Commensal bacteria produce antimicrobial peptides (AMPs) to maintain microbiome homeostasis, yet the traits underlying this resilience and their translation into biotherapeutics remain understudied. Phosphorylated lantibiotics (pLANs) are a recently identified class of ribosomally synthesized and post-translationally modified peptides (RiPPs), with dual antimicrobial and pro-immune activities. In this manuscript, we explore the potential of commensals’ pLANs biosynthesis as a mechanism for pathogen suppression and microbiome homeostasis. Subgingival metagenomics revealed that oral health correlates with Streptococcus salivarius enrichment and an increased prevalence of streptococcal RiPP biosynthetic gene clusters. Guided by these associations, we screened 80 S. salivarius isolates, identifying a small subset producing pLANs with potent activity against Porphyromonas gingivalis, vancomycin-resistant Enterococcus faecium, and multidrug-resistant Streptococcus pneumoniae. A representative lead strain, SALI-10, exhibited robust epithelial adhesion and a sorbitol-driven metabolic adaptation that enhances pLANs expression. In human-derived dysbiotic biofilms, SALI-10 stably engrafted, suppressed periopathogens, reduced antibiotic-resistance genes, and enriched acid-buffering pathways. In a first-in-human feasibility trial, daily oral administration of SALI-10 for one week yielded increased pLANs signals, pathogen depletion, and reduced oral neutrophil counts. Ultimately, pLANs-producing S. salivarius acts as a precision commensal to restore ecological balance, defining a mechanistically grounded and microbiota-mediated strategy to prevent oral and respiratory infections.

This review highlights several basic problems associated with bacterial drug resistance, including the decreasing efficacy of commercially available antimicrobials as well as the related problem of microbiome irregularity and dysbiosis. The article explains that this present situation is addressable through LAB species, such as Streptococcus salivarius and Ligilactobacillus salivarius, which are well established synthesizers of both broad- and narrow-spectrum antimicrobials. The sheer number of antimicrobials produced by LAB species and the breadth of their biological effects, both in terms of their bacteriostatic/bactericidal abilities and their immunomodulation, make them prime candidates for new probiotics and antibiotics. Given the ease with which several of the molecules can be biochemically engineered and the fact that many of these compounds target evolutionarily constrained target sites, it seems apparent that these compounds and their producing organisms ought to be looked at as the next generation of robust dual action symbiotic drugs.

Biofilms are an important colonization mechanism employed by several microbial species to better establish themselves and monopolize the acquisition of resources across different environs. Some bacteria have evolved specialized metabolites that, when secreted, disrupt the formation and stability of biofilms generated by competing heterospecies, providing the producing organism with an ecological advantage. Soil-derived species are probable candidates for the identification of such compounds, given the intense level of competition that occurs within the terrestrial ecosystem. The MS14 strain of Burkholderia contaminans isolated from soil in Mississippi has previously been shown to produce antimicrobial compounds like occidiofungin and ornibactin. In this report, we demonstrate that this strain also produces 4-hydroxy-3-methyl-2-alkenylquinoline (HMAQ-7), an alkaloid-based metabolite structurally similar to others produced by Burkholderia. HMAQ-7 was isolated and purified in sufficient quantities to enable the elucidation of its covalent structure and the evaluation of its biological effects. The compound was found to possess a unique ability to inhibit biofilm biosynthesis in several species, including opportunistic pathogens like Staphylococcus haemolyticus and within saliva-derived multispecies biofilms. HMAQ-7 also demonstrated an ability to modulate additional cellular behaviors in Bacillus subtilis, including motility and sporulation, suggesting that this molecule is important to the interspecies dynamics present across many diverse microenvironments.IMPORTANCEThe present study furthers our understanding of the structural complexity and the biological functions of the 2-alkyl-4(1H)-quinolone metabolites produced by Burkholderia spp. Low micromolar concentrations of HMAQ-7' induced observable bacterial growth morphology differences. The antibiofilm properties of the HMAQ-7' characterized in this study will promote future investigations into possible biological and applied roles. The ability to alter biofilm formation using HMAQ-7' may facilitate Burkholderia spp. colonization in a multitude of environments, that is, aquatic, soil, and possibly during infection. HMAQ may subvert competition by potential competitor species in natural environments of Burkholderia spp. and possibly lung infections of cystic fibrosis patients.

