Antimicrobial peptides are host-encoded immune effectors that combat pathogens and shape the microbiome in plants and animals. However, little is known about how the host antimicrobial peptide repertoire is adapted to its microbiome. Here, we characterized the function and evolution of the Diptericin antimicrobial peptide family of Diptera. Using mutations affecting the two Diptericins (Dpt) of Drosophila melanogaster, we reveal the specific role of DptA for the pathogen Providencia rettgeri and DptB for the gut mutualist Acetobacter. The presence of DptA- or DptB-like genes across Diptera correlates with the presence of Providencia and Acetobacter in their environment. Moreover, DptA- and DptB-like sequences predict host resistance against infection by these bacteria across the genus Drosophila. Our study explains the evolutionary logic behind the bursts of rapid evolution of an antimicrobial peptide family and reveals how the host immune repertoire adapts to changing microbial environments.

Apiadecin 1b (Api1b), produced by the honeybee Apis mellifera, is a Proline-rich Anti-microbial Peptide (PrAMP), that enters Gram-negative bacteria via inner membrane transporters and arrests ribosomes at translation termination, causing cell death. Homologs of Api1b are found in genomes of several members of the bee and wasp families, often, with multiple isoforms found within the same insect. These homologs differ both in their lengths and amino acid composition, especially in the N-terminal part of the peptide. Apidaecins are synthesized by the insect as prepropeptides, which are proteolytically cleaved to yield biologically active peptides, making it challenging to identify the sequence of the active PrAMP from the gene sequence. Our bioinformatics analyses identified several potential homologs of Api1b from genomes of multiple species of bees and wasps. Peptide sequences that differed significantly from Api1b were chemically synthesized and tested against several Gram negative bacteria. Strikingly, a few of the selected peptides showed increased antimicrobial activity compared to Api1b as well as the more potent synthetic variant, Api137. Our in vitro experiments demonstrated that, despite differences in sequence, these active peptides maintain the ability to arrest ribosomes at stop codons, as we described for Api1b and Api137. Interestingly, some of the identified peptides also arrest the terminating ribosomes during in vitro translation but were unable to kill the tested bacteria, suggesting they are unable to penetrate these cells. Future studies may help uncover the sequence requirements for transporter recognition and elucidate whether these peptides have evolved to target specific bacterial species.

Introduction: Bacteriocins are ribosomal-synthesized peptides with antimicrobial activity, produced by different groups of bacteria, including lactic acid bacteria (LAB). Most of the produced by LAB bacteriocins can be described with rather broad spectra of inhibition and they offer suggested applications in food preservation and pharmaceutical sector. An iconic example for bacteriocins is nisin, produced by different strains of Lactococcus spp., however, to our best of knowledge this is the first report on strains of Lactococcus lactis isolated from kimchi, producer of nisin.

Aim: The goal of this study was to explore bacteriocinogenic properties for LAB isolated from kimchi.

Methods: Different LAB were isolated from kimchi, obtained from the region of Pohang, Korea and identified based on physiological, biochemical, and molecular methods. The promising isolate (Lactococcus lactis subsp. lactis 431) was evaluated for production of bacteriocin, including stability in presence of enzymes, chemicals, pH and temperatures. Adherence properties for the expressed bacteriocins by Lactococcus lactis subsp. lactis 431 were evaluated at presence of selected chemicals, pH and temperatures. The presence of bacteriocin genes in strain 431 was investigated and analyzed. Growth of Lactococcus lactis subsp. lactis 431 in different pH values and presence of ox-bile was explored. Safety properties for Lactococcus lactis subsp. lactis 431 were evaluated.

Results: Selected isolate 431 was identified as Lactococcus lactis subsp. lactis based on biochemical, physiological, and biomolecular 16S rRNA gene sequencing. The bacterial effect of bacteriocin produced by strain 431 on Listeria spp. and Staphylococcus spp., has been shown for actively growing and stationary cells. Similar growth and bacteriocin production were observed when Lactococcus lactis subsp. lactis 431 was cultured in MRS at 30^oC or 37^oC. The presence of nisin operon with some point mutations on the genomic DNA was recorded based on the performed PCR reactions targeting different genes associated with nisin expression. Good growth for strain 431 was recorded in MRS broth with pH 5.0 to 9.0 and in absence of oxbile or concentration below 0.8%. Lactococcus lactis subsp. lactis 431 was shown that can be considered as safe for human and animals application based on negative reaction for production of biogenic amines, lipolytic, proteolytic and γ-hemolytic activity and susceptibility to different antibiotics and growth not affected by different commercial drugs.

Significance: Lactococcus lactis subsp. lactis 431 shows to have potential beneficial properties: production of nisin, be safe and suitable to GIT environment.

Acknowledgment: Handong Global University, FAPESP (Grant 2013/07914-8) and Centre for Research and Development in Agrifood Systems and Sustainability, funded by FCT (UIDB/05937/2020 and UIDP/05937/2020), Fundação para a Ciência e a Tecnologia, Portugal for providing financial support.

Antarctic fishes, living in an extreme environment and normally exposed to pathogens, are a promising source for antimicrobial peptides (AMPs), fundamental for the innate immune responses of these vertebrates. These natural peptides are emerging as next-generation therapeutics due to their action against bacteria, viruses, yeasts and protozoa. As they show a broad spectrum of activity against multidrug resistant (MDR) bacteria, strong efforts are in progress to bring AMPs into clinical use, in order to counteract the increasing resistance to classical antibiotics. Beyond intrinsic/acquired resistance, MDR species also uses virulence factors (like biofilm formation and protease secretion) to infect hosts. Hence, there is a need for innovative approaches targeting these virulence factors especially in the case of bacteria involved in chronic pathogenesis.

In our research, we used a mutant peptide, named KHS-Cnd, that was obtained from the scaffold of the chionodracine (Cnd), a natural peptide identified in the icefish Chionodraco hamatus. Among virulence factors, we investigated the effect of KHS-Cnd on protease production of two model Gram-negative/positive bacteria, Escherichia coli and Bacillus cereus. The peptide was tested both at minimum inhibitory concentrations (MICs) and 2x MICs previously determined for the two bacterial strains. A significant reduction in protease activity was observed for both bacteria at the tested concentrations within 1-3 h from the treatment. Moreover, we determined that KHS-Cnd has low cytotoxicity on human primary cells and no hemolytic activity on mammalian erythrocytes at concentrations displaying anti-virulence activity, thus confirming the interesting potential of the peptide as a new drug.

Antimicrobial peptides are important candidates for developing new antibiotics against drug-resistant pathogens. There is a high interest in engineering human cathelicidin LL-37 to overcome its shortcomings such as high cost and lack of protease stability. Our previous studies have identified major antimicrobial peptides (SK-24, GI-20, and GF-17) as well as the smallest antibacterial peptide (KR-12) within LL-37. It is well-known that rich media (100%) could mask the activity of LL-37 against methicillin-resistant Staphylococcus aureus (MRSA). This poster found that dilution of Muller Hinton medium (MHB) led to a sensitive medium to observe LL-37 activity against MRSA. This diluted medium also allowed us to screen a library of ultrashort overlapping LL-37 peptides, leading to the identification of KR-8, which is four-residue shorter than KR-12. Based on the KR-8 template, we have engineered LL-37mini, which was potent against MRSA, Escherichia coli, and Pseudomonas aeruginosa, but was not toxic to mammalian cells. It also disrupted preformed biofilms in vitro and killed MRSA in murine wound biofilms. Consistent with membrane targeting mechanism of the peptide, S. aureus USA300 was unable to develop resistance to LL-37minin in a multiple passage experiment. The peptide is stable when made in D-amino acids. Because LL-37mini possesses numerous desired properties, it is a promising lead for developing new peptide antibiotic.

The skin wound healing process is complicated and vulnerable to factors such as accidental injury, microbial infection, skin diseases and metabolic dysfunction. Amphibian skin peptides have great potential in the treatment of acute and chronic skin wounds. We firstly cloned a novel group of bioactive peptides from frog Fejervarya moodiei and F. multistriata, showing no amino acid sequence similarities with known bioactive peptides, which is named as Fejervarin (Fej). Mature Fej-1a, Fej-1b, Fej-1c and Fej-1d are composed of 8 amino acid residues, including 2 or 3 negative amino acid residues, with -1 or -2 net negative charges under physiological conditions. In vitro, the Fej peptides significantly promoted proliferation and migration of RAW264.7, HaCaT and HSF cells assessed by CCK-8, EdU proliferation and scratch assays. In vivo, they exhibited the efficacy on promoting acute and chronic skin wound healing on mouse models of full-thickness skin injury and second-degree deep scald, after the mice were treated for 12 and 21 days, respectively. MASSON and H&E stains showed that in the peptide treated group, fibroblasts were formed; inflammation was relieved and the wounds were completely covered by the newly generated epidermis. IHC, WB and RT-PCR assays revealed that the Fej peptides up-regulated EGF and TGF-β expressions to promote the proliferation and migration of epithelial keratinocytes and skin fibroblasts. Collectively, the Fej peptides are promising multifunctional wound healing peptides to reshape the damaged tissue environment, providing effective alternative candidates for the repair and regeneration of acute and chronic cutaneous wounds.

Klebsiella pneumoniae is a dangerous pathogen that has gained much notoriety due to its extreme rate of resistance development. K. pneumoniae acute and chronic wound treatment is complicated by the presence of biofilm formation, which contributes to drug resistance, persistence, tolerance, and slow wound healing. In addition to wound infections, biofilm formation abilities of K. pneumoniae lead to persistent, drug resistant catheter infections. Antimicrobial peptides have shown promise in recent years as a topical treatment and as a device coating to eradicate drug resistant K. pneumoniae. Previous work from our lab revealed a mechanism used by antimicrobial peptides to disrupt the capsule of K. pneumoniae. Recently, we have found that a polyproline peptide bac7 (1-35) can disrupt pre-formed biofilms of hypervirulent K. pneumoniae. Using confocal microscopy of K. pneumoniae NTUH K2044 biofilms with bacteria constitutively expressing GFP and matrix polysaccharides labeled with Texas red tagged Concanavalin A, we show bac7 (1-35) decreases the biofilm matrix material and releases the cells from their protective encasing. Live/dead staining images then revealed the dispersed cells were eradicated by the antimicrobial properties of the peptide. Interestingly, bac7 (1-35) treatment was found to decrease the mucoid phenotype of this species that is provided by capsular polysaccharides. Our results show that in addition to capsular polysaccharides, biofilm matrix polysaccharides are targeted by peptides to potentially sensitize hypervirulent K. pneumoniae to the host immune system and antibiotic therapies.