RESUMEN
Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.
Asunto(s)
Bacteriocinas , Humanos , Bacteriocinas/farmacología , Bacteriocinas/química , Bacterias , Antibacterianos/farmacología , Antibacterianos/química , PéptidosRESUMEN
The rise of multidrug resistant fungal infections highlights the need to identify and develop novel antifungal agents. Occidiofungin is a nonribosomally synthesized glycolipopeptide that has a unique mechanism of action, disrupting actin-mediated functions and inducing cellular apoptosis. Antifungal activity has been observed in vitro against various fungal species, including multidrug resistant Candida auris, and in vivo efficacy has been demonstrated in a murine vulvovaginal candidiasis model. Occidiofungin, a cyclic glycolipopeptide, is composed of eight amino acids and in previous studies, an asparagine residue was assigned at position 7 (ASN7). In this study, new structural variants of occidiofungin have been characterized which have aspartic acid (ASP7), glutamine (GLN7), or glutamic acid (GLU7) at position 7. The side chain of the ASP7 variant contains the only terminal carboxylic acid in the peptide and provides a useful site for selective chemical modifications. Analogues were synthesized at the ASP7 position and tested for antifungal activity. These analogues were shown to be more active as compared to the ASP7 variant against a panel of Candida species. The naturally occurring variants of occidiofungin with a side chain containing a carboxylic acid at the seventh amino acid position can be used to develop semisynthetic analogues with enhanced therapeutic properties.
Asunto(s)
Antifúngicos , Burkholderia , Glicopéptidos , Péptidos Cíclicos , Ratones , Animales , Antifúngicos/química , Burkholderia/química , Ácidos Carboxílicos , Pruebas de Sensibilidad MicrobianaRESUMEN
Burkholderia contaminans MS14, isolated from a soil sample in Mississippi, is known for producing the novel antifungal compound occidiofungin. In addition, MS14 exhibits a broad range of antibacterial activities against common plant pathogens. Random mutagenesis and gene complementation indicate that four genes are required for antibacterial activity of strain MS14 against the fire blight pathogen Erwinia amylovora. With the aim of finding the biosynthetic gene cluster for the unknown antibacterial compound, we used RNA-seq to analyze the transcriptome of MS14 wild type and mutants lacking antibacterial activity. The twofold lower expressed genes in all mutants were studied, and a polyketide synthase (PKS) gene cluster was predicted to be directly involved in MS14 antibacterial activities. The nptII-resistance cassette and CRISPR-Cas9 systems were used to mutate the PKS gene cluster. Plate bioassays showed that either insertion or frame-shifting one of the PKS genes resulted in a loss of antibacterial activity. Considering that the antibacterial-defective mutants maintain the same antifungal activities as the wild-type strain, the results suggest that this PKS gene cluster is highly likely to be involved in or directly responsible for the production of MS14 antibacterial activity. Purification efforts revealed that the antibacterial activity of the compound synthesized by the gene cluster is sensitive to UV radiation. Nevertheless, these findings have provided more insights to understand the antibacterial activity of strain MS14.
