok njegove toksičnosti i stvaranja ROS tijekom redukcije selenata

The Effect of ATP Sulphurylase on the Prooxidant Properties of Selenate in Yeast Schizosaccharomyces pombe

Selenium is an essential microelement in human and animal nutrition, whose intake can be in inorganic (e.g. selenite, selenate) or organic form (e.g. selenomethionine). The prooxidant effect of inorganic selenium sources in the animal nutrition has been found as a great disadvantage. Therefore, in this study the effect of the ATP sulphurylase on the prooxidant properties and toxicity of selenate in the fission yeast model organism Schizosaccharomyces pombe has been studied. Two strains of yeast Schizosaccharomyces pombe were used, selenate resistant (SeR) ZIM 1889 and selenate sensitive (SeS) ZIM 1878 strains, with inactive and active ATP sulphurylase, respectively. During the yeasts’ exposure to selenate growth, intracellular oxidation, cell viability and antioxidative defence systems were determined. Also, activities of antioxidative enzymes (catalase, superoxide dismutases, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase) and intracellular content of reduced glutathione were measured. The results show altered enzymatic activities and decreased intracellular content of reduced form of glutathione in the selenate sensitive strain as a consequence of active ATP sulphurylase, which enables selenate reduction leading to oxidative stress. During the selenate reduction, reactive oxygen species (ROS) are generated and therefore antioxidative defence systems are induced. In contrast, in the selenate resistant strain with inactive ATP sulphurylase, where selenate reduction does not occur, no induction of antioxidative defence systems was found. Consequently, the active ATP sulphurylase is the key enzyme for the prooxidant properties of selenate and it seems to be the main reason for selenate toxicity and ROS formation during the selenate reduction

Production of extracellular heterologous proteins in Streptomyces rimosus, producer of the antibiotic oxytetracycline

Among the Streptomyces species, Streptomyces lividans has often been used for the production of heterologous proteins as it can secrete target proteins directly into the culture medium. Streptomyces rimosus, on the other hand, has for long been used at an industrial scale for oxytetracycline production, and it holds ‘Generally Recognised As Safe’ status. There are a number of properties of S. rimosus that make this industrial strain an attractive candidate as a host for heterologous protein production, including (1) rapid growth rate(2) growth as short fragments, as for Escherichia coli(3) high efficiency of transformation by electroporationand (4) secretion of proteins into the culture medium. In this study, we specifically focused our efforts on an exploration of the use of the Sec secretory pathway to export heterologous proteins in a S. rimosus host. We aimed to develop a genetic tool kit for S. rimosus and to evaluate the extracellular production of target heterologous proteins of this industrial host. This study demonstrates that S. rimosus can produce the industrially important enzyme phytase AppA extracellularly, and analogous to E. coli as a host, application of His-Tag/Ni-affinity chromatography provides a simple and rapid approach to purify active phytase AppA in S. rimosus. We thus demonstrate that S. rimosus can be used as a potential alternative protein expression system

Elementi koji reguliraju biosintezu tetraciklinskih antibiotika u genskim nakupinama: otcG gen pozitivno regulira proizvodnju oksitetraciklina u vrste Streptomyces rimosus

The expression of bacterial polyketide synthase gene clusters is often controlled by a number of different families of regulatory proteins that can have either a pathway-specific or a pleiotropic mode of action, e.g. the SARP family (Streptomyces antibiotic regulatory proteins), ribosome-associated ppGpp synthetase, γ-butyrolactone-binding regulatory proteins, and two-component regulatory proteins. The molecular genetics of such regulatory mechanisms that govern the biosynthesis of tetracyclines is poorly understood. In this work, a comparative bioinformatic analysis of regulatory genes present in three tetracycline antibiotic gene clusters, namely oxytetracycline (OTC), chlortetracycline and recently cloned chelocardin gene clusters of S. rimosus, S. aureofaciens and Amycolatopsis sulphurea has been performed. A SARP family regulatory protein is located in the chlortetracycline gene cluster, but is not detected in the gene cluster encoding OTC biosynthesis. Interestingly, the only regulatory element identified in chelocardin gene cluster was chdA, an otrR and ctcR homologue from the TetR family of regulators that regulates the expression of the otrB and ctc05 exporter genes in the oxytetracycline and chlortetracycline gene clusters. In the oxytetracycline gene cluster, a new LAL (LuxR) family regulatory gene homologue, otcG, was identified. This homologue is also present in the ctc gene cluster. By gene disruption and overexpression experiments, a \u27conditionally positive\u27 role of otcG in OTC biosynthesis has been demonstrated. The observation, the bioinformatics data and the previous work on phosphate regulation suggest the presence of a more complex, fine tuning role of the otcG gene product in overall expression of genes for OTC biosynthesis.Ekspresiju bakterijskih genskih nakupina poliketidnih sintaza obično reguliraju različiti vrlo specifični proteini koji su uglavnom usmjereni samo na ciljane gene u nakupini gena ili je riječ o regulatornim genima što djeluju šire na stanične procese, takozvani pleiotropni regulatori. Prema načinu djelovanja ti se regulatorni proteini mogu podijeliti u više skupina, kao što su SARP (engl. Streptomyces Antibiotic Regulatory Proteins), skupina ribosomski povezanih regulatornih proteina nazvanih ppGpp sintaze, γ-butirolaktonskih veznih i dvokomponentnih regulatornih proteina. Molekularna genetika takvih regulatornih mehanizama, koji utječu na biosintezu tetraciklina, još je i danas slabo istražena. U ovom je radu prikazana komparativna bioinformatička studija regulatornih proteina triju genskih nakupina koje kodiraju biosintezu oksitetraciklina (OTC) u vrste Streptomyces rimosus, klortetraciklina (u vrste Streptomyces aureofaciens), a i nedavno klonirane genske nakupine za biosintezu kelokardina (CHD) u vrste Amycolatopsis sulphurea. U genskoj nakupini koja kodira biosintezu klortetraciklina nalazi se takozvani SARP regulatorni gen. Međutim, homolog toga gena ne postoji u nakupini gena za biosintezu oksitetraciklina. U nakupini gena za biosintezu kelokardina pronađen je samo jedan regulatorni gen nazvan chdA, homolog gena otrR i ctcR iz skupine TetR regulatornih proteina, koji regulira ekspresiju otrB i ctc05 gena u nakupinama gena za biosintezu oksitetraciklina i klortetraciklina. U genskoj nakupini za biosintezu oksitetraciklina identificiran je novi regulatorni protein iz skupine LAL (LuxR), nazvan OtcG. Taj se genski homolog nalazi i u genskoj nakupini za biosintezu klortetraciklina. Inaktivacijom i povećanom ekspresijom gena otcG eksperimentalno je utvrđeno njegovo djelovanje kao uvjetno pozitivnog regulatora u biosintezi oksitetraciklina. Takvi eksperimentalni rezultati bioinformatičke studije i rezultati koji se odnose na prijašnje istraživanje utjecaja fosfata na reguliranje biosinteze oksitetraciklina upućuju na vrlo kompleksnu organizaciju regulatornih elemenata i njihova djelovanja u procesu biosinteze oksitetraciklina, u kojima sudjeluje i novi regulatorni element, produkt gena otcG

Production of extracellular heterologous proteins in Streptomyces rimosus, producer of the antibiotic oxytetracycline

Among the Streptomyces species, Streptomyces lividans has often been used for the production of heterologous proteins as it can secrete target proteins directly into the culture medium. Streptomyces rimosus, on the other hand, has for long been used at an industrial scale for oxytetracycline production, and it holds ‘Generally Recognised As Safe’ status. There are a number of properties of S. rimosus that make this industrial strain an attractive candidate as a host for heterologous protein production, including (1) rapid growth rate; (2) growth as short fragments, as for Escherichia coli; (3) high efficiency of transformation by electroporation; and (4) secretion of proteins into the culture medium. In this study, we specifically focused our efforts on an exploration of the use of the Sec secretory pathway to export heterologous proteins in a S. rimosus host. We aimed to develop a genetic tool kit for S. rimosus and to evaluate the extracellular production of target heterologous proteins of this industrial host. This study demonstrates that S. rimosus can produce the industrially important enzyme phytase AppA extracellularly, and analogous to E. coli as a host, application of His-Tag/Ni-affinity chromatography provides a simple and rapid approach to purify active phytase AppA in S. rimosus. We thus demonstrate that S. rimosus can be used as a potential alternative protein expression system.This study was funded by the Ministry of Higher Education, Science and Technology, Slovenian Research Agency (grant no. L4-7117), grant no. C3330-17-529038 ‘Raziskovalci-2.0-UL-BF-529038’ by the Ministry of Higher Education, Science and Technology—Republic of Slovenia, Slovene Human Resources, Development and Scholarship Funds (grant no. 58-T-003) and ‘Augusto González Linares’ program (University of Cantabria, Spain)