The flavoenzyme d-amino acid oxidase (DAAO) is deputed to the degradation of d-enantiomers of amino acids. DAAO plays various relevant physiological roles in different organisms and tissues. Thus, it has been recently suggested that the goblet cells of the mucosal epithelia secrete into the lumen of intestine, a processed and active form of DAAO that uses the intestinal d-amino acids to generate hydrogen peroxide (H₂O₂), an immune messenger that helps fighting gut pathogens, and by doing so controls the homeostasis of gut microbiota. Here, we show that the DAAO form lacking the 1–16 amino acid residues (the putative secretion signal) is unstable and inactive, and that DAAO is present in the epithelial layer and the mucosa of mouse gut, where it is largely proteolyzed. In silico predicted DAAO-derived antimicrobial peptides show activity against various Gram-positive and Gram-negative bacteria but not on Lactobacilli species, which represent the commensal microbiota. Peptidomic analysis reveals the presence of such peptides in the mucosal fraction. Collectively, we identify a novel mechanism for gut microbiota selection implying DAAO-derived antimicrobial peptides which are generated by intestinal proteases and that are secreted in the gut lumen. In conclusion, we herein report an additional, ancillary role for mammalian DAAO, unrelated to its enzymatic activity.

With the increasing need of effective antibiotics against multi-drug resistant pathogens, lantibiotics are an attractive option of a new class of molecules. They are ribosomally synthetized and posttranslationally modified peptides possessing potent antimicrobial activity against aerobic and anaerobic Gram-positive pathogens, including those increasingly resistant toβ-lactams and glycopeptides. Some of them (actagardine, mersacidin, planosporicin, and microbisporicin) inhibit cell wall biosynthesis in pathogens and their effect is not antagonized by vancomycin. Hereby, we apply an efficient strategy for lantibiotic screening to 240 members of a newly described genus of filamentous actinomycetes, namedActinoallomurus, that is considered a yet-poorly-exploited promising source for novel bioactive metabolites. By combining antimicrobial differential assay againstStaphylococcus aureusand its L-form (also in the presence of aβ-lactamase cocktail or Ac-Lys-D-alanyl-D-alanine tripeptide), with LC-UV-MS dereplication coupled with bioautography, a novel producer of the potent microbisporicin complex was rapidly identified. Under the commercial name of NAI-107, it is currently in late preclinical phase for the treatment of multi-drug resistant Gram-positive pathogens. To our knowledge, this is the first report on a lantibiotic produced by anActinoallomurussp. and on a microbisporicin producer not belonging to theMicrobisporagenus.

Background: Orthopedic implant-associated infections are difficult to eradicate because of biofilm formation and impaired healing. FK-16 and GF-17 are short synthetic antimicrobial fragments of the human cathelicidin LL-37; FK-16 consists of 16 amino acids and differs from the 17-amino-acid GF-17 by the absence of its N-terminal glycine. This study aimed to assess their acute tolerability following single-dose intravenous administration in mice and to evaluate the local antibacterial efficacy of FK-16 delivered via peptide-enriched silk fibroin (PSF) scaffolds in a rat model of methicillin-resistant Staphylococcus epidermidis (MRSE)-associated femoral nonunion. Methods: Thirty-six BALB/c mice (18 males and 18 females) received vehicle, FK-16, or GF-17 (0.1 or 1 mg/kg for each peptide) and underwent clinical and functional assessments, hemolysis testing, biochemical analyses, organ-weight measurements, and histopathological examination. Thirty-two male Wistar rats were assigned to a noninfected control group or infected groups treated with unloaded PSF, vancomycin-loaded PSF, or FK-16-loaded PSF. On day 56, antibacterial activity and bone healing were evaluated using microbiological culture, micro-computed tomography, and histopathology. Results: No mortality, persistent neurobehavioral abnormalities, or treatment-related organ lesions occurred in mice. Transient hind-limb paralysis occurred in one mouse treated with FK-16 and one treated with GF-17 at 1 mg/kg. Biochemical and hemolysis-related changes were isolated and did not follow a consistent dose–response pattern; no corresponding treatment-related organ lesions were observed. In rats, viable MRSE was recovered from 3/4 untreated specimens and none from those treated with vancomycin or FK-16. FK-16 was associated with partial normalization of the circulating ratio of osteoprotegerin to receptor activator of nuclear factor-κB ligand without significantly reducing early systemic inflammation. Bone mineral density, tissue mineral density, and bone volume fraction increased numerically, but no quantitative micro-computed tomography healing parameter improved significantly; the radiographic osteomyelitis score decreased significantly. Untreated PSF showed absent bridging, abundant fibrovascular tissue, chronic inflammation, rare Gram-positive cocci, and higher histological scores than noninfected controls; both treated groups showed minimal inflammation but incomplete bridging. Conclusions: FK-16 and GF-17 showed generally acceptable acute tolerability despite transient high-dose neurological effects. Locally delivered FK-16 demonstrated antibacterial activity, reduced osteomyelitis severity, and was associated with minimal histological evidence of inflammation, but did not significantly improve quantitative bone healing or restore complete bridging by day 56.