Asunto(s)
Burkholderia , Policétidos , Antifúngicos , Ligasas/genética , Enfermedades de las Plantas/microbiología , Burkholderia/genética , Antibacterianos/farmacología , Familia de MultigenesRESUMEN
Novel antiparasitic activity was observed for the antifungal occidiofungin. It efficaciously and irreversibly inhibited the zoonotic enteric parasite Cryptosporidium parvumin vitro with limited cytotoxicity (50% effective concentration [EC50] = 120 nM versus 50% cytotoxic concentration [TC50] = 988 nM), and its application disrupted the parasite morphology. This study expands the spectrum of activity of a glycolipopeptide named occidiofungin. Occidiofungin has poor gastrointestinal tract absorption properties, supporting future investigations into its potential activities on other enteric parasites.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Antifúngicos/farmacología , Antiparasitarios/farmacología , Glicopéptidos , Humanos , Péptidos CíclicosRESUMEN
Occidiofungin is a nonribosomally synthesized cyclic lipopeptide that possesses broad-spectrum antifungal properties at submicromolar concentrations. This report explores multiple routes of administration and formulations of occidiofungin, as well as its toxicity in mice. Further, infection studies were performed in mice to assess the application of occidiofungin for treating systemic and intravaginal yeast infections. Formulations for intravenous and intravaginal administration of occidiofungin were prepared. Pharmacokinetic analyses were performed in a murine model, and a liquid chromatography-mass spectrometry (LC-MS) method was developed and used to quantify occidiofungin in mouse plasma samples. Toxicological and histopathological analyses of two repeat-dose studies using occidiofungin were performed. In these animal models, following intravenous administration, a liposomal formulation of occidiofungin improved the half-life and peak plasma drug concentration over that with a liposome-free formulation. Two long-term repeat-dosing toxicity studies of occidiofungin indicated the absence of toxicity in organ tissues. Murine models of a systemic yeast infection and a vulvovaginal yeast infection were performed. The findings of the systemic infection study revealed limitations in the use of occidiofungin that may be alleviated with the development of novel structural analogs or with further formulation studies. The gel formulation of occidiofungin demonstrated improved efficacy over that of the commercial product Monistat 3 in a vulvovaginal candidiasis study. This report outlines the optimal routes of administration of occidiofungin and demonstrates minimal toxicity following chronic exposure. Further, the results of these studies provide a clear indication for the use of occidiofungin for the treatment of recurrent vulvovaginal candidiasis (RVVC), which is a serious and clinically relevant issue.
Asunto(s)
Antifúngicos , Candidiasis Vulvovaginal , Animales , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Candidiasis Vulvovaginal/tratamiento farmacológico , Femenino , Glicopéptidos , Humanos , Ratones , Péptidos CíclicosRESUMEN
Occidiofungin is produced by the soil bacterium Burkolderia contaminans MS14 and is structurally similar or identical to the burkholdines, xylocandins, and cepacidines. This study identified the primary cellular target of occidiofungin, which was determined to be actin. The modification of occidiofungin with a functional alkyne group enabled affinity purification assays and localization studies in yeast. Occidiofungin has a subtle effect on actin dynamics that triggers apoptotic cell death. We demonstrate the highly specific localization of occidiofungin to cellular regions rich in actin in yeast and the binding of occidiofungin to purified actin in vitro Furthermore, a disruption of actin-mediated cellular processes, such as endocytosis, nuclear segregation, and hyphal formation, was observed. All of these processes require the formation of stable actin cables, which are disrupted following the addition of a subinhibitory concentration of occidiofungin. We were also able to demonstrate the effectiveness of occidiofungin in treating a vulvovaginal yeast infection in a murine model. The results of this study are important for the development of an efficacious novel class of actin binding drugs that may fill the existing gap in treatment options for fungal infections or different types of cancer.
Asunto(s)
Actinas/metabolismo , Antifúngicos/uso terapéutico , Burkholderia/metabolismo , Candidiasis Vulvovaginal/tratamiento farmacológico , Glicopéptidos/metabolismo , Glicopéptidos/uso terapéutico , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/uso terapéutico , Animales , Candida/efectos de los fármacos , Femenino , Glicopéptidos/química , Ratones , Ratones Endogámicos BALB C , Péptidos Cíclicos/químicaRESUMEN
Mutacin 1140, a member of the epidermin family of type AI lantibiotics, has a broad spectrum of activity against Gram-positive bacteria. It blocks cell wall synthesis by binding to lipid II. Although it has rapid bactericidal effects and potent activity against Gram-positive pathogens, its rapid clearance and short half-life in vivo limit its development in the clinic. In this study, we evaluated the effect of charged and dehydrated residues on the pharmacokinetics of mutacin 1140. The dehydrated residues were determined to contribute to the stability of mutacin 1140, while alanine substitutions for the lysine or arginine residues improved the pharmacological properties of the antibiotic. Analogs K2A and R13A had significantly lower clearances, leading to higher plasma concentrations over time. They also had improved bioactivities against several pathogenic bacteria. In a murine systemic methicillin-resistant Staphylococcus aureus (MRSA) infection model, a 10-mg/kg single intravenous bolus injection of the K2A and R13A analogs (1:1 ratio) protected 100% of the infected mice, while a 2.5-mg/kg dose resulted in 50% survival. The 10-mg/kg treatment group had a significant reduction in bacteria load in the livers and kidneys compared to that in the vehicle control group. The study provides lead compounds for the future development of antibiotics used to treat systemic Gram-positive infections.