Fungal infections are caused by opportunistic pathogens that can be life threatening and have been growing in prevalence. Many clinically relevant pathogens have resistance to or are developing resistance to the commonly used antifungal treatments. Occidiofungin (OCF) is a unique cyclic lipoglycopeptide with a novel structure that includes noncanonical amino acid in its covalent structure. It exhibits broad spectrum antifungal activity and has activity against drug resistant Candida species. Occidiofungin is a fungicidal compound that has a novel mechanism of action in which it disrupts higher order actin structures. Currently, occidiofungin is being developed for use in treating vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). This study describes the development and application of a bioanalytical method for the quantification of occidiofungin in rabbit plasma. Method development was performed to quantify occidiofungin in rabbit plasma after intravaginal administration of a hydrogel containing occidiofungin. The method was validated with a linear range of 30-15 000 ng/mL in rabbit plasma. Precision, accuracy, calibration curve linearity, and stability of drug in plasma were established in quality controls. Extract stability, matrix effects, and recovery of drug in the extract were also determined. This study supported a repeat dose toxicity study in rabbits to determine occidiofungin pharmacokinetics and toxicokinetics. The pharmacokinetic and toxicokinetic primarily showed plasma concentrations of occidiofungin below the limit of quantification (BLOQ), suggesting that OCF-B does not readily cross the vaginal epithelial membrane.

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.

INTRODUCTION:The emergence of antibiotic resistance among the clinically important bacterial pathogens has increased healthcare costs and reduced patient safety and quality of life. Lantibiotics is a large class of ribosomally synthesized, and posttranslationally modified peptides have been the primary focus of numerous research aimed at discovering compounds for treating bacterial infections. AREAS COVERED:The article explains the most up to date hierarchy of methods followed in the field for high throughput screening of lantibiotics/analogs with improved therapeutic properties. Herein, we explain how the structure and the biosynthesis of lantibiotics can be manipulated for the expansion of the horizon of lantibiotic potency. Furthermore, we discuss the lantibiotic analogs that have demonstrated the efficacy against bacterial pathogens of interest in animal models. EXPERT OPINION:In this current age of rapidly advancing antimicrobial resistance, there is a dire need for the development of therapeutic agents that possess distinct mechanisms of action to existing modes of treatment. Recent advances in the understanding of many of the lantibiotic biosynthesis systems and the discovery of new analogs with superior properties to the native compound may have paved the way for the development of a much-needed novel potent class of antibiotic.

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 β-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.

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 micro-biome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first com-mensal 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 bio-films. 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.

Occidiofungin is a broad-spectrum antifungal compound produced by Burkholderia contaminans MS14. It is a cyclic glycol-lipopeptide with a novel beta-amino acid (NAA2) containing a hydroxylated C18 fatty acid chain with a xylose sugar. This study reports a strategy to produce semisynthetic analogs of occidiofungin to further explore the structure activity relationships of this class of compounds. Oxidative cleavage of the diol present on carbons five C(5) and six C(6) removes the xylose and twelve carbons of the fatty acid chain. The resulting cyclic peptide product, occidiofungin aldehyde, is devoid of antifungal activity. However, the free aldehyde group on this product can be subjected to reductive amination reactions to provide interesting semisynthetic analogs. This chemistry allows the quick generation of analogs to study the structure activity relationships of this class of compounds. Despite restoring the length of the aliphatic side chain by reductive amination addition with undecylamine or dodecylamine to the free aldehyde group, the obtained analogs did not demonstrate any antifungal activity. The antifungal activity was partially restored by the addition of a DL-dihydrosphingosine. The dodecylamine analog was demonstrated to still bind to the cellular target actin, suggesting that the diol on the side chain of native occidiofungin is important for entry into the cell enabling access to cellular target F-actin. These results show that the alkyl side chain on NAA2 along with the diol present on this side chain is important for occidiofungin's antifungal activity.

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.

Mutacin 1140 (Mu1140) is a potent antibiotic against Gram-positive bacteria, such as Staphylococcus aureus. The antibiotic is produced by the oral bacterium Streptococcus mutans and is a member of the epidermin family of type AI lantibiotics. The antibiotic exerts its inhibitory activity by binding to the cell wall precursor lipid II, blocking cell wall synthesis, and by disrupting bacterial membranes. In previous studies, the novel K2A and R13A analogs of Mu1140 have been identified to have superior pharmacokinetic properties compared to native Mu1140. In this study, the use of a combined formulation of the Mu1140 K2A and R13A analogs was shown to be more effective at treating MRSA bacteremia than the native Mu1140 or vancomycin. The analogs were also shown to be effective in treating an MRSA skin infection. The use of K2A and R13A analogs may provide a future alternative for treating serious Gram-positive bacterial infections. In a previous study, the Mu1140 analogs were shown to have significantly longer drug clearance times, leading to higher plasma concentrations over time. These properties warranted further testing to determine whether the analogs are effective for the treatment of systemic MRSA and acute skin infections. In this study, Mu1140 analogs were shown to be more effective than currently available treatments for systemic and skin MRSA infections. Further, the study clearly shows that the new analogs are superior to native Mu1140 for the treatment of a systemic MRSA infection. These findings support continued drug product development efforts using the K2A and R13A Mu1140 analogs, and that these analogs may ameliorate the outcome of serious bacterial infections.

