Rationally Designed Minimal Bioactive Domains of AS-48 Bacteriocin Homologs Possess Potent Antileishmanial Properties.

Leishmaniasis, a category I neglected tropical disease, is a group of diseases caused by the protozoan parasite Leishmania species with a wide range of clinical manifestations. Current treatment options can be highly toxic and expensive, with drug relapse and the emergence of resistance. Bacteriocins, antimicrobial peptides ribosomally produced by bacteria, are a relatively new avenue for potential antiprotozoal drugs. Particular interest has been focused on enterocin AS-48, with previously proven efficacy against protozoan species, including Leishmania spp. Sequential characterization of enterocin AS-48 has illustrated that antibacterial bioactivity is preserved in linearized, truncated forms; however, minimal domains of AS-48 bacteriocins have not yet been explored against protozoans. Using rational design techniques to improve membrane penetration activity, we designed peptide libraries using the minimal bioactive domain of AS-48 homologs. Stepwise changes to the charge (z), hydrophobicity (H), and hydrophobic dipole moment (µH) were achieved through lysine and tryptophan substitutions and the inversion of residues within the helical wheel, respectively. A total of 480 synthetic peptide variants were assessed for antileishmanial activity against Leishmania donovani. One hundred seventy-two peptide variants exhibited 50% inhibitory concentration (IC50) values below 20 µM against axenic amastigotes, with 60 peptide variants in the nanomolar range. Nine peptide variants exhibited potent activity against intracellular amastigotes with observed IC50 values of <4 µM and limited in vitro host cell toxicity, making them worthy of further drug development. Our work demonstrates that minimal bioactive domains of naturally existing bacteriocins can be synthetically engineered to increase membrane penetration against Leishmania spp. with minimal host cytotoxicity, holding the promise of novel, potent antileishmanial therapies. IMPORTANCE Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. There are three primary clinical forms, cutaneous, mucocutaneous, and visceral, with visceral leishmaniasis being fatal if left untreated. Current drug treatments are less than ideal, especially in resource-limited areas, due to the difficult administration and treatment regimens as well as the high cost and the emergence of drug resistance. Identifying potent antileishmanial agents is of the utmost importance. We utilized rational design techniques to synthesize enterocin AS-48 and AS-48-like bacteriocin-based peptides and screened these peptides against L. donovani using a fluorescence-based phenotypic assay. Our results suggest that bacteriocins, specifically these rationally designed AS-48-like peptides, are promising leads for further development as antileishmanial drugs.

A Seed-Endophytic Bacillus safensis Strain With Antimicrobial Activity Has Genes for Novel Bacteriocin-Like Antimicrobial Peptides.

Bacteriocins are a highly diverse group of antimicrobial peptides that have been identified in a wide range of commensal and probiotic organisms, especially those resident in host microbiomes. Rising antibiotic resistance have fueled renewed research into new drug scaffolds such as antimicrobial peptides for use in therapeutics. In this investigation, we examined mung bean seeds for endophytes possessing activity against human and plant pathogens. We isolated a novel strain of Bacillus safensis, from the contents of surface-sterilized mung bean seed, which we termed B. safensis C3. Genome sequencing of C3 identified three distinct biosynthetic systems that produce bacteriocin-based peptides. C3 exhibited antibacterial activity against Escherichia coli, Xanthomonas axonopodis, and Pseudomonas syringae. Robust antimicrobial activity of B. safensis C3 was observed when C3 was co-cultured with Bacillus subtilis. Using the cell-free supernatant of C3 and cation exchange chromatography, we enriched a product that retained antimicrobial activity against B. subtilis. The peptide was found to be approximately 3.3 kDa in size by mass spectrometry, and resistant to proteolysis by Carboxypeptidase Y and Endoproteinase GluC, suggesting that it is a modified variant of an AS-48 like bacteriocin. Our findings open new avenues into further development of novel bacteriocin-based scaffolds for therapeutic development, as well as further investigations into how our discoveries of bacteriocin-producing plant commensal microorganisms may have the potential for an immediate impact on the safety of food supplies.

Safety assessment of Streptococcus salivarius DB-B5 as a probiotic candidate for oral health.