Asunto(s)
Bacteriocinas/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Péptidos/farmacología , Ingeniería de Proteínas/métodos , Infecciones Estafilocócicas/tratamiento farmacológico , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Arginina/metabolismo , Bacteriocinas/sangre , Bacteriocinas/síntesis química , Bacteriocinas/farmacocinética , Diseño de Fármacos , Femenino , Riñón/efectos de los fármacos , Riñón/microbiología , Riñón/patología , Hígado/efectos de los fármacos , Hígado/microbiología , Hígado/patología , Lisina/metabolismo , Meticilina/farmacología , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Péptidos/sangre , Péptidos/síntesis química , Péptidos/farmacocinética , Estabilidad Proteica , Infecciones Estafilocócicas/sangre , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/mortalidad , Electricidad Estática , Relación Estructura-Actividad , Análisis de SupervivenciaRESUMEN
Mutacin 1140 belongs to the epidermin family of type AI lantibiotics. This family has a broad spectrum of activity against Gram-positive bacteria. The binding of mutacin 1140 to lipid II leads to the inhibition of cell wall synthesis. Pharmacokinetic experiments with type AI lantibiotics are generally discouraging for clinical applications due to the short half-life of these compounds. The unprotected dehydrated and protease-susceptible residues outside the lanthionine rings may play a role in the short half-life in physiological settings. Previous mutagenesis work on mutacin 1140 has been limited to the lanthionine-forming residues, the C-terminally decarboxylated residue, and single amino acid substitutions at residues Phe1, Trp4, Dha5, and Arg13. To study the importance of the dehydrated (Dha5 and Dhb14) and protease-susceptible (Lys2 and Arg13) residues within mutacin 1140 for stability and bioactivity, each of these residues was evaluated for its impact on production and inhibitory activity. More than 15 analogs were purified, enabling direct comparison of the activities against a select panel of Gram-positive bacteria. The efficiency of the posttranslational modification (PTM) machinery of mutacin 1140 is highly restricted on its substrate. Analogs in the various intermediate stages of PTMs were observed as minor products following single point mutations at the 2nd, 5th, 13th, and 14th positions. The combination of alanine substitutions at the Dha5 and Dhb14 positions abolished mutacin 1140 production, while the production was restored by substitution of a Gly residue at one of these positions. Analogs with improved activity, productivity, and proteolytic stability were identified.IMPORTANCE Our findings show that the efficiency of mutacin 1140 PTMs is highly dependent on the core peptide sequence. Analogs in various intermediate stages of PTMs can be transported by the bacterium, which indicates that PTMs and transport are finely tuned for the native mutacin 1140 core peptide. Only certain combinations of amino acid substitutions at the Dha5 and Dhb14 dehydrated residue positions were tolerated. Observation of glutamylated core peptide analogs shows that dehydrations occur in a glutamate-dependent manner. Interestingly, mutations at positions outside rings A and B, the lipid II binding domain, would interfere with lipid II binding. Purified mutacin 1140 analogs have various activities and selectivities against different genera of bacteria, supporting the effort to generate analogs with higher specificity against pathogenic bacteria. The discovery of analogs with improved inhibitory activity against pathogenic bacteria, increased stability in the presence of protease, and higher product yields may promote the clinical development of this unique antimicrobial compound.
