The human intestinal tract is colonized with microorganisms, which present a diverse array of immunological challenges. A number of antimicrobial mechanisms have evolved to cope with these challenges. A key defense mechanism is the expression of inducible antimicrobial peptides (AMPs), such as beta-defensins, which rapidly inactivate microorganisms. We currently have a limited knowledge of mechanisms regulating the inducible expression of AMP genes, especially factors from the host required in these regulatory mechanisms. To identify the host factors required for expression of the beta-defensin-2 gene (HBD2) in intestinal epithelial cells upon a bacterial challenge, we performed a RNAi screen using a siRNA library spanning the whole human genome. The screening was performed in duplicate to select the strongest 79 and 110 hit genes whose silencing promoted or inhibited HBD2 expression, respectively. A set of 57 hits selected among the two groups of genes was subjected to a counter-screening and a subset was subsequently validated for its impact onto HBD2 expression. Among the 57 confirmed hits, we brought out the TLR5-MYD88 signaling pathway, but above all new signaling proteins, epigenetic regulators and transcription factors so far unrevealed in the HBD2 regulatory circuits, like the GATA6 transcription factor involved in inflammatory bowel diseases. This study represents a significant step toward unveiling the key molecular requirements to promote AMP expression in human intestinal epithelial cells, and revealing new potential targets for the development of an innovative therapeutic strategy aiming at stimulating the host AMP expression, at the era of antimicrobial resistance.

Beta-defensins, identified from fishes, constitute a crucial category of antimicrobial peptides important in combating bacterial fish pathogens. The present investigation centers on the molecular and functional characterization of CsDef, a 63-amino acid beta-defensin antimicrobial peptide derived from snakehead murrel (Channa striata). The physicochemical attributes of CsDef align with the distinctive characteristics observed in AMPs. CsDef was recombinantly produced, and the recombinant peptide, rCsDef, exhibited notable antibacterial efficacy against bacterial fish pathogens with an MIC of 16 μM for V. proteolyticus. A. hydrophila exhibited 91% inhibition, E. tarda 92%, and V. harveyi 53% at 32 μM of rCsDef. The rCsDef exhibited a multifaceted mechanism of action against bacteria, i.e., through membrane depolarization, membrane permeabilization, and generation of ROS. The rCsDef was non-hemolytic to hRBCs and non-cytotoxic to normal mammalian cell line CHO-K1. However, it exhibited anticancer properties in MCF-7. rCsDef demonstrated notable stability with respect to pH, temperature, salt, metal ions, and proteases. These findings suggest it is a potential candidate molecule for prospective applications in aquaculture.

A simple method to generate antibacterial peptides by alkaline hydrolysis of hen egg whites is reported. The method reproducibly generates short peptides with molecular weight of less than 14.4 kDa that exhibit low to no cytotoxicity on RAW 264.7 macrophage cells, but do inhibit the bacterial growth of Cutibacterium acnes (C. acnes), Staphylococcus aureus (S. aureus) and antibiotic-resistant S. aureus (MRSA), while also reducing nitric oxide production from heat-killed C. acnes-treated RAW 264.7 cells. Peptidomics revealed at least thirty peptides within the complex mixture, of which eight were evaluated individually. Three peptides (PK8, EE9 and RP8) were potent anti-inflammation and antibacterial agents, but notably the complex egg white hydrolysate (EWH) was more effective than the individual peptides. Electron microscopy suggests the antibacterial mechanism of both the hydrolysate and the selected peptides is through disruption of the cell membrane of C. acnes. These findings suggest that EWH and EWH-derived peptides are promising candidates for infection and inflammation treatment, particularly in managing acne and combating antibiotic-resistant bacteria like MRSA.

