ABSTRACT
In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, ß-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The ß-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 ß-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 ß-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 ß-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.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , DNA Polymerase III/antagonists & inhibitors , Peptides/chemistry , Peptides/pharmacology , Animals , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/therapeutic use , DNA Polymerase III/chemistry , DNA Replication/drug effects , DNA-Directed DNA Polymerase , Female , Methicillin-Resistant Staphylococcus aureus/drug effects , Methicillin-Resistant Staphylococcus aureus/genetics , Methicillin-Resistant Staphylococcus aureus/growth & development , Mice, Inbred BALB C , Mutagenesis/drug effects , Nucleic Acid Synthesis Inhibitors/chemistry , Nucleic Acid Synthesis Inhibitors/pharmacology , Nucleic Acid Synthesis Inhibitors/therapeutic use , Peptides/metabolism , Peptides/therapeutic use , Proliferating Cell Nuclear Antigen/metabolism , Protein Interaction Domains and Motifs , Staphylococcal Skin Infections/drug therapy , Staphylococcus epidermidis/drug effects , Staphylococcus epidermidis/genetics , Staphylococcus epidermidis/growth & developmentABSTRACT
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.
Subject(s)
DNA Damage/radiation effects , DNA Replication , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Protein Interaction Domains and Motifs , Ultraviolet Rays/adverse effects , Biomarkers , Cell Line , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Fluorescent Antibody Technique , Humans , Mutation , Mutation Rate , Protein Binding , Protein Sorting Signals , Protein TransportABSTRACT
Proliferating cell nuclear antigen (PCNA), commonly known as a nuclear protein essential for regulation of DNA replication, DNA repair, and epigenetics, has recently been associated with multiple cytosolic functions. Many proteins containing one of the two known PCNA-interacting motifs, the AlkB homologue 2 PCNA interacting motif (APIM) and the PCNA-interacting peptide (PIP)-box, are considered to be mainly cytosolic. APIM is found in more than 20 kinases and/or associated proteins including several direct or indirect members of the mitogen-activated protein kinase (MAPK) and PI3K/Akt pathways. Mass spectrometry analysis of PCNA-pull downs verified that many cytosolic proteins involved in the MAPK and PI3K/Akt pathways are in complex with PCNA. Furthermore, treatment of cells with a PCNA-interacting APIM-containing peptide (APIM-peptide) reduced Akt phosphorylation in human peripheral blood monocytes and a human keratinocyte cell line (HaCaT). Additionally, the APIM-peptide strongly reduced the cytokine secretion from monocytes stimulated with toll like receptor (TLR) ligands and potentiated the effects of MAPK and PI3K/Akt inhibitors. Interestingly, the protein level of the APIM-containing PKR/RIG-1 activator protein (PACT) was initially strongly reduced in HaCaT cells stimulated with APIM-peptide in combination with the TLR ligand polyinosinic-polycytidylic acid (polyIC). Our results suggest that PCNA has a platform role in cytosol affecting cellular signaling.
Subject(s)
Cytokines/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Toll-Like Receptors/metabolism , Amino Acid Sequence , Cell Survival/drug effects , Cells, Cultured , Humans , Immunoprecipitation , Keratinocytes/cytology , Keratinocytes/metabolism , Microscopy, Confocal , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , Peptides/chemistry , Peptides/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Proliferating Cell Nuclear Antigen/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , RNA-Binding Proteins/metabolism , Signal Transduction/drug effects , Toll-Like Receptors/chemistryABSTRACT
Seven polyene macrolides with alterations in the polyol region and exocyclic carboxy group were obtained via genetic engineering of the nystatin biosynthesis genes in Streptomyces noursei. In vitro analyses of the compounds for antifungal and hemolytic activities indicated that combinations of several mutations caused additive improvements in their activity-toxicity properties. The two best analogs selected on the basis of in vitro data were tested for acute toxicity and antifungal activity in a mouse model. Both analogs were shown to be effective against disseminated candidosis, while being considerably less toxic than amphotericin B. To our knowledge, this is the first report on polyene macrolides with improved in vivo pharmacological properties obtained by genetic engineering. These results indicate that the engineered nystatin analogs can be further developed into antifungal drugs for human use.
Subject(s)
Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Genetic Engineering/methods , Nystatin/biosynthesis , Nystatin/pharmacology , Polyenes/chemistry , Streptomyces/genetics , Animals , Antifungal Agents/chemistry , Antifungal Agents/toxicity , Base Sequence , Candida albicans/drug effects , Genes, Bacterial/genetics , Hemolysis/drug effects , Humans , Male , Mice , Nystatin/analogs & derivatives , Nystatin/chemistry , Nystatin/toxicity , Polymers/chemistry , Streptomyces/metabolism , Structure-Activity RelationshipABSTRACT
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 beta-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.