Asunto(s)
Antibacterianos/química , Antibacterianos/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Péptidos/química , Péptidos/genética , Streptococcus mutans/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacteriocinas/metabolismo , Bacteriocinas/farmacología , Estabilidad de Medicamentos , Mutagénesis Sitio-Dirigida , Péptidos/metabolismo , Péptidos/farmacología , Streptococcus mutans/química , Streptococcus mutans/genéticaRESUMEN
Lantibiotics are a class of lanthionine-containing, ribosomally synthesized, and posttranslationally modified peptides (RiPPs) produced by Gram-positive bacteria. Salivaricin A2 belongs to the type AII lantibiotics, which are generally considered to kill Gram-positive bacteria by binding to the cell wall precursor lipid II via a conserved ring A structure. Salivaricin A2 was first reported to be isolated from a probiotic strain, Streptococcus salivarius K12, but the structural and bioactivity characterizations of the antibiotic have remained limited. In this study, salivaricin A2 was purified and its covalent structure was characterized. N-terminal analogues of salivaricin A2 were generated to study the importance for bioactivity of the length and charge of the N-terminal amino acids. Analogue salivaricin A2(3-22) has no antibacterial activity and does not have an antagonistic effect on the native compound. The truncated analogue also lost its ability to bind to lipid II in a thin-layer chromatography (TLC) assay, suggesting that the N-terminal amino acids are important for binding to lipid II. The creation of N-terminal analogues of salivaricin A2 promoted a better understanding of the bioactivity of this antibiotic and further elucidated the structural importance of the N-terminal leader peptide. The antibacterial activity of salivaricin A2 is due not only to the presence of the positively charged N-terminal amino acid residues, but to the length of the N-terminal linear peptide.IMPORTANCE The amino acid composition of the N-terminal linear peptide of salivaricin A2 is crucial for function. Our study shows that the length of the amino acid residues in the linear peptide is crucial for salivaricin A2 antimicrobial activity. Very few type AII lantibiotic covalent structures have been confirmed. The characterization of the covalent structure of salivaricin A2 provides additional support for the predicted lanthionine and methyl-lanthionine ring formations present in this structural class of lantibiotics. Removal of the N-terminal Lys1 and Arg2 residues from the peptide causes a dramatic shift in the chemical shift values of amino acid residues 7 through 9, suggesting that the N-terminal amino acids contribute to a distinct structural conformer for the linear peptide region. The demonstration that the bioactivity could be partially restored with the substitution of N-terminal alanine residues supports further studies aimed at determining whether new analogues of salivaricin A2 for novel applications can be synthesized.
Asunto(s)
Bacterias/efectos de los fármacos , Bacteriocinas/farmacología , Streptococcus salivarius/química , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Bacteriocinas/químicaRESUMEN
Mutacin 1140 belongs to the epidermin group of lantibiotics. Epidermin class lantibiotics are ribosomally synthesized and posttranslationally modified antibiotics with potent activity against Gram-positive bacteria. In particular, this class is effective at targeting drug-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium tuberculosis, and Clostridium difficile A C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) residue is derived from a decarboxylation of a terminal cysteine that is involved in lanthionine ring formation. Studies on mutacin 1140 have revealed new insight into the structural importance of the C-terminal AviCys residue. A C-terminal carboxyl analogue of mutacin 1140 was engineered. Capping the C-terminal carboxyl group with a primary amine restores bioactivity and affords a novel opportunity to synthesize new analogues. A C-terminal fluorescein-labeled mutacin 1140 analogue traps lipid II into a large lipid II lantibiotic complex, similar to that observed in vivo for the lantibiotic nisin. A C-terminal carboxyl analogue of mutacin 1140 competitively inhibits the activity of native mutacin 1140 and nisin. The presence of a C-terminal carboxyl group prevents the formation of the large lipid II lantibiotic complexes but does not prevent the binding of the lantibiotic to lipid II.IMPORTANCE This study addressed the importance of the C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) residue for antibacterial activity. We have learned that the posttranslational modification for making the AviCys residue is presumably important for the lateral assembly mechanism of activity that traps lipid II into a large complex. The C-terminal carboxyl analogue of this class of lantibiotics is agreeable to the addition of a wide variety of substrates. The addition of fluorescein enabled in vivo visualization of the epidermin class of lantibiotics in action. These results are significant because, as we demonstrate, the presence of the AviCys residue is not essential for bioactivity, but, more importantly, the removal of the carboxyl group is essential. The ability to make a C-terminal carboxyl analogue that is modifiable will facilitate the synthesis of novel analogues of the epidermin class of lantibiotics that can be developed for new applications.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Bacteriocinas/química , Bacteriocinas/farmacología , Péptidos/química , Péptidos/farmacología , Clostridioides difficile/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Streptococcus mutans/efectos de los fármacosRESUMEN