Streptococcus salivarius DB-B5 was previously isolated from the supragingival plaque of a healthy female adult and selected for development as a probiotic candidate for oral health. Probiotics are an important emerging therapeutic method for preventing, treating, and maintaining oral health. Although S. salivarius is a predominant member of the commensal oral microbiota and generally regarded as a safe species, it is recognized that each strain needs to be comprehensively assessed for safety. This study describes the in silico, in vitro, and clinical testing that were conducted to evaluate the safety of S. salivarius DB-B5. Both 16S rRNA and multi-gene phylogenetic reconstruction was used to confirm the taxonomic identity of this strain. Bioinformatic analysis of the genome demonstrated the absence of transmissible antibiotic resistance genes or virulence factors. Phenotypic testing further showed S. salivarius DB-B5 to be susceptible to clinically relevant antibiotics. S. salivarius DB-B5 displayed weak alpha-hemolysis, and does not produce biogenic amines. In a randomized, double-blind, placebo-controlled clinical study, consumption of S. salivarius DB-B5 at 10 billion CFU/day for 4 weeks by healthy adults was safe and well-tolerated (ClinicalTrials.gov registry number NCT04492631). This work has indicated that S. salivarius DB-B5 is a safe probiotic candidate.

Synthetic Peptide Libraries Designed From a Minimal Alpha-Helical Domain of AS-48-Bacteriocin Homologs Exhibit Potent Antibacterial Activity.

The circularized bacteriocin enterocin AS-48 produced by Enterococcus sp. exhibits antibacterial activity through membrane disruption. The membrane-penetrating activity of enterocin AS-48 has been attributed to a specific alpha-helical region on the circular peptide. Truncated, linearized forms containing these domains have been shown to preserve limited bactericidal activity. We utilized the amino acid sequence of the active helical domain of enterocin AS-48 to perform a homology-based search of similar sequences in other bacterial genomes. We identified similar domains in three previously uncharacterized AS-48-like bacteriocin genes in Clostridium sordellii, Paenibacillus larvae, and Bacillus xiamenensis. Enterocin AS-48 and homologs from these bacterial species were used as scaffolds for the design of a minimal peptide library based on the active helical domain of each bacteriocin sequence. 95 synthetic peptide variants of each scaffold peptide, designated Syn-enterocin, Syn-sordellicin, Syn-larvacin, and Syn-xiamensin, were designed and synthesized from each scaffold sequence based on defined biophysical parameters. A total of 384 total peptides were assessed for antibacterial activity against Gram-negative and Gram-positive bacteria. Minimal Inhibitory Concentrations (MICs) as low as 15.6 nM could be observed for the most potent peptide candidate tested, with no significant cytotoxicity to eukaryotic cells. Our work demonstrates for the first time a general workflow of using minimal domains of natural bacteriocin sequences as scaffolds to design and rapidly synthesize a library of bacteriocin-based antimicrobial peptide variants for evaluation.

Algorithmic assessment of missense mutation severity in the Von-Hippel Lindau protein.

Von Hippel-Lindau disease (VHL) is an autosomal dominant rare disease that causes the formation of angiogenic tumors. When functional, pVHL acts as an E3 ubiquitin ligase that negatively regulates hypoxia inducible factor (HIF). Genetic mutations that perturb the structure of pVHL result in dysregulation of HIF, causing a wide array of tumor pathologies including retinal angioma, pheochromocytoma, central nervous system hemangioblastoma, and clear cell renal carcinoma. These VHL-related cancers occur throughout the lifetime of the patient, requiring frequent intervention procedures, such as surgery, to remove the tumors. Although VHL is classified as a rare disease (1 in 39,000 to 1 in 91,000 affected) there is a large heterogeneity in genetic mutations listed for observed pathologies. Understanding how these specific mutations correlate with the myriad of observed pathologies for VHL could provide clinicians insight into the potential severity and onset of disease. Using a select set of 285 ClinVar mutations in VHL, we developed a multiparametric scoring algorithm to evaluate the overall clinical severity of missense mutations in pVHL. The mutations were assessed according to eight weighted parameters as a comprehensive evaluation of protein misfolding and malfunction. Higher mutation scores were strongly associated with pathogenicity. Our approach establishes a novel in silico method by which VHL-specific mutations can be assessed for their severity and effect on the biophysical functions of the VHL protein.

