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  [...]a mounting body of evidence indicates the presence of additional or complementary mechanisms such as intracellular targeting of cytoplasmic components crucial to proper cellular physiology (Figure 2) [7], [8]. [...]the initial interaction between the peptides and the microbial cell membrane would allow them to penetrate into the cell to bind intracellular molecules, resulting in the inhibition of cell wall biosynthesis and DNA, RNA, and protein synthesis. [...]insights into the mechanisms employed by AMPs will facilitate new approaches to discover and develop pharmacologic agents.

Defensins combat pathogenic bacteria invading the mammalian intestine. Salzman and co-workers find that defensins influence the composition of the small intestinal commensal microbiota and the presence of interleukin 17–producing T cells in the lamina propria. Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal kingdoms. In the mammalian small intestine, Paneth cell α-defensins are antimicrobial peptides that contribute to host defense against enteric pathogens. To determine if α-defensins also govern intestinal microbial ecology, we analyzed the intestinal microbiota of mice expressing a human α-defensin gene ( DEFA5 ) and in mice lacking an enzyme required for the processing of mouse α-defensins. In these complementary models, we detected significant α-defensin-dependent changes in microbiota composition, but not in total bacterial numbers. Furthermore, DEFA5 -expressing mice had striking losses of segmented filamentous bacteria and fewer interleukin 17 (IL-17)-producing lamina propria T cells. Our data ascribe a new homeostatic role to α-defensins in regulating the makeup of the commensal microbiota.

Sepsis claims an estimated 30 million episodes and 6 million deaths per year, and treatment options are rather limited. Human neutrophil peptides 1–3 (HNP1–3) are the most abundant neutrophil granule proteins but their neutrophil content varies because of unusually extensive gene copy number polymorphism. A genetic association study found that increased copy number of the HNP-encoding gene DEFA1/DEFA3 is a risk factor for organ dysfunction during sepsis development. However, direct experimental evidence demonstrating that these risk alleles are pathogenic for sepsis is lacking because the genes are present only in some primates and humans. Here, we generate DEFA1/DEFA3 transgenic mice with neutrophil-specific expression of the peptides. We show that mice with high copy number of DEFA1/DEFA3 genes have more severe sepsis-related vital organ damage and mortality than mice with low copy number of DEFA1/DEFA3 or wild-type mice, resulting from more severe endothelial barrier dysfunction and endothelial cell pyroptosis after sepsis challenge. Mechanistically, HNP-1 induces endothelial cell pyroptosis via P2X7 receptor-mediating canonical caspase-1 activation in a NLRP3 inflammasome-dependent manner. Based on these findings, we engineered a monoclonal antibody against HNP-1 to block the interaction with P2X7 and found that the blocking antibody protected mice carrying high copy number of DEFA1/DEFA3 from lethal sepsis. We thus demonstrate that DEFA1/DEFA3 copy number variation strongly modulates sepsis development in vivo and explore a paradigm for the precision treatment of sepsis tailored by individual genetic information.

Antimicrobial peptides, in particular α-defensins expressed by Paneth cells, control microbiota composition and play a key role in intestinal barrier function and homeostasis. Dynamic conditions in the local microenvironment, such as pH and redox potential, significantly affect the antimicrobial spectrum. In contrast to oxidized peptides, some reduced defensins exhibit increased vulnerability to proteolytic degradation. In this report, we investigated the susceptibility of Paneth-cell–specific human α-defensin 5 (HD-5) and -6 (HD-6) to intestinal proteases using natural human duodenal fluid. We systematically assessed proteolytic degradation using liquid chromatography–mass spectrometry and identified several active defensin fragments capable of impacting bacterial growth of both commensal and pathogenic origins. Of note, incubation of mucus with HD-5 resulted in 255–8,000 new antimicrobial combinations. In contrast, HD-6 remained stable with consistent preserved nanonet formation. In vivo studies demonstrated proof of concept that a HD-5 fragment shifted microbiota composition (e.g., increases of Akkermansia sp.) without decreasing diversity. Our data support the concept that secretion of host peptides results in an environmentally dependent increase of antimicrobial defense by clustering in active peptide fragments. This complex clustering mechanism dramatically increases the host’s ability to control pathogens and commensals. These findings broaden our understanding of host modulation of the microbiome as well as the complexity of human mucosal defense mechanisms, thus providing promising avenues to explore for drug development.

Objective Although polymorphisms of the NOD2 gene predispose to the development of ileal Crohn's disease, the precise mechanisms of this increased susceptibility remain unclear. Previous work has shown that transcript expression of the Paneth cell (PC) antimicrobial peptides (AMPs) α-defensin 4 and α-defensin-related sequence 10 are selectively decreased in Nod2−/− mice. However, the specific mouse background used in this previous study is unclear. In light of recent evidence suggesting that mouse strain strongly influences PC antimicrobial activity, we sought to characterise PC AMP function in commercially available Nod2−/− mice on a C57BL/6 (B6) background. Specifically, we hypothesised that Nod2−/− B6 mice would display reduced AMP expression and activity. Design Wild-type (WT) and Nod2−/− B6 ileal AMP expression was assessed via real-time PCR, acid urea polyacrylamide gel electrophoresis and mass spectrometry. PCs were enumerated using flow cytometry. Functionally, α-defensin bactericidal activity was evaluated using a gel-overlay antimicrobial assay. Faecal microbial composition was determined using 454-sequencing of the bacterial 16S gene in cohoused WT and Nod2−/− littermates. Results WT and Nod2−/− B6 mice displayed similar PC AMP expression patterns, equivalent α-defensin profiles, and identical antimicrobial activity against commensal and pathogenic bacterial strains. Furthermore, minimal differences in gut microbial composition were detected between the two cohoused, littermate mouse groups. Conclusions Our data reveal that Nod2 does not directly regulate PC antimicrobial activity in B6 mice. Moreover, we demonstrate that previously reported Nod2-dependent influences on gut microbial composition may be overcome by environmental factors, such as cohousing with WT littermates.