Burkholderia contaminans MS14 was isolated from soil in Mississippi. When it is cultivated on nutrient broth-yeast extract agar, the colonies exhibit bactericidal activity against a wide range of plant-pathogenic bacteria. A bacteriostatic compound with siderophore activity was successfully purified and was determined by nuclear magnetic resonance spectroscopy to be ornibactin. Isolation of the bactericidal compound has not yet been achieved; therefore, the exact nature of the bactericidal compound is still unknown. During an attempt to isolate the bactericidal compound, an interesting relationship between the production of ornibactin and the bactericidal activity of MS14 was characterized. Transposon mutagenesis resulted in two strains that lost bactericidal activity, with insertional mutations in a nonribosomal peptide synthetase (NRPS) gene for ornibactin biosynthesis and a luxR family transcriptional regulatory gene. Coculture of these two mutant strains resulted in restoration of the bactericidal activity. Furthermore, the addition of ornibactin to the NRPS mutant restored the bactericidal phenotype. It has been demonstrated that, in MS14, ornibactin has an alternative function, aside from iron sequestration. Comparison of the ornibactin biosynthesis genes in Burkholderia species shows diversity among the regulatory elements, while the gene products for ornibactin synthesis are conserved. This is an interesting observation, given that ornibactin is thought to have the same defined function within Burkholderia species. Ornibactin is produced by most Burkholderia species, and its role in regulating the production of secondary metabolites should be investigated.IMPORTANCE Identification of the antibacterial product from strain MS14 is not the key feature of this study. We present a series of experiments that demonstrate that ornibactin is directly involved in the bactericidal phenotype of MS14. This observation provides evidence for an alternative function for ornibactin, aside from iron sequestration. Ornibactin should be further evaluated for its role in regulating the biosynthesis of secondary metabolites in other Burkholderia species.
Asunto(s)
Antibacterianos/metabolismo , Antibiosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia/genética , Burkholderia/fisiología , Sideróforos/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Burkholderia/química , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Hierro/metabolismo , Mutagénesis , Mutagénesis Insercional , Péptido Sintasas/genética , Proteínas Represoras/genética , Sideróforos/química , Sideróforos/farmacología , Transactivadores/genéticaRESUMEN
UNLABELLED: Lantibiotics are ribosomally synthesized peptide antibiotics composed of an N-terminal leader peptide that is cleaved to yield the active antibacterial peptide. Significant advancements in molecular tools that promote the study of lantibiotic biosynthesis can be used in Streptococcus mutans. Herein, we further our understanding of leader peptide sequence and core peptide structural requirements for the biosynthesis and transport of the lantibiotic mutacin 1140. Our study on mutacin 1140 biosynthesis shows a dedicated secondary cleavage site within the leader peptide and the dependency of transport on core peptide posttranslational modifications (PTMs). The secondary cleavage site on the leader peptide is found at the -9 position, and secondary cleavage occurs before the core peptide is transported out of the cell. The coordinated cleavage at the -9 position was absent in a lanT deletion strain, suggesting that the core peptide interaction with the LanT transporter enables uniform cleavage at the -9 position. Following transport, the LanP protease was found to be tolerant to a wide variety of amino acid substitutions at the primary leader peptide cleavage site, with the exception of arginine at the -1 position. Several leader and core peptide mutations produced core peptide variants that had intermediate stages of PTM enzyme modifications, supporting the concept that PTM enzyme modifications, secondary cleavage, and transport are occurring in a highly coordinated fashion. IMPORTANCE: Mutacin 1140 belongs to the class I lantibiotic family of ribosomally synthesized and posttranslationally modified peptides (RiPPs). The biosynthesis of mutacin 1140 is a highly efficient process which does not lead to a discernible level of production of partially modified core peptide variants. The products isolated from an extensive mutagenesis study on the leader and core peptides of mutacin 1140 show that the posttranslational modifications (PTMs) on the core peptide occur under a highly coordinated dynamic process. PTMs are dictated by the distance of the core peptide modifiable residues from PTM enzyme active sites. The formation of lanthionine rings aids in the formation of successive PTMs, as was observed in a peptide variant lacking a C-terminal decarboxylation.
