LL-37

Antimicrobial peptides in malaria and tuberculosis management: a systematic review of emerging evidence.

Neha Sylvia Walter, Varun Gorki, Bikash Medhi +1 more

AMPs (Antimicrobial peptides) are small molecules that are crucial components of biological activities, viz wound healing, angiogenesis, antimicrobial activity, immune regulation, and exhibit anticancer properties. This systematic review summarizes the origin, sequence, efficacy, and structure-activity mechanisms of AMPs against tuberculosis and malaria, the world's leading killer infectious diseases, employing the AMP database (APD3) (till June 2025) and PubMed search (January 1965 to June 2025). The comprehension is well addressed by covering the inhibitory activity, SAR (structure-activity relationships), and action mechanisms of AMPs (natural and synthetic) against Mycobacterium tuberculosis and Plasmodium spp., as both pathogens exhibit multidrug resistance. The review also illustrates how these specific motifs, along with their properties such as hydrophobicity, charge, or amphipathicity, influence the AMPs activity against the pathogens. Moreover, challenges in the AMPs use and the way forward, including the recent breakthroughs employing nanocarriers, inhalable formulations for TB, and targeted erythrocyte delivery for malaria, are also highlighted. AMPs have illustrated promising anti-TB and antimalarial efficacy owing to their unique structure and novel mode of action, despite exhibiting varying extents of toxicity. Anti-malarial AMPs are grouped in defensins and cecropins, while the majority of natural anti-TB AMPs are classified as bacteriocins, defensins, and cathelicidins. Synthetic AMPs, in contrast, are designed to enhance activity and selectivity while reducing toxicity and the development of resistance. AMPs have α-helical, β-sheet, or random-coil structures and are cationic, amphipathic, and highly hydrophobic, attributes that contribute to observed anti-TB and anti-malarial potential. A comprehensive understanding of AMP's structure and function, along with the use of artificial intelligence (AI) and machine learning, can provide impetus for AMP therapy as an alternative to tackle tuberculosis and malaria.

Design and Screening of the Peptide SAMP-12aa Derived from LL-37, Which Exhibits Anti-H. Pylori Activity and Immunomodulatory Effects.

Jianliang Lu, Qingyu Wang, Meisong Qin +3 more

The appearance of antibiotic-resistant strains of Helicobacter pylori (H. pylori) is leading to a decreased eradication rate of H. pylori infection. There is an urgent need to find new agents with antimicrobial mechanisms different from those of antibiotics, with therapeutic potential to clear colonization of H. pylori in the stomach. Some antimicrobial peptides (AMPs) possess bactericidal activity by enhancing the permeability of the outer membrane and damaging the integrity of the cell membrane. Bacteria are not susceptible to drug resistance through this antimicrobial mechanism. In this study, 28 short peptides containing 12 amino acid residues were designed based on nine amino acid fragments (KRIVQRIKD) from human cathelicidin LL-37, which is stable in gastric juice, and 3 amino acids were added at the C-terminus of the peptide. These designed peptides were not digested and degraded by pepsin at low pH values. The peptides were predicted using the online tool platform. Then, the strongest antimicrobial peptide, named SAMP-12aa (KRIVQRIKDVIR), was screened from 28 short peptides. Further studies found that SAMP-12aa retained anti-H. pylori activity after incubation in simulated gastric juice. The MIC and MBC of SAMP-12aa were 8 μg/mL and 32 μg/mL, respectively. SAMP-12aa showed good bactericidal kinetics. SAMP-12aa was found to have cell selectivity, penetrating and damaging bacterial cell membranes and exhibiting almost no toxicity to human cells at a relatively high concentration (128 μg/mL). Regulatory T (Treg) cells express CD25High with immunosuppressive activity that induces immune tolerance in response to H. pylori. Molecular docking prediction revealed that SAMP-12aa could target the active center of Foxp3. Flow cytometry analysis revealed that SAMP-12aa can inhibit Foxp3 activity and downregulate CD25 protein expression on CD4+ T cells, thereby reducing the development and differentiation of CD4+Foxp3+CD25High Treg cells with immunosuppressive effects. Further research revealed that the levels of the cytokine interferon-γ (IFN-γ), which activates CD8+ T-cell activity, were significantly elevated, and the levels of transforming growth factor-β (TGF-β), which inhibits CD8+ T-cell activity, were significantly reduced. The results of this study reveal that SAMP-12aa not only possesses antibacterial activity but also has immunomodulatory effects.

