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The human defensins are a group of cationic antimicrobial peptides that range in size from 2 to 5 kDa and share a common structural motif of six disulphide-linked cysteines. Several naturally occurring human α- and β-defensins have been identified over the past two decades. They have a wide variety of antimicrobial effects, and their potential to avoid the development of resistance to antimicrobial treatment makes them attractive as therapeutic agents. Human defensins have recently been the focus of medical and molecular biology studies due to their promising application in medicine and the pharmaceutical industry. This work aims to provide a comprehensive summary of the current developments of human defensins, including their identification, categorization, molecular features, expression, modes of action, and potential application in medical settings. Current obstacles and future opportunities for using human defensins are also covered. Furthermore, we shed light on the potential of this class as an antiviral agent, particularly against SARS CoV-2, by providing an in silico-based investigation of their plausible mechanisms of action.

Big defensins are antimicrobial polypeptides believed to be the ancestors of β-defensins, the most evolutionary conserved family of host defense peptides (HDPs) in vertebrates. Nevertheless, big defensins underwent several independent gene loss events during animal evolution, being only retained in a limited number of phylogenetically distant invertebrates. Here, we explore the evolutionary history of this fascinating HDP family and investigate its patchy distribution in extant metazoans. We highlight the presence of big defensins in various classes of lophotrochozoans, as well as in a few arthropods and basal chordates (amphioxus), mostly adapted to life in marine environments. Bivalve mollusks often display an expanded repertoire of big defensin sequences, which appear to be the product of independent lineage-specific gene tandem duplications, followed by a rapid molecular diversification of newly acquired gene copies. This ongoing evolutionary process could underpin the simultaneous presence of canonical big defensins and non-canonical (β-defensin-like) sequences in some species. The big defensin genes of mussels and oysters, two species target of in-depth studies, are subjected to gene presence/absence variation (PAV), i.e., they can be present or absent in the genomes of different individuals. Moreover, big defensins follow different patterns of gene expression within a given species and respond differently to microbial challenges, suggesting functional divergence. Consistently, current structural data show that big defensin sequence diversity affects the 3D structure and biophysical properties of these polypeptides. We discuss here the role of the N-terminal hydrophobic domain, lost during evolution toward β-defensins, in the big defensin stability to high salt concentrations and its mechanism of action. Finally, we discuss the potential of big defensins as markers for animal health and for the nature-based design of novel therapeutics active at high salt concentrations.

The last decade led to the discovery and characterization of several human beta-defensins. Analysis of genomic information indicates that the number of beta-defensin-like molecules encoded by the human genome may number in the tens. Growing interest in beta-defensins steadily enhances our knowledge about various aspects of their gene location, expression patterns and the transcription factors involved in their regulation in vivo. The hallmark property of beta-defensins, their antimicrobial activity, is clearly only the tip of the iceberg in the extensive network of inter-relations within the immune system in which these peptides function. Structural studies of beta-defensins provide the molecular basis for a better understanding of their properties, functions and their potential for practical applications. In this review, we present some recent advances in the studies of human beta-defensins, with an emphasis on possible correlations between their structural and functional properties.

Cyclic peptides are receiving significant attention thanks to their antimicrobial activity and high serum stability, which is useful to develop and design novel antimicrobial agents. Antimicrobial peptides appear to be key components of innate defences against bacteria, viruses, and fungi. Among the others, defensins possess a strong microbicidial activity. Defensins are cationic and amphipathic peptides with six cysteine residues connected by three disulfide bonds found in plants, insects, and mammals; they are divided in three families: α-, β-, and θ-defensins. α-Defensins are contained in the primary granules of human neutrophils; β-defensins are expressed in human epithelia; and θ-defensins are pseudo-cyclic defensins not found in humans, but in rhesus macaques. The structural diversities among the three families are reflected in a different antimicrobial action as well as in serum stability. The engineering of these peptides is an exciting opportunity to obtain more functional antimicrobial molecules highlighting their potential as therapeutic agents. The present review reports the most recent advances in the field of cyclic peptides with a specific regard to defensin analogs.

The defensin and defensin-like proteins are an extensive group of small, cationic, disulfide-rich proteins found in animals, plants, and fungi and mostly perform roles in host defense. The term defensin was originally used for small mammalian proteins found in neutrophils and was subsequently applied to insect proteins and plant γ-thionins based on their perceived sequence and structural similarity. Defensins are often described as ancient innate immunity molecules and classified as a single superfamily and both sequence alignments and phylogenies have been constructed. Here, we present evidence that the defensins have not all evolved from a single ancestor. Instead, they consist of two analogous superfamilies, and extensive convergent evolution is the source of their similarities. Evidence of common origin necessarily gets weaker for distantly related genes, as is the case for defensins, which are both divergent and small. We show that similarities that have been used as evidence for common origin are all expected by chance in short, constrained, disulfide-rich proteins. Differences in tertiary structure, secondary structure order, and disulfide bond connectivity indicate convergence as the likely source of the similarity. We refer to the two evolutionarily independent groups as the cis-defensins and trans-defensins based on the orientation of the most conserved pair of disulfides.