Asunto(s)
Bacteriocinas/biosíntesis , Regulación Bacteriana de la Expresión Génica/fisiología , Streptococcus mutans/metabolismo , Secuencia de Aminoácidos , Bacteriocinas/genética , Bacteriocinas/metabolismo , Transporte Biológico/fisiología , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Conformación Proteica , Streptococcus mutans/genéticaRESUMEN
Burkholderia contaminans strain MS14 produces the antifungal compound occidiofungin, which is responsible for significant antifungal activities against a broad range of plant and animal fungal pathogens. Occidiofungin is a cyclic glycolipopeptide made up of eight amino acids and one xylose. A 56-kb ocf gene cluster was determined to be essential for occidiofungin production. In this study, the ocfC gene, which is located downstream of ocfD and upstream of the ocfB gene in the ocf gene cluster, was examined. Antifungal activity of the ocfC gene mutant MS14KC1 was reduced against the indicator fungus Geotrichum candidum compared with that of the wild-type strain. Furthermore, the analysis of the protein sequence suggests that the ocfC gene encodes a glycosyltransferase. Biochemical analyses using nuclear magnetic resonance (NMR) and mass spectroscopy revealed that the ocfC mutant produced the occidiofungin without the xylose. The purified ocfC mutant MS14KC1 product had a level of bioactivity similar to that of the wild-type product. The revertant MS14KC1-R of the ocfC mutant produced the same antifungal activity level on plate assays and the same antifungal compound based on high-performance liquid chromatography (HPLC) and mass spectroscopy analysis as wild-type strain MS14. Collectively, the study demonstrates that the ocfC gene encodes a glycosyltransferase responsible to add a xylose to the occidiofungin molecule and that the presence of the xylose is not important for antifungal activity against Candida species. The finding provides a novel variant for future studies aimed at evaluating its use for inhibiting clinical and agricultural fungi, and the finding could also simplify the chemical synthesis of occidiofungin variants.
Asunto(s)
Antifúngicos/metabolismo , Burkholderia/enzimología , Glicopéptidos/metabolismo , Pentosiltransferasa/genética , Péptidos Cíclicos/metabolismo , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Burkholderia/química , Burkholderia/efectos de los fármacos , Burkholderia/genética , Candida/efectos de los fármacos , Candida/crecimiento & desarrollo , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Genotipo , Geotrichum/efectos de los fármacos , Geotrichum/crecimiento & desarrollo , Glicopéptidos/química , Glicopéptidos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Pentosiltransferasa/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Análisis de Secuencia de ADN , Xilosa/metabolismo , UDP Xilosa Proteína XilosiltransferasaRESUMEN
The oral bacterium Streptococcus mutans, strain JH1140, produces the antibiotic mutacin 1140. Mutacin 1140 belongs to a group of antibiotics called lanthipeptides. More specifically, mutacin 1140 is related to the epidermin type A(I) lanthipeptides. Mutagenesis experiments of this group of lanthipeptides have been primarily restricted to the posttranslationally modified meso-lanthionine and 3-methyllanthionine residues. Site-directed mutagenesis of the core peptide of mutacin 1140 was performed using the suicide vector pVA891. Substitutions of the N-terminal residue, the charged residue in the hinge region, and residues in ring A and intertwined rings C and D were investigated. A truncation and insertion of residues in ring A and intertwined rings C and D were also performed to determine whether or not they would alter the antimicrobial activity of the producing strain. Bioassays revealed that five of 14 mutants studied had improved antimicrobial activity against the indicator strain Micrococcus luteus ATCC 10240. MICs against Streptococcus mutans UA159, Streptococcus pneumoniae ATCC 27336, Staphylococcus aureus ATCC 25923, Clostridium difficile UK1, and Micrococcus luteus ATCC 10240 were determined for three mutacin 1140 variants that had the most significant increases in bioactivity in the M. luteus bioassay. This mutagenesis study of the epidermin group of lanthipeptides shows that antimicrobial activity can be significantly improved.