Development of nanobody-conjugated LL37 for synergistic therapy against MDR Acinetobacter baumannii.

Apisitt Thaiprayoon, Worrapoj Oonanant, Siriphan Boonsilp +10 more

Multidrug resistance (MDR) of the pathogen Acinetobacter baumannii is a major challenge to global healthcare due to the limited treatment options. The emergence of MDR bacteria necessitates innovative therapeutic approaches, especially given the associated economic burden and the rapid spread of infections. Conventional treatments such as antibiotics and vaccines face significant obstacles. Antimicrobial peptides (AMPs) such as LL37 have potential as an alternative treatment due to their broad-spectrum activity and ability to target specific bacterial structures such as the outer membrane protein A (OmpA). The efficacy of AMPs can be enhanced by using nanobodies (Nbs) that bind to bacterial OmpA, guiding LL37 precisely to its target. In this study, A. baumannii OmpA (AbOmpA)-specific Nbs (NbO7 and NbO13) were efficiently isolated through magnetic-activated cell sorting-based screening of a yeast surface display library, eliminating the need for specialized equipment. Nbs exhibited specific, dose-dependent binding to the target. Conjugation of Nbs with LL37 effectively inhibited the growth of MDR A. baumannii. This approach leverages the natural antimicrobial properties of AMPs and enhances their specificity and effectiveness by targeting bacterial cell surface proteins. LL37-conjugated AbOmpA-Nbs present a promising therapeutic strategy against MDR A. baumannii and other resistant pathogens.IMPORTANCEMultidrug-resistant (MDR) Acinetobacter baumannii poses a major global health threat due to its resistance to nearly all available antibiotics and its persistence in hospital settings. This challenge underscores the urgent need for new therapeutic approaches beyond conventional drugs. In this study, we developed an innovative strategy that combines the human antimicrobial peptide LL37 with nanobodies (Nbs) targeting the outer membrane protein A (OmpA), a key virulence and survival factor of A. baumannii. OmpA-specific Nbs were efficiently isolated from a fully synthetic library using a simple, low-cost selection approach without animal immunization. When conjugated with LL37, these Nbs bound specifically to OmpA and strongly inhibited MDR A. baumannii growth in vitro. Our findings introduce a simple yet powerful platform for generating targeted Nb-peptide conjugates, offering strong potential for adaptation against other antibiotic-resistant pathogens and contributing to the development of next-generation biologics to overcome antibiotic limitations.

Cathelicidin Links Visceral Fat Accumulation and Coronary Artery Disease.

Motoki Taniguchi, Akira Taruya, Chie Kitahara +20 more

Visceral fat (VF), particularly epicardial adipose tissue (EAT), plays a crucial role in the development of coronary artery disease (CAD). Cathelicidin (LL37) is an antimicrobial peptide involved in innate immunity and has been implicated in inflammatory processes. However, the relationship between VF accumulation, cathelicidin, and atherosclerosis remains unclear.

Novel Hybrid Peptide ML-2 with Antibacterial and Antibiofilm Activity Against Pseudomonas aeruginosa.

Shenghua Wu, Yanping Shi, Yang He +4 more

Antimicrobial resistance significantly contributes to treatment failures in bacterial infections and poses a major global public health challenge, urgently necessitating the development of new antimicrobial agents, such as antimicrobial peptides (AMPs), which represent a promising drug class. This study aimed to develop a novel, highly effective, low-toxicity antimicrobial agent employing a hybridization strategy to fuse the broad-spectrum AMP LL-37 with the low-toxicity Musca domestica antifungal peptide-1 A. The resulting hybrid peptides (ML-1, ML-2, and ML-3) exhibited markedly reduced cytotoxicity and hemolytic activity relative to LL-37. Notably, ML-2 demonstrated broad-spectrum antimicrobial activity, with particularly exerted effective antibacterial and antibiofilm effects against Pseudomonas aeruginosa in vitro. Moreover, with excellent stability, ML-2 retained activity against P. aeruginosa following pepsin treatment and thermal stress. Importantly, P. aeruginosa developed resistance to ML-2 more slowly than to the potent antibiotic ciprofloxacin. Mechanistically, ML-2 killed bacteria by increasing cell membrane permeability and disrupting cell integrity. Altogether, these findings suggest that the hybrid peptide ML-2 has potential as a novel antimicrobial agent against P. aeruginosa.