Complete Genome Sequence of Streptococcus salivarius DB-B5, a Novel Probiotic Candidate Isolated from the Supragingival Plaque of a Healthy Female Subject.

Streptococcus salivarius DB-B5 was isolated from the supragingival plaque of a healthy female subject. The complete 2.3-Mb genome consists of one circular chromosome, two circular plasmids (including a megaplasmid), and one linear phage-like episome. The genome possesses two separate loci encoding bacteriocins.

Synthetic Antimicrobial Peptide Tuning Permits Membrane Disruption and Interpeptide Synergy.

The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy.

Novel antimicrobial peptide discovery using machine learning and biophysical selection of minimal bacteriocin domains.

Bacteriocins, the ribosomally produced antimicrobial peptides of bacteria, represent an untapped source of promising antibiotic alternatives. However, bacteriocins display diverse mechanisms of action, a narrow spectrum of activity, and inherent challenges in natural product isolation making in vitro verification of putative bacteriocins difficult. A subset of bacteriocins exert their antimicrobial effects through favorable biophysical interactions with the bacterial membrane mediated by the charge, hydrophobicity, and conformation of the peptide. We have developed a pipeline for bacteriocin-derived compound design and testing that combines sequence-free prediction of bacteriocins using machine learning and a simple biophysical trait filter to generate 20 amino acid peptides that can be synthesized and evaluated for activity. We generated 28,895 total 20-mer candidate peptides and scored them for charge, α-helicity, and hydrophobic moment. Of those, we selected 16 sequences for synthesis and evaluated their antimicrobial, cytotoxicity, and hemolytic activities. Peptides with the overall highest scores for our biophysical parameters exhibited significant antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Our combined method incorporates machine learning and biophysical-based minimal region determination to create an original approach to swiftly discover bacteriocin candidates amenable to rapid synthesis and evaluation for therapeutic use.

Rational design of syn-safencin, a novel linear antimicrobial peptide derived from the circular bacteriocin safencin AS-48.

Bacteriocins hold unprecedented promise as a largely untapped source of antibiotic alternatives in the age of multidrug resistance. Here, we describe the first approach to systematically design variants of a novel AS-48 bacteriocin homologue, which we have termed safencin AS-48, from Bacillus safensis, to gain insights into engineering improved activity of bacteriocins. A library of synthetic peptides in which systematic amino acid substitutions to vary the periodicity and abundance of polar, acidic, aliphatic, and hydrophobic residues were generated for a total of 96 novel peptide variants of a single bacteriocin candidate. Using this method, we identified nine synthetic safencin (syn-safencin) variants with broad and potent antimicrobial activities with minimal inhibitory concentrations (MIC) as low as 250 nM against E. coli, P. aeruginosa, X. axonopodis, and S. pyogenes with minimal cytotoxicity to mammalian cells. It is anticipated that the strategies we have developed will serve as general guides for tuning the specificity of a given natural bacteriocin compound for therapeutic specificity.

Using bacterial genomes and essential genes for the development of new antibiotics.

The shrinking antibiotic development pipeline together with the global increase in antibiotic resistant infections requires that new molecules with antimicrobial activity are developed. Traditional empirical screening approaches of natural and non-natural compounds have identified the majority of antibiotics that are currently available, however this approach has produced relatively few new antibiotics over the last few decades. The vast amount of bacterial genome sequence information that has become available since the sequencing of the first bacterial genome more than 20years ago holds potential for contributing to the discovery of novel antimicrobial compounds. Comparative genomic approaches can identify genes that are highly conserved within and between bacterial species, and thus may represent genes that participate in key bacterial processes. Whole genome mutagenesis studies can also identify genes necessary for bacterial growth and survival under different environmental conditions, making them attractive targets for the development of novel inhibitory compounds. In addition, transcriptomic and proteomic approaches can be used to characterize RNA and protein levels on a cellular scale, providing information on bacterial physiology that can be applied to antibiotic target identification. Finally, bacterial genomes can be mined to identify biosynthetic pathways that produce many intrinsic antimicrobial compounds and peptides. In this review, we provide an overview of past and current efforts aimed at using bacterial genomic data in the discovery and development of novel antibiotics.