Asunto(s)
Bacteriocinas/genética , Biotecnología/métodos , Mutagénesis Sitio-Dirigida/métodos , Péptidos/genética , Streptococcus mutans/genética , Bacteriocinas/aislamiento & purificación , Bacteriocinas/farmacología , Clostridioides difficile/efectos de los fármacos , Genes Transgénicos Suicidas/genética , Vectores Genéticos/genética , Micrococcus luteus/efectos de los fármacos , Estructura Molecular , Péptidos/aislamiento & purificación , Péptidos/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Staphylococcus aureus/efectos de los fármacos , Streptococcus/efectos de los fármacos , Streptococcus mutans/metabolismoRESUMEN
Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity produced by the Gram-negative bacterium Burkholderia contaminans. The biosynthetic gene cluster was confirmed to contain two cyclase thioesterases. NMR analysis revealed that the presence of both thioesterases is used to increase the conformational repertoire of the cyclic peptide. The loss of the OcfN cyclic thioesterase by mutagenesis results in a reduction of conformational variants and an appreciable decrease in bioactivity against Candida species. Presumably, the presence of both asparagine and ß-hydroxyasparagine variants coordinates the enzymatic function of both of the cyclase thioesterases. OcfN has presumably evolved to be part of the biosynthetic gene cluster due to its ability to produce structural variants that enhance antifungal activity against some fungi. The enhancement of the antifungal activity from the incorporation of an additional cyclase thioesterase into the biosynthetic gene cluster of occidiofungin supports the need to explore new conformational variants of other therapeutic or potentially therapeutic cyclic peptides.
Asunto(s)
Antifúngicos/aislamiento & purificación , Burkholderia/química , Burkholderia/genética , Péptidos Cíclicos/aislamiento & purificación , Tioléster Hidrolasas/metabolismo , Antifúngicos/química , Antifúngicos/farmacología , Candida/efectos de los fármacos , Glicopéptidos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Familia de Multigenes , Resonancia Magnética Nuclear Biomolecular , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Tioléster Hidrolasas/genéticaRESUMEN
Occidiofungin is a nonribosomally synthesized cyclic peptide having a base mass of 1200 Da. It is naturally produced by the soil bacterium Burkholderia contaminans MS14 and possesses potent broad-spectrum antifungal properties. The mechanism of action of occidiofungin is unknown. Viability, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), reactive oxygen species (ROS) detection, membrane and cell wall stability, and membrane mimetic assays were used to characterize the effect of occidiofungin on yeast cells. Confocal and electron microscopy experiments were used to visualize morphological changes within treated cells. TUNEL and ROS detection assays revealed an increase in fluorescence with increasing concentrations of the antifungal. Yeast cells appeared to shrink in size and showed the presence of 'dancing bodies' at low drug concentrations (1 µg/mL). A screen carried out on Saccharomyces cerevisiae gene deletion mutants in the apoptotic and autophagy pathways identified the apoptotic gene for YCA1, as having an important role in occidiofungin response as cells deleted for this gene exhibit a 2-fold increase in resistance. Results from our experiments demonstrate that the mechanism of action for occidiofungin in yeast is different from that of the common classes of antifungals used in the clinic, such as azoles, polyenes, and echinocandins. Our study also indicates that occidiofungin causes cell death in yeast through an apoptotic mechanism of action.