Endogenous cathelicidin protects against Toxoplasma gondii-associated liver damage.

Yi Lin Tan, Paloma Cavalcante, Karina M Cirone +6 more

Toxoplasmosis, a disease caused by apicomplexan Toxoplasma gondii (Tg), is associated with various neuropsychiatric and behavioral conditions. Toxoplasmosis can cause serious complications for those with weakened immune systems and during pregnancy. Cathelicidins, peptides with antimicrobial and immunomodulatory functions, are critical factors in host defense against microbes, but their role in parasitic infections is less well understood. This study demonstrates the protective function of endogenous cathelicidin against hepatic damage caused by Tg infection in an oral infection model. We challenged wild-type (Camp+/+) and cathelicidin-deficient (Camp-/-) mice with low-virulent Type II strain of Tg (ME-49) cysts. Our findings demonstrate that Camp-/- mice exhibited more severe clinical manifestations, higher mortality rates, and more pronounced hepatic damage compared to their Camp+/+ counterparts. Histological liver examinations indicated significant necrotic hepatitis in Camp-/- mice, correlating with increased local concentrations of pro-inflammatory cytokines and proteomic upregulation of poly(ADP-ribose) polymerase 3 and guanylate-binding proteins. Increased cerebral inflammation and Tg cystogenesis were also observed in Camp-/- mice. Systemically, Camp-/- mice presented elevated levels of pro-inflammatory mediators, specifically interferon-gamma (Ifn-γ) and tumor necrosis factor-alpha (Tnf-α). In cultured macrophages, endogenous cathelicidin increased after Tg challenge, while Camp-/- bone marrow-derived macrophages released higher amounts of Tnf-α than their counterparts. We conclude that cathelicidin protects against liver injury and systemic deterioration induced by Tg infection by downregulating the synthesis of pro-inflammatory cytokines.

LL-37 and bacterial DNA complexes in dental plaque: Implications for biofilm structure, innate immunity, and periodontal pathogenesis.

Gen Tanabe, Taiki Mori, Mariko Hanaoka +2 more

Dental plaque is a highly organized polymicrobial biofilm, in which extracellular DNA serves as a vital structural and functional component of the extracellular matrix. The human antimicrobial peptide LL-37 plays an important role in oral innate defense, exhibiting both antimicrobial and immunomodulatory activities. Our recent study indicated that LL-37 forms stable complexes with bacterial DNA in dental plaque. This review summarizes current knowledge of the molecular mechanisms and immunological consequences of LL-37-bacterial DNA interactions in dental plaque, highlighting their potential implications in biofilm structure, innate immunity, and periodontal pathogenesis.

LL-37 selectively targets Plasmodium-infected erythrocytes and exhibits antimalarial activity.

Xiaoqin He, Yutong Zhang, Junchao Lou +6 more

Malaria control is challenged by the emergence of resistance to virtually all antimalarial drugs, from the frontline artemisinin to other classes, highlighting the critical need for new therapies. This study demonstrates that the human antimicrobial peptide LL-37 exhibits antiplasmodial activity against both drug-sensitive and drug-resistant parasites in vitro. LL-37 selectively targets infected red blood cells through membrane disruption mediated by phosphatidylserine externalization and cholesterol depletion. Elevated plasma LL-37/CRAMP levels were observed in malaria patients and infected mice, and exogenous LL-37/CRAMP administration reduced parasitemia, improved survival, and modulated pro-inflammatory cytokine levels in a mouse model. CRAMP-deficient mice showed higher susceptibility to infection, underscoring its role in host defense. Our findings reveal a naturally occurring host defense mechanism centered on LL-37/CRAMP, which acts through direct targeting of the infected erythrocyte membrane. However, therapeutic administration after infection establishment showed limited efficacy, likely due to rapid peptide degradation in vivo, and the effective concentrations required for direct killing in vitro are substantially higher than endogenous systemic levels. The reduction in systemic cytokines observed in treated mice is likely primarily attributable to decreased parasite burden rather than direct immunomodulation. Further studies are needed to evaluate stabilized analogs, optimized delivery strategies, and combination approaches before therapeutic applications can be considered.