Acinetobacter baumannii is the causative agent of a wide range of nosocomial and community-acquired infections that remain extremely difficult to treat due largely to its antibiotic resistance contributed, in part, by biofilm formation. We find that the prototypic human neutrophil α-defensin HNP1, present in the bronchoalveolar lavage fluids from Acinetobacter baumannii -infected patients, promotes Acinetobacter baumannii biofilm formation through interactions with the bacterial outer membrane protein OmpA. As a result of HNP1-enhanced biofilm formation, Acinetobacter baumannii becomes more tolerant to antibiotics and more readily colonizes host cells and tissues. These unexpected findings contrast the protective roles HNP1 plays in innate immunity against microbial infection, showcasing an example of the host-pathogen arms race where a host defense peptide is exploited by a microbe for pathogenicity. Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial and community-acquired infections. Here, the authors determine that human neutrophil α-defensin HNP1 can promote A. baumannii biofilm formation.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies, driven by late diagnosis, limited therapeutic options, and high metastatic potential. Beyond their canonical roles in hemostasis, platelets have emerged as active modulators of tumor progression and promising noninvasive biomarkers. Among platelet-associated molecules, α-defensins, particularly Defensin Alpha 1/3 (DEFA1/3), have been implicated in inflammation and immunity; however, their contribution to PDAC pathogenesis remains unclear. We combined bioinformatic analysis of platelet transcriptomes with functional and in vivo zebrafish xenograft validation to investigate the impact of DEFA1/3 on PDAC aggressiveness. DEFA1/3 was significantly upregulated in PDAC-derived platelets. Defensin-enriched platelet-like particles (defensin-rich platelets, DRPs) and recombinant DEFA1/3 enhanced pancreatic cancer cell proliferation, migration, and three-dimensional growth in vitro and promoted tumor dissemination in zebrafish xenografts. Transcriptomic profiling revealed the upregulation of SPARC, KDM6A, and GATA6, whereas clinical data from The Cancer Genome Atlas (TCGA)-PDAC linked high DEFA1/3 expression to poor survival, increased immune infiltration, and activation of epithelial–mesenchymal transition (EMT). Platelet-derived DEFA1/3 acts as a functional modulator of PDAC progression, linking platelet granule content to tumor aggressiveness and highlighting a potential biomarker and therapeutic target within the platelet–tumor axis.

Anti-β 2 glycoprotein I (Anti-β 2 GPI) antibodies are a heterogeneous group of antiphospholipid antibodies targeting β 2 glycoprotein I (β 2 GPI). High titer of anti-β 2 GPI antibodies is a risk factor for thrombosis in antiphospholipid syndrome (APS). Although it has been shown that anti-β 2 GPI antibodies can induce neutrophil activation involved in thrombosis, the underlying mechanism remains unclear. In this study, we analyzed the clinical data of thrombotic patients who were positive or negative for anti-β 2 GPI antibodies, as well as healthy individuals. The results showed that the percentage and absolute count of neutrophils, serum levels of human neutrophil peptides (HNPs), and HNP mRNA levels were significantly higher in the anti-β 2 GPI-positive patient group compared to the healthy control group. Notably, when compared to the anti-β 2 GPI-negative patient group with similar neutrophil percentages and counts, the serum HNPs levels were also significantly elevated in the anti-β 2 GPI-positive patient group. In vitro, we further showed that anti-β 2 GPI and β 2 GPⅠ complex (anti-β 2 GPI/ β 2 GPⅠ complex) induced a concentration - and time-dependent increase in HNPs, which was mediated through P2Y 2 receptors on the surface of neutrophils. Meanwhile, we found that intracellular signaling pathways P38MAPK (P38 mitogen-activated protein kinase) and ERK (extracellular signal-regulated kinase) were also involved in the generation of HNPs. We also found that high levels of human neutrophil peptide-1 (HNP-1) could induce the production of procoagulant factors von Willebrand factor (vWF) and P-selectin in endothelial cells through the nuclear factor-κB (NF-κB) signaling pathway, which increased the risk of thrombosis.

In mammals, Paneth cells, located in the crypts of the small intestine, produceantimicrobial peptides that serve to keep the intestinal microbiome under control. a-Defensins are the primary antimicrobial peptides produced by these cells. We used 148 publicly available bulk RNA-seq samples on purified PCs, proteomics on enriched purified PC proteins and peptide activity assays to detect all transcrips, including potential chimeric transcrips. We identified 28 expressed genes in mice, with up to 85% of Paneth cell RNA reads mapping to these genes. Chimeric mRNAs, involving sequences from two different genes, were detected in most experiments. Despite their low abundance (less than 0.3%), mass spectrometry confirmed the presence of chimeric peptides. Synthetic versions of these peptides demonstrated antibacterial activity against multiple bacterial species. We show the existence of chimeric transcripts and peptides in mice that are biologically active. We propose a possible stochatic mechanism or that the activation of the UPR patway may play a role in their production.

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a β-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.