Rainbow trout β-defensin 3 ( rt Defb3) is identified as a small cationic antimicrobial peptide, but its recombinant expression and functional characterization have not been reported. In this study, the mature rt Defb3 Komagataella phaffii coding sequence was cloned from trout liver cDNA and heterologously expressed in GS115 using the pPIC9K with an α-factor secretion signal. Expression condition was optimized by adjusting methanol concentration (0.5–1.25%), induction temperature (26–30 °C), and induction duration (24–168 h). Optimized induction with 1.0% methanol at 30 ℃ for 96 h produced ~ 7 mg/mL of secreted peptide, which was purified to > 90% purity using one-step Ni-IDA affinity chromatography. The identity and purity of the recombinant rt Defb3 were confirmed by Tricine-SDS-PAGE, HPLC (~ 97% purity), and MALDI-TOF mass spectrometry. Functional assays revealed potent broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. These findings demonstrate, for the first time, the successful production of active rt Defb3 in Komagataella phaffii , establishing a scalable expression platform for fish β-defensins. The recombinant rt Defb3 peptide holds promise as a natural antimicrobial agent for aquaculture and potential therapeutic applications, addressing the urgent demand for alternatives to conventional antibiotics. Key points • First successful recombinant production of rainbow trout β-defensin 3 in Komagataella phaffii • via optimized induction strategy. • High-yield secretory expression (~ 7 mg/mL) with > 90% purity confirmed by HPLC and biochemical analysis. • Recombinant rtDefb3 displayed potent broad-spectrum antibacterial activity, offering a repeatable and scalable system and functional validation. Graphical Abstract Heterologous expression of rainbow trout β-defensin 3 ( rt Defb3) in Komagataella phaffii enabled efficient secretion, one-step purification, and functional validation. The recombinant peptide displayed broad-spectrum antimicrobial activity, highlighting its potential as a natural alternative to antibiotics in aquaculture and biomedical applications.

Defensins are small, cysteine-rich peptides involved in plant defense, though their insecticidal properties remain largely unexplored. Previously, based on transcriptome we identified a defensin gene in black gram in response to bruchid ( Callosobruchus maculatus ) infestation. In the present study, we cloned and sequenced full-length cDNAs of defensin genes from multiple legumes and conducted phylogenetic analyses. Two sequence variants were identified, exhibiting 95–98% homology with a previously reported insecticidal defensin gene (Accession no. AF326687). Variant 1 (DefV1) was present in black gram, pea, cowpea, and common bean, whereas variant 2 (DefV2) was identified in mung bean, chickpea, and pigeon pea. Computational analysis, including molecular docking, visualization, and molecular dynamics (MD) simulations, demonstrated enhanced interactions between DefV1 and bruchid α-amylase, suggesting a “Cork in the Bottle” inhibitory mechanism. Additionally, insect bioassays using artificial seeds supplemented with DefV1 showed no adult emergence. These findings highlight black gram defensin as a promising insecticidal agent and a potential candidate for genetic improvement of bruchid resistance in legumes.

Aims/Hypothesis Defensins play a crucial role in the innate immune system's first defense against microbial threats. However, little is known about the defensin system in the pancreas, especially in relation to Type 1 diabetes. We explore the expression of defensins in different disease stages of Type 1 diabetes and correlated obtained findings to the degree of inflammation, providing new insights into the disease and the innate immune system. Material and methods Pancreases from non-diabetic human organ donors of different age groups and donors with Type 1 diabetes with different disease duration were examined. Sections from head, body and tail of the pancreas were stained for eight different defensins and for immune cells; CD3+, CD45+, CD68+ and NES+ (granulocytes). Results In non-diabetic adult controls the level of expression for defensins Beta-1,Alpha-1, Cathelicidin and REG3A correlated with the level of inflammation. In contrast, individuals with Type  1 diabetes exhibit a reduction or absence of several central defensins regardless of the level of inflammation in their pancreas. The expression of Cathelicidin is present in neutrophils and macrophages but not in T-cells in subjects with Type 1 diabetes. Conclusions Obtained findings suggest a pancreatic dysfunction in the innate immune system and the bridging to the adaptive system in Type 1 diabetes. Further studies on the role of the local innate immune system in Type 1 diabetes is needed.

Defensins are a well-characterised group of small, disulphide-rich, cationic peptides that are produced by essentially all eukaryotes and are highly diverse in their sequences and structures. Most display broad range antimicrobial activity at low micromolar concentrations, whereas others have other diverse roles, including cell signalling (e.g. immune cell recruitment, self/non-self-recognition), ion channel perturbation, toxic functions, and enzyme inhibition. The defensins consist of two superfamilies, each derived from an independent evolutionary origin, which have subsequently undergone extensive divergent evolution in their sequence, structure and function. Referred to as the cis - and trans -defensin superfamilies, they are classified based on their secondary structure orientation, cysteine motifs and disulphide bond connectivities, tertiary structure similarities and precursor gene sequence. The utility of displaying loops on a stable, compact, disulphide-rich core has been exploited by evolution on multiple occasions. The defensin superfamilies represent a case where the ensuing convergent evolution of sequence, structure and function has been particularly extreme. Here, we discuss the extent, causes and significance of these convergent features, drawing examples from across the eukaryotes.

SmD1 isolated from seeds of common chickweed Stellaria media has strong inhibitory activity against phytopathogenic fungi and oomycetes in the micromolar range (IC 50 ≤ 1 μM). However, the low production of plant defensins in natural strains limits their large-scale actual production. In this study, defensin gene SmD1 was successfully heterologously expressed in Escherichia coli BL21 (DE3) for efficient production of plant defensins. The defensin gene SmD1 fused with thioredoxin was cloned into pET22b (+) vector. Then, it was transformed into E. coli BL21 (DE3) and expressed solubly after induction of isopropyl-β-D-thiogalactopyranoside (IPTG). At 50 °C, active SmD1 was released by 50% (v/v) formic acid hydrolysis of the cleavage of Asp-Pro bond between fused proteins. The recombinant protein SmD1 was purified by Ni-IDA column and showed significant antifungal activities against fungi. The induction conditions was optimized, and the results showed that the antimicrobial activity reached its maximum when the IPTG had a concentration of 0.6 mmol/L, a temperature of 25 °C, an induction time of 12 h and an OD 600 of 0.8.