Introduction: The increasing threat of antibiotic-resistant pathogens makes it imperative that new antibiotics to combat them are discovered. Burkholderia is a genus of Gram-negative, non-sporulating bacteria. While ubiquitous and capable of growing within plants and groundwater, they are primarily soil-dwelling organisms. These include the more virulent forms of Burkholderia such as Burkholderia mallei, Burkholderia pseudomallei, and the Burkholderia cepacia complex (Bcc).Areas covered: This review provides a synopsis of current research on the natural products isolated from the genus Burkholderia. The authors also cover the research on the drug discovery efforts that have been performed on the natural products derived from Burkholderia.Expert opinion: Though Burkholderia has a small number of pathogenic species, the majority of the genus is avirulent and almost all members of the genus are capable of producing useful antimicrobial products that could potentially lead to the development of novel therapeutics against infectious diseases. The need for discovery of new antibiotics is urgent due to the ever-increasing prevalence of antibiotic-resistant pathogens, coupled with the decline in the discovery of new antibiotics.

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.

Novel antiparasitic activity was observed for the antifungal occidiofungin. It efficaciously and irreversibly inhibited the zoonotic enteric parasite Cryptosporidium parvum in vitro with limited cytotoxicity (50% effective concentration [EC 50 ] = 120 nM versus 50% cytotoxic concentration [TC 50 ] = 988 nM), and its application disrupted the parasite morphology. This study expands the spectrum of activity of a glycolipopeptide named occidiofungin.

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.

Lantibiotic salivaricins are polycyclic peptides containing lanthionine and/or β-methyllanthionine residues produced by certain strains of Streptococcus salivarius, which almost exclusively reside in the human oral cavity. The importance of these molecules stems from their antimicrobial activity towards relevant oral pathogens which has so far been applied through the development of salivaricin-producing probiotic strains. However, salivaricins may also prove to be of great value in the development of new and novel antibacterial therapies in this era of emerging antibiotic resistance. In this review, we describe the biosynthesis, antimicrobial activity, structure, and mode of action of the lantibiotic salivaricins characterized to date. Moreover, we also provide an expert opinion and suggestions for future development of this important field of microbiology.

Streptococcus salivarius is a prevalent commensal species of human oral mucosal surfaces. S. salivarius strain HS0302 produces the type AII lantibiotic salivaricin A2. Here, we report its draft genome sequence, revealing its potential to produce a variety of bacteriocins.

Current antifungal treatment options are plagued with rapidly increasing occurrence of resistance, high degree of toxicity and a limited spectrum of activity. The need to develop a novel antifungal with a unique target, wider spectrum of activity, and reduced toxicity to the host, is urgent. We have identified and characterized one such compound named occidiofungin that is produced by the soil bacterium Burkholderia contaminans MS14. This study identifies the primary cellular target of the antifungal, which was determined to be actin. Actin binding metabolites are generally characterized by their ability to inhibit polymerization or depolymerization of actin filaments, which presumably accounts for their severe toxicity. Occidiofungin, instead, has a subtler effect on actin dynamics that triggers apoptotic cell death. We were able to demonstrate the effectiveness of the antifungal in treating a vulvovaginal yeast infection in a murine model. This discovery puts occidiofungin in a unique class of actin-binding antifungal compounds with minimal reported toxicity to the host. The results of this study are important for the development of a novel class of antifungals that could fill the existing gap in treatment options for fungal infections.Author summary Widespread resistance to antifungal compounds currently in use has been alarming. Identification and development of a new class of antifungals with a novel cellular target is desperately needed. This study describes the assays carried out to determine the molecular target and evaluate efficacy of one such novel antifungal compound called occidiofungin. Occidiofungin modified with a functional alkyne group enabled affinity purification assays and localization studies in yeast. These studies led to the identification of the actin binding property of occidiofungin. Actin-binding by secondary metabolites often exhibit severe host toxicity, but this does not appear to be the case for occidiofungin. We have previously been able to administer occidiofungin to mice at concentrations in the range of 5 mg/kg without any serious complications. We were able to demonstrate the effectiveness of the antifungal in treating a vaginal fungal infection in a murine model. The results outlined in this manuscript establish that occidiofungin is an efficacious compound with a novel molecular target, putting it in a completely new class of antifungals.

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.

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.