Asunto(s)
Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Burkholderia/química , Glicopéptidos/aislamiento & purificación , Glicopéptidos/farmacología , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Antifúngicos/química , Apoptosis/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , ADN Nucleotidilexotransferasa/metabolismo , Glicopéptidos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Péptidos Cíclicos/química , Especies Reactivas de Oxígeno/análisis , Saccharomyces cerevisiae/genéticaRESUMEN
Fungal infections are caused by opportunistic pathogens that can be life threatening or debilitating. Candida spp. are becoming increasingly resistant to current clinically approved antifungal therapeutics. Candida infections afflict not only immunosuppressed but also immunocompetent individuals. Recurrent vulvovaginal candidiasis (RVVC) is a disease that afflicts 5-9% of women. Occidiofungin is a novel cyclic peptide that has a broad spectrum of antifungal activity with a novel fungicidal mechanism of action. A gel formulation containing occidiofungin (OCF001) is being developed for use to treat vulvovaginal candidiasis. The formulated gel for intravaginal application used hydroxyethyl cellulose as the primary gelling agent and hydroxypropyl ß-cyclodextrin as a solubilizing agent for occidiofungin. Franz cells and LC-MS/MS were used to determine the rate of drug substance diffusion in the gel formulation. The formulation was tested in an ex vivo mouse skin efficacy study, and the safety was tested following repeat intravaginal administration in rabbits. In this study, the gel formulation was shown to reduce the drug substance rate of diffusion across a skin memetic membrane. The study showed that the formulation extends exposure time to inhibitory concentrations of occidiofungin over a 24-h period and supports a single daily application for the treatment of RVVC.
RESUMEN
Occidiofungin is a cyclic glyco-lipopeptide produced by Burkholderia contaminans. MICs against Candida species were between 0.5 and 2.0 µg/ml. Occidiofungin retains its in vitro potency in the presence of 5% and 50% human serum with a minimal lethal concentration (MLC) of 2 and 4 µg/ml, respectively. Time-kill and postantifungal effect (PAFE) experiments of occidiofungin against Candida albicans were performed. The results demonstrate that occidiofungin is fungicidal. Occidiofungin was also found to be a very stable molecule. It is resistant to extreme temperatures and pH and maintains its activity following exposure to gastric proteases.
Asunto(s)
Antifúngicos/química , Antifúngicos/farmacología , Candida/clasificación , Candida/efectos de los fármacos , Lipopéptidos/química , Lipopéptidos/farmacología , Burkholderia/metabolismo , Candida albicans/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad Microbiana/normas , Péptido Hidrolasas/metabolismo , TemperaturaRESUMEN
Occidiofungin, a glycolipopeptide obtained from the liquid culture of Burkholderia contaminans MS14, has been identified as a novel fungicide. The present study was designed to initially assess the in vitro toxicity in a rat hepatoma (H4IIE) cell line and acute toxicological effects of occidiofungin using a mouse model. In vitro toxicity was observed in all variables at 5 µmol/L. B6C3F1 mice were given single and repeat doses of occidiofungin up to 20 mg/kg. Key effects were a reduction in body and organ weights. However, no significant decrease in body weight was noted at a dose of 1 mg/kg, which is comparable to the dose level of other cyclic glycopeptide antifungal agents currently approved for human use. Microscopic examination of treated mice did not identify any signs of organ-specific toxicity at the dose levels tested.
Asunto(s)
Antifúngicos/farmacología , Toxinas Bacterianas/farmacología , Glicopéptidos/farmacología , Péptidos Cíclicos/farmacología , Animales , Peso Corporal/efectos de los fármacos , Burkholderia/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos/efectos de los fármacos , Ratas , Pruebas de Toxicidad AgudaRESUMEN
The identification and development of natural products into novel antimicrobial agents is crucial to combat the rise of multidrug-resistant microorganisms. Clinical fungal isolates have been identified, which have shown resistance to all current clinical antifungals, highlighting a significant need to develop a novel antifungal agent. One of the natural products produced by the bacterium Burkholderia contaminans MS14 is the glycolipopeptide occidiofungin. Occidiofungin has demonstrated in vitro activity against a multitude of fungal species, including multidrug-resistant Candida auris strains, and in vivo effectiveness in treating vulvovaginal candidiasis. Characterization of occidiofungin revealed the mechanism of action as binding to actin to disrupt higher-order actin-mediated functions leading to the induction of apoptosis in fungal cells. Occidiofungin is the first small molecule capable of disrupting higher-order actin functions and is a first-in-class compound that is able to circumvent current antifungal resistant mechanisms by fungal species. Anticancer properties and antiparasitic activities, against Cryptosporidium parvum, have also been demonstrated in vitro. The novel mechanism of action and wide spectrum of activity highlights the potential of occidiofungin to be developed for clinical use.