Exploring the role of cathelicidin LL-37 and ceragenins in wound healing processes.

Milena Łuckiewicz, Urszula Wnorowska, Magdalena Zakrzewska +6 more

Wound healing and tissue regeneration are essential for maintaining skin integrity in the face of mechanical, chemical, or thermal damage. In adult mammals, the wound healing process may include complications leading to chronic wounds and hypertrophic scars. Recently, particular attention has been paid to governing wound healing using antimicrobial peptides (AMPs), such as human cathelicidin LL-37, and its synthetic analogs, ceragenins (CSAs). These substances are gaining interest due to their versatile properties that support healing and regeneration. This work reviews literature describing the role of AMPs, particularly LL-37 and synthetic ceragenins, in wound healing. We focus on their complex mechanisms of action, including strong antimicrobial properties preventing infections and the ability to support cell migration and proliferation, highlighting their potential to enhance tissue repair. The interactions of these agents with skin cells and skin homeostasis-associated signaling pathways have been discussed. Ceragenins stand out as non-peptide mimics of AMPs characterized by resistance to proteolysis, anti-biofilm activity, and stability in varying environmental conditions. Additionally, modulation of inflammatory responses by these compounds aids healing and reduces complications associated with excessive inflammation. These properties make ceragenins promising candidates for treating infected wounds and for skin regenerative therapy. As research progresses, the integration of LL-37 and ceragenins into clinical practice could revolutionize current therapeutic strategies, offering new hope to patients with chronic wounds and skin injuries. Continuing research on these compounds will be crucial for understanding their full potential and optimizing their applications in regenerative medicine.

Amoebicidal effect of Antimicrobial Peptides against Acanthamoeba castellanii: A study of cell death using Fluorescence imaging systems.

Ines Sifaoui, Rubén L Rodríguez-Expósito, María Reyes-Batlle +6 more

Acanthamoeba infections are hindered by limitations including prolonged treatment duration, cytotoxicity, limited efficacy against the resistant cystic stage, and a high rate of recurrence. Antimicrobial peptides (AMPs) demonstrate broad-spectrum antimicrobial activity, rapid mechanism of action, low potential for inducing resistance, and efficacy against both trophozoite and cyst forms, highlighting their potential as promising alternatives in the treatment of these infections. Herein, we report the amoebicidal activity of various peptides derived from cathelicidins and piscidins against Acanthamoeba castellanii Neff. Afterwards, the impact of the most effective peptide on the clinical features of a clinical strain of Acanthamoeba castellanii was evaluated using a fluorescence imaging system. Peptide OH_KR34 was the most active against A. castellanii Neff with an IC₅₀ values of 2.00 ± 0.58 µM. OH_KR34 was able to inhibit the clinical strain of A. castellanii (L10) with an IC50 of 5.75 ± 0.30 μM. We demonstrated that the peptide OH_KR34 (related to OH-CATH) induces cytoskeletal damage and trigger apoptosis-like process by disrupting cell membrane permeability, mitochondrial dysfunction and DNA fragmentation. This antimicrobial peptide could therefore offer a potential target to develop new anti-parasitic drugs.

CRISPR/Cas9-engineered Salmonella phage displaying antimicrobial peptide LL37 for enhanced antibacterial activity.

Su Jin Jo, Se Chang Park, Sang Guen Kim

The increasing prevalence of antibiotic-resistant Salmonella Typhimurium has highlighted the urgent need for alternative therapeutic strategies. This study engineered a lytic S. Typhimurium bacteriophage to present the antimicrobial peptide LL-37 on the virion surface, followed by evaluation of its enhanced antibacterial efficacy.

Influence of N- and O-glycosylation on structural properties and biological activity of a C-terminal LL-37 fragment.

Daria Grzywacz, Francesca Nuti, Krzysztof Żamojć +5 more

Glycosylation represents a versatile strategy in peptide glycoengineering to modulate the structural, physicochemical, and biological properties of antimicrobial peptides (AMPs). In this study, we report the synthesis and characterization of O- and N-glycosylated analogues of the C-terminal fragment of human cathelicidin LL-37 (Ac-DFLRNLVPRTES-COOH), achieved via solid-phase peptide synthesis using Fmoc-Thr(β-d-Glc) and Fmoc-Asn(β-d-Glc) as glycosylated building blocks. The metal-binding affinity of these glycopeptides (N163 and T168) and the unmodified reference peptide (hCAP) toward Cu2+ and Mn2+ ions were evaluated by steady-state fluorescence spectroscopy. All peptides formed non-permanent complexes (Ka = 102 -104 M-1), with the native hCAP exhibiting the highest affinity. Molecular dynamics (MD) simulations revealed that glycosylation reduced conformational flexibility without significantly altering the overall shape of the peptide. Occasional sugar-metal contacts were observed, indicating transient carbohydrate participation in metal coordination. Cytotoxicity studies using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays confirmed the biocompatibility of all tested peptides, showing low toxicity against human fibroblasts, keratinocytes, and T-cells up to 100 μM. Our results demonstrate that carbohydrate conjugation subtly reshapes peptide-metal ion interactions, and structural dynamics. In this study, glycosylation serves primarily as a chemical model of post-translational modification, designed to probe how the presence of a carbohydrate moiety influences peptide conformation and coordination behaviour, rather than a strategy for enhancing antimicrobial function.

Structural determinants of glycosaminoglycan oligosaccharides as LL-37 inhibitors in breast cancer.

Chloe Le Fournis, Martyna Maszota-Zieleniak, Adam Kulesza +6 more

The human antimicrobial peptide LL-37 exerts a dual role in cancer, promoting tumor progression in breast carcinoma by stimulating calcium influx and enhancing cell migration. Its interactions with glycosaminoglycans (GAGs) provide an attractive therapeutic avenue for intervention. In this study, we investigated the ability of structurally distinct GAG oligosaccharides to inhibit LL-37-mediated responses in MDA-MB-231 breast cancer cells. Functional assays were performed to evaluate membrane depolarization, intracellular calcium mobilization, and migration capacity. When applied at a 1:1 stoichiometric ratio with LL-37, heparin emerged as the most effective inhibitor. This inhibitory activity required a minimum oligosaccharide length of 18 monosaccharide units and was strongly dependent on the degree of sulfation. Heparin displayed significantly higher efficacy than either chondroitin sulfate or dermatan sulfate. Binding studies using microscale thermophoresis demonstrated nanomolar affinity of LL-37 for heparin, whereas interactions with chondroitin and dermatan sulfate were weaker. Complementary molecular dynamics simulations reinforced these findings by highlighting the predominant role of electrostatic interactions in stabilizing LL-37/GAG complexes and by providing atomistic models of the binding interfaces. Binding studies on a heparin-derived synthetic oligosaccharide array revealed that, apart from the degree of sulfation, structural components in heparin contribute to its affinity for LL-37. Collectively, our results suggest that the structural optimization of GAG mimetics may represent a promising targeted strategy to block LL-37-driven tumor progression.

Non-specific Protein and Peptide Antibacterial Factors of Mammals.

Pavel Levashov, Ilia Zaitsev, Sergei Zaitsev +1 more

The review summarises the basic information on non-specific factors of mammals that potentially have antimicrobial action. A comparison of previously known factors with the latest literature data is carried out. The following peptide and protein factors are considered: lysozymes, transferrins, interferons, interleukin-2, antimicrobial peptides (defensins, cathelicidins, histatins) and protective glycoproteins (mucins, lectins). These major antibacterial factors perform regulatory functions in the immune system, and some are also able to resist viral and fungal infections or oncological pathologies. The study of the internal antibacterial factors of mammals and the mechanisms of their activation is of great importance for the fight against bacterial infections, including antibiotic-resistant ones. This knowledge is necessary for the development of new approaches to the treatment of humans and farm animals.

A hedgehog cathelicidin-derived peptide exhibits antiviral activity against herpes simplex virus type 1 infection.

Enjie Deng, Yaping Pei, Kun Yuan +7 more

Herpes simplex virus type 1 (HSV-1) is a widespread infectious virus that poses a substantial public health burden. With no specific curative therapy available and current antivirals providing only symptomatic relief, the development of new effective antiviral agents remains imperative. Antimicrobial peptides, particularly cathelicidins, are key immune effector molecules with antiviral and immunomodulatory properties. In this study, we found that European hedgehog skin extract (HSE) exhibited potent anti-HSV-1 activity, prompting us to investigate the contribution of the cathelicidin peptide CathEE. Based on this observation, three CathEE-derived antiviral peptides (CathEE-1, CathEE-2, and CathEE-3) were designed and further optimized to yield CathEE-2a and CathEE-2b. Notably, CathEE-2a displayed pronounced antiviral activity in vitro and significantly reduced brain viral loads in an HSV-1 mouse infection model. Histopathological analyses further revealed that CathEE-2a attenuated HSV-1-induced brain damage. Mechanistically, CathEE-2a suppressed HSV-1 infection by upregulating type I interferons and downstream antiviral genes. Collectively, these findings identify CathEE-2a as an immunomodulatory antiviral peptide and a promising lead candidate for the development of novel therapeutics against HSV-1 infection, highlighting the therapeutic potential of hedgehog-derived antimicrobial peptides in antiviral drug discovery.

Vitamin D in Infectious Diseases: A Narrative Review Focusing on COVID-19, Long COVID, and Influenza.

Olga Adriana Caliman-Sturdza, Roxana Elena Gheorghita, Iuliana Soldanescu +2 more

Vitamin D is a secosteroid hormone traditionally recognized for its role in bone and mineral metabolism, but it is increasingly understood to also function as an important immunomodulator influencing susceptibility to and outcomes of infectious diseases. This narrative review summarizes current evidence on the immunological, clinical, and preventive effects of vitamin D in the context of novel coronavirus disease (COVID-19), post-acute sequelae of SARS-CoV-2 infection (long COVID), and influenza. Mechanistically, vitamin D enhances innate immune defenses through the induction of antimicrobial peptides, including cathelicidin and defensins, and modulates adaptive immunity by suppressing maladaptive Th1/Th17 responses while promoting regulatory T-cell activity. Observational studies have frequently associated vitamin D deficiency with more severe COVID-19 outcomes; however, these associations may be influenced by confounding factors and reverse causality. Some meta-analyses suggest that vitamin D supplementation reduced rates of intensive care unit admission and ventilatory support, particularly among older adults and individuals with low baseline serum 25-hydroxyvitamin D concentrations. Emerging evidence also indicates that inadequate vitamin D status may be associated with an increased risk and symptom burden of long COVID, although causality has not been established. In the case of influenza, a limited number of randomized controlled trials (RCTs) and meta-analyses report a modest but statistically significant reduction in infection risk, especially with daily or weekly vitamin D supplementation in populations with low baseline vitamin D levels. Clinical guidelines consistently recommend maintaining adequate vitamin D status for general health but do not endorse high-dose vitamin D as a treatment for COVID-19 due to inconsistent trial findings. Overall, vitamin D should not be considered a standalone therapeutic agent; rather, maintaining sufficient vitamin D levels represents a low-risk, potentially beneficial strategy to support immune resilience against respiratory viral infections.

Analysis of Antimicrobial Peptide Expression Under Acute and Chronic Alcohol Exposure: A Cross-Sectional Study and a Systematic Review of the Literature.

Maura Rojas-Pirela, Cristian Herrera-Flores, Pilar Costa-Alba +10 more

Alcohol exposure affects immune regulation and tissue homeostasis. Antimicrobial peptides (AMPs) are essential components of innate immunity, not only defending against pathogens but also modulating processes such as inflammation. However, their tissue-specific regulation in response to alcohol remains poorly characterized, particularly in humans after acute intoxication. We evaluated the expression of AMPs in the peripheral blood of patients with alcohol use disorder (AUD, n = 9), individuals with acute alcohol consumption (AAC, n = 9), and controls using quantitative polymerase chain reaction (qPCR). Additionally, we analyzed AMP expression in selected tissues of mice exposed to chronic ethanol feeding (National Institute on Alcohol Abuse and Alcoholism model for 5 days) and performed a systematic review of AMP regulation in alcohol-related disorders (2005-2025; n = 36 studies, reflecting a limited and heterogeneous body of available evidence). Human cathelicidin antimicrobial peptide (LL-37), lipopolysaccharide-binding protein (LBP), and bactericidal/permeability-increasing protein (BPI) were significantly upregulated in patients with AUD, whereas LL-37 and LBP were significantly upregulated in AAC. In the livers of ethanol-fed mice, LEP2, LCN2, and LBP levels were markedly increased, whereas LL-37 and LEP1 were downregulated. Duodenal tissue exhibited upregulation of DEFB1. In adipose tissue, DEFA2 was significantly increased in peripheral depots, whereas only LCN2 was upregulated in brain tissue. The systematic review demonstrated complex, heterogeneous, and organ-dependent AMP regulation and also highlighted the paucity of human data on AAC, a gap that our study partially addresses. Our results are consistent with the hypothesis that selected AMPs may serve as candidate markers of organ damage or microbial translocation and as possible therapeutic targets, a hypothesis that requires confirmation in larger, adequately powered studies.

Investigation of Regulation and Binding Patterns of the Human Cathelicidin Peptide LL-37 in Complexation with Nucleic Acids, and its Impact on Neutrophil Extracellular Traps.

Claudia Zielke, Behzad Rad, Josefine E Nielsen +6 more

The human cathelicidin host defense peptide LL-37 forms complexes with nucleic acids that can have either beneficial or detrimental health effects. We suggest that these differential impacts are directly connected to dsDNA binding by LL-37 and to complex formation between protomers. Here, we show using phage λ DNA that LL-37 binds non-specifically to dsDNA, condensing it, followed by complex formation between LL-37 peptides. We find that complex formation is concentration-dependent, with low LL-37 amounts yielding loosely aggregated DNA structures, while higher LL-37 concentrations lead to well-defined, disc-like structures of about 150 nm in diameter. The condensation of the nucleic acids, which causes a loss of the characteristic B-DNA features, results from interactions of the phosphodiester backbone with protonated amino acid side chains of the peptide at physiological pH, predominantly in A-T rich sequences of the nucleic acid. However, in our studies, electrostatic interactions did not appear to be the driving force for complexation, but rather we found the α-helical structure of the peptide with its amphipathic and hydrophobic surfaces to be essential. Further, we show that LL-37 also interacts with nucleic acids from neutrophil extracellular traps (NETs) in a concentration-dependent way, causing a reduction in NET aggregate area, which may offer new biophysical insights into diseases such as systemic lupus erythematosus (SLE), which involve slower-than-normal NET clearance. Our results indicate the key importance of LL-37 expression levels for regulation of the innate immune system for optimal human health, since the relative amounts of expressed LL-37 present to interact with extracellular DNA will determine the extent to which the DNA can be condensed, which in turn will affect the ability of the body to clear the NETs before they can cause inflammatory conditions.

Injectable self-healing hydrogel loaded with a self-assembling LL-37 derivative for treating infected skin wounds.

Qi Ba, Jiaxin Yao, Hao Tian +10 more

Effective management of infected skin wounds remains a major clinical challenge, particularly as rising antibiotic resistance compromises conventional therapies. Antimicrobial peptides (AMPs) have emerged as promising alternatives, but their clinical translation is hindered by poor stability and potential toxicity. In this study, we report the rational design of a novel antimicrobial peptide, FR-20, a self-assembling derivative of the human cathelicidin LL-37, engineered for enhanced structural stability and antimicrobial efficacy. Using dynamic Schiff base cross-linking, we developed an injectable, self-healing chitosan hydrogel (CS/FR-20) loaded with FR-20. The hydrogel exhibited robust mechanical integrity, excellent injectability, and a porous microarchitecture, while maintaining strong cytocompatibility and hemocompatibility. CS/FR-20 demonstrated potent bactericidal activity against both Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). In a murine full-thickness wound infection model, it effectively eradicated bacteria, reduced inflammation, accelerated tissue regeneration, and showed no detectable systemic toxicity. These findings establish CS/FR-20 as a versatile, biocompatible platform for treating infected wounds, offering a clinically translatable strategy to overcome antibiotic resistance and promote effective wound healing.

Discovery and classification of new reptile cathelicidins by genome mining: study of their structure and genomic organization in Testudines and Squamata.

Andrea Otazo-Pérez, Manuel R López, Sergio González-Acosta +2 more

Cathelicidins, a diverse family of host defence peptides (HDPs) in vertebrates, are recognized for their potential in combating resistant microorganisms and their varied biological functions. While extensive studies have focused on mammalian cathelicidins, those in reptiles remain largely unexplored. In this study, we conducted a genome mining analysis that identified 287 cathelicidin genes across reptilian orders Testudines and Squamata. Of these genes, we identified 219 complete cathelicidin protein sequences and 68 cathelicidin-like gene copies that appear to be pseudogenes or gene fragments lacking intact open reading frames. We established for the first time a classification for reptile cathelicidins of the cited orders based on their sequence and structure, observing six types for Testudines and six for Squamata, suggesting a common ancestral lineage. Furthermore, we investigated the genomic arrangement of these cathelicidin genes, uncovering that they are grouped into clusters with variability in the gene number and in their organization. Cathelicidin derived mature peptides were classified into nine groups based on their sequences and physicochemical properties. This comprehensive study enhances the understanding of the cathelicidin family in reptiles, clarifying their genomic organization and describing the different types present. These classifications pave the way for future studies on the functions and specialization of each identified cathelicidin group. Moreover, they enable potential structure-activity studies of the peptides, establishing a foundation for differentiating their key features.

Cathelicidin CATH-2 suppresses the NF-κB/ROS/NLRP3 signaling pathway via regulating mTOR-dependent autophagy during Streptococcus suis infection.

Liuyi Xu, Yilin Lu, Shichao Xu +10 more

Cathelicidin CATH-2 has been reported to exert potent anti-inflammatory activity in different species though neutralizing stimuli such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA). CATH-2 has been shown to inhibit Streptococcus suis (S. suis)-induced activation of dendritic cells and macrophages by binding to LTA. However, the exact mechanism of this prophylactically anti-inflammatory activity remains unclear. Therefore, we investigated the anti-inflammatory activity and mechanism of CATH-2 in mice peritoneal macrophages pretreated with CATH-2 followed by S. suis infection. The results showed that CATH-2 pretreatment significantly reduced S. suis-induced transcription and secretion of interleukin (IL)-1β, IL-6, and IL-12. CATH-2 also downregulated NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) expression and apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, and inhibited the maturation of IL-1β, suggesting that CATH-2 inhibits NLRP3 activation. In addition, CATH-2 significantly inhibited S. suis-induced phosphorylation of p65 and extracellular signal-regulated kinase (ERK). Further study showed that CATH-2 inhibited S. suis-induced reactive oxygen species (ROS) by upregulating the expression of ROS scavenging genes including catalase (CAT) and superoxide dismutase 1 (SOD1). Mechanistically, transcriptome analysis revealed that CATH-2 regulated the protein kinase B (ATK)/mammalian target of rapamycin (mTOR) pathway, which was evident by the downregulation of phosphorylated (p)-ATK and p-mTOR induced by CATH-2. Notably, CATH-2 induced autophagy and autophagic flux. Inhibition of mTOR using rapamycin enhanced the CATH-2-induced autophagic efficacy, demonstrating that CATH-2 induces mTOR-dependent autophagy. However, inhibition of autophagy using 3-methyladenine (3-MA) reversed the reduction in the expression of p-p65, p-ERK, and IL-1β induced by CATH-2. Our study reveals that CATH-2 inhibits the nuclear factor kappa-B (NF-κB)/NLRP3-mediated inflammatory response through the induction of mTOR-dependent autophagy during S. suis infection, which provides new insight into the anti-inflammatory pathways of antimicrobial peptides.

Immunomodulatory Effects of the Antimicrobial Peptