Peptides containing the PCNA interacting motif APIM bind to the β-clamp and inhibit bacterial growth and mutagenesis

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents

Post-PKS modifications in the biosynthesis of the antifungal antibiotic nystatin

The antifungal polyene macrolide nystatin is produced by Streptomyces noursei ATCC 11455. The nystatin biosynthesis gene cluster of Streptomyces noursei has been cloned and sequenced, and a biosynthesis route has been predicted. In the present work, investigation of genes presumably involved in post-PKS modifications of nystatin is described. The aim of this work was to better understand the nystatin biosynthesis and to further use this information for generation of novel nystatin analogues. Two PKS-modifications of the nystatin molecule were targeted in this study: glycosylation with mycosamine at C-19 and oxidation of the exocyclic methyl group at C-16. Two genes putatively involved in mycosamine biosynthesis (NysDIII and NysDII) and one in attachment of mycosamine to the nystatin aglycone (nysDI) have been identified in the nystatin gene cluster. Their functions have been suggested, respectively, as a putative mannose dehydratase, aminotransferase and a glycosyltransferase. The deoxysugar mycosamine is proposed to have an important function for the activity of nystatin. To better understand the biosynthesis and importance of mycosamine and to perform modifications of nystatin via this post-PKS modifying step, the mycosamine biosynthesis was studied. The NysDIII protein was overexpressed in Escherichia coli and purified, and its in vitro mannose 4,6-dehydratase activity was confirmed. To study the function of nysDII and nysDI, the genes were individually deleted from the S. noursei chromosome. Both mutants were shown to produce a mixture of nystatinolide and 10-deoxynystatinolide, albeit at considerably different levels. Complementation experiments unequivocally confirmed the involvement of these two in mycosamine biosynthesis and attachement. Both antifungal and hemolytic activity of the purified nystatinolides were tested, and were found to be strongly reduced compared to nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin. A gene for putative P450 monooxyganse NysN has been identified in the nystatin biosynthesis gene cluster. The function of NysN has been predicted to be oxidation of an exocyclic C16 methyl group on the nystatin molecule in order to afford a C16 carboxyl. The latter group has been implicated in selective toxicity of other polyene macrolides, and thus is considered an important target for manipulation. The nysNgene was inactivated in S. noursei by both in-frame deletion and site-specific mutagenesis, and the resulting mutants were shown to produce 16-decarboxy-16-methylnystatin, supporting the suggested biological role of NysN as C16 methyl oxidase. The recombinant NysN protein was also expressed in Escherichia coli, but its C16-methyl oxidase activity in vitro could not be demonstrated. 16-decarboxy-16-methylnystatin was purified from the nysN mutant, and its antifungal activity was identical with nystatin whereby the toxicit was reduced compared to nystatin. In the work of developing new methods for obtaining nystatin analogues, bioconversion of nystatinolide was performed as a means to modify nystatin aglycone. For this purpose a sub-library of 35 different Streptomyces strains isolated from the Trondheims fjord was selected. One strain was shown to be able to add a water molecule (presumed epoxidation) and another strain was able to chlorinate the nystatinolides. An attempt on alternative glycosylation of nystatinolide was performed by using glycosyltransferase hybrids and deoxysugar biosynthesis gene cassettes. However, these experiments did not afford novel nystatin analogues, suggesting strong preference of the NysDI glycosyltyransferase for its natural sugar substrate GDP-mycosamine

APIM-Mediated REV3L-PCNA Interaction Important for Error Free TLS Over UV-Induced DNA Lesions in Human Cells

Proliferating cell nuclear antigen (PCNA) is essential for the organization of DNA replication and the bypass of DNA lesions via translesion synthesis (TLS). TLS is mediated by specialized DNA polymerases, which all interact, directly or indirectly, with PCNA. How interactions between the TLS polymerases and PCNA affects TLS specificity and/or coordination is not fully understood. Here we show that the catalytic subunit of the essential mammalian TLS polymerase POLζ, REV3L, contains a functional AlkB homolog 2 PCNA interacting motif, APIM. APIM from REV3L fused to YFP, and full-length REV3L-YFP colocalizes with PCNA in replication foci. Colocalization of REV3L-YFP with PCNA is strongly reduced when an APIM-CFP construct is overexpressed. We also found that overexpression of full-length REV3L with mutated APIM leads to significantly altered mutation frequencies and mutation spectra, when compared to overexpression of full-length REV3L wild-type (WT) protein in multiple cell lines. Altogether, these data suggest that APIM is a functional PCNA-interacting motif in REV3L, and that the APIM-mediated PCNA interaction is important for the function and specificity of POLζ in TLS. Finally, a PCNA-targeting cell-penetrating peptide, containing APIM, reduced the mutation frequencies and changed the mutation spectra in several cell lines, suggesting that efficient TLS requires coordination mediated by interactions with PCNA.publishedVersion© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

Analysis of the Mycosamine Biosynthesis and Attachment Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455▿

The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the β-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin

Peptides containing the PCNA interacting motif APIM bind to the beta-clamp and inhibit bacterial growth and mutagenesis

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents

SARS-CoV-2 vaccination modelling for safe surgery to save lives: data from an international prospective cohort study

Background Preoperative SARS-CoV-2 vaccination could support safer elective surgery. Vaccine numbers are limited so this study aimed to inform their prioritization by modelling. Methods The primary outcome was the number needed to vaccinate (NNV) to prevent one COVID-19-related death in 1 year. NNVs were based on postoperative SARS-CoV-2 rates and mortality in an international cohort study (surgical patients), and community SARS-CoV-2 incidence and case fatality data (general population). NNV estimates were stratified by age (18-49, 50-69, 70 or more years) and type of surgery. Best- and worst-case scenarios were used to describe uncertainty. Results NNVs were more favourable in surgical patients than the general population. The most favourable NNVs were in patients aged 70 years or more needing cancer surgery (351; best case 196, worst case 816) or non-cancer surgery (733; best case 407, worst case 1664). Both exceeded the NNV in the general population (1840; best case 1196, worst case 3066). NNVs for surgical patients remained favourable at a range of SARS-CoV-2 incidence rates in sensitivity analysis modelling. Globally, prioritizing preoperative vaccination of patients needing elective surgery ahead of the general population could prevent an additional 58 687 (best case 115 007, worst case 20 177) COVID-19-related deaths in 1 year. Conclusion As global roll out of SARS-CoV-2 vaccination proceeds, patients needing elective surgery should be prioritized ahead of the general population.The aim of this study was to inform vaccination prioritization by modelling the impact of vaccination on elective inpatient surgery. The study found that patients aged at least 70 years needing elective surgery should be prioritized alongside other high-risk groups during early vaccination programmes. Once vaccines are rolled out to younger populations, prioritizing surgical patients is advantageous