UNASSIGNED: The emergence of multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) and the decline of effective antibiotics lead to the urgent need for new antibacterial agents. The aim of this study is to investigate the therapeutic effect of antimicrobial peptides against gentamicin-resistant (RT) K. pneumoniae and to screen effective antimicrobial peptides.
UNASSIGNED: In this study, the RT strains were induced by gradient gentamicin, and the RT strains were selected by detecting the expression levels of efflux pump genes, porin genes, and biofilm formation genes of the strains combined with their effects on the cells. Then the effects of four antimicrobial peptides on the efflux pump activity, biofilm formation level and cell condition after infection were detected to explore the effects of antimicrobial peptides on RT strains. Finally, the RT strain was used to induce a mouse model of pneumonia, and the four antimicrobial peptides were used to treat pneumonia mice for in vivo experiments. The pathological changes in lung tissues in each group were detected to explore the antimicrobial peptide with the most significant effect on the RT strain in vivo.
UNASSIGNED: The results showed that the minimal inhibitory concentrations of the RT strains (strain C and strain I) were significantly higher than those of the wild-type strain, and the expression of efflux pump, porin and biofilm formation genes was significantly increased. The antimicrobial peptides could effectively inhibit the biofilm formation and efflux pump protein function of the RT strains. In addition, the antimicrobial peptides showed promising antibacterial effects both in vitro and in vivo.
UNASSIGNED: Our study provided a theoretical basis for the treatment of gentamicin resistant K. pneumoniae infection with antimicrobial peptides, and found that KLA was significantly superior to LL37, Magainin I, KLA and Dermaseptin (10 μg/mL in cells, 50 μg in mice).

Vitamin A and its biologically active derivative, retinoic acid (RA), are important for many immune processes. RA, in particular, is essential for the development of immune cells, including neutrophils, which serve as a front-line defense against infection. While vitamin A deficiency has been linked to higher susceptibility to infections, the precise role of vitamin A/RA in host-pathogen interactions remains poorly understood. Here, we provided evidence that RA boosts neutrophil killing of methicillin-resistant Staphylococcus aureus (MRSA). RA treatment stimulated primary human neutrophils to produce reactive oxygen species, neutrophil extracellular traps, and the antimicrobial peptide cathelicidin (LL-37). Because RA treatment was insufficient to reduce MRSA burden in an in vivo murine model of skin infection, we expanded our analysis to other infectious agents. RA did not affect the growth of a number of common bacterial pathogens, including MRSA, Escherichia coli K1 and Pseudomonas aeruginosa; however, RA directly inhibited the growth of group A Streptococcus (GAS). This antimicrobial effect, likely in combination with RA-mediated neutrophil boosting, resulted in substantial GAS killing in neutrophil killing assays conducted in the presence of RA. Furthermore, in a murine model of GAS skin infection, topical RA treatment showed therapeutic potential by reducing both skin lesion size and bacterial burden. These findings suggest that RA may hold promise as a therapeutic agent against GAS and perhaps other clinically significant human pathogens.

Antimicrobial peptide (AMP) is an important component of crustaceans\' innate immune system. In this study, a short neuropeptide F (sNPF) gene (Pc-sNPF) and a Forkhead box O (FOXO) gene (PcFOXO) from Procambarus clarkii were identified. Analysis findings showed that the expression level of AMP genes differed between male and female P. clarkii. Furthermore, Pc-sNPF and PcFOXO were related to the sex dimorphism of AMP. Knockdown of Pc-sNPF in the eyestalk significantly upregulated the expression of PcFOXO and two anti-lipopolysaccharide factors (PcALF4 and PcALFL) in the intestine of P. clarkii. The expression of PcFOXO in the intestine of female P. clarkii was higher than in that of males. Results from RNA interference revealed that PcFOXO positively regulated the expression of PcALF4 and PcALFL in the intestine of male and female P. clarkii. In summary, our study showed that differences in Pc-sNPF expression in eyestalk of male and female P. clarkii leading to sex dimorphism of AMP expression in the intestine are mediated by the sNPF-FOXO-AMP signal pathway called the eyestalk-intestine axis.

The problem of drug resistance caused by long-term use of antibiotics has been a concern for many years. As this problem worsens, there are various bacterial-induced infections that have a serious impact on human health. Currently, antimicrobial peptides are good alternatives to antibiotics, which have powerful antimicrobial activity and unique antimicrobial mechanisms. Developing bacterial resistance is not easy. In addition, how to reduce the production cost of antimicrobial peptides and improve the screening efficiency are the problems that must be solved for antimicrobial peptide application. In this study, we employed cell membrane chromatography linked with the one-bead-one-substance approach to screen and prepare the antimicrobial peptide (SALSP), which offers the benefits of fast synthetic screening and easy operation. Meanwhile, the antimicrobial peptide showed great antimicrobial activity and biocompatibility. We prepared a conjugated sodium alginate/gelatin hydrogel wound dressing incorporating antimicrobial peptides to promote wound healing. In conclusion, this research provides solutions for the development and application of antimicrobial peptides.

The alarming rise of antibiotic-resistant bacterial infections is driving efforts to develop alternatives to conventional antibiotics. In this context, antimicrobial peptides (AMPs) have emerged as promising candidates for their ability to target a broad range of microorganisms. However, the development of AMPs with optimal potency, selectivity, and/or stability profiles remains a challenge. To address it, computational tools for predicting AMP properties and designing novel peptides have gained increasing attention. PyAMPA is a novel platform for AMP discovery. It consists of five modules, namely AMPScreen, AMPValidate, AMPSolve, AMPMutate, and AMPOptimize, that allow high-throughput proteome inspection, candidate screening, and optimization through point-mutation and genetic algorithms. The platform also offers additional tools for predicting and evaluating AMP properties, including antimicrobial and cytotoxic activity, and peptide half-life. By providing innovative and accessible inroads into AMP motifs in proteomes, PyAMPA will enable advances in AMP development and potential translation into clinically useful molecules. PyAMPA is available at: https://github.com/SysBioUAB/PyAMPA.
OBJECTIVE: This paper introduces PyAMPA, a new bioinformatics platform designed for the discovery and optimization of antimicrobial peptides (AMPs). It addresses the urgent need for new antimicrobials due to the rise of antibiotic-resistant infections. PyAMPA, with its five predictive modules -AMPScreen, AMPValidate, AMPSolve, AMPMutate and AMPOptimize, enables high-throughput screening of proteomes to identify potential AMP motifs and optimize them for clinical use. Its unique approach, combining prediction, design, and optimization tools, makes PyAMPA a robust solution for developing new AMP-based therapies, offering a significant advance in combatting antibiotic resistance.

Leucine residues are commonly found in the hydrophobic face of antimicrobial peptides (AMPs) and are crucial for membrane permeabilization, leading to the cell death of invading pathogens. Melittin, which contains four leucine residues, demonstrates broad-spectrum antimicrobial properties but also significant cytotoxicity against mammalian cells. To enhance the cell selectivity of melittin, this study synthesized five analogs by replacing leucine with its structural isomer, 6-aminohexanoic acid. Among these analogs, Mel-LX3 exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria. Importantly, Mel-LX3 displayed significantly reduced hemolytic and cytotoxic effects compared to melittin. Mechanistic studies, including membrane depolarization, SYTOX green uptake, FACScan analysis, and inner/outer membrane permeation assays, demonstrated that Mel-LX3 effectively permeabilized bacterial membranes similar to melittin. Notably, Mel-LX3 showed robust antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Furthermore, Mel-LX3 effectively inhibited biofilm formation and eradicated existing biofilms of MDRPA. With its improved selective antimicrobial and antibiofilm activities, Mel-LX3 emerges as a promising candidate for the development of novel antimicrobial agents. We propose that the substitution of leucine with 6-aminohexanoic acid in AMPs represents a significant strategy for combating resistant bacteria.

Rattusin, an α-defensin-related antimicrobial peptide isolated from the small intestine of rats, has been previously characterized through NMR spectroscopy to elucidate its three-dimensional structure, revealing a C2 homodimeric scaffold stabilized by five disulfide bonds. This study aimed to identify the functional region of rattusin by designing and synthesizing various short analogs, subsequently leading to the development of novel peptide-based antibiotics. The analogs, designated as F1, F2, F3, and F4, were constructed based on the three-dimensional configuration of rattusin, among which F2 is the shortest peptide and exhibited superior antimicrobial efficacy compared to the wild-type peptide. The central cysteine residue of F2 prompted an investigation into its potential to form a dimer at neutral pH, which is critical for its antimicrobial function. This activity was abolished upon the substitution of the cysteine residue with serine, indicating the necessity of dimerization for antimicrobial action. Further, we synthesized β-hairpin-like analogs, both parallel and antiparallel, based on the dimeric structure of F2, which maintained comparable antimicrobial potency. In contrast to rattusin, which acts by disrupting bacterial membranes, the F2 dimer binds directly to DNA, as evidenced by fluorescence assays and DNA retardation experiments. Importantly, F2 exhibited negligible cytotoxicity up to 515 μg/mL, assessed via hemolysis and MTT assays, underscoring its potential as a lead compound for novel peptide-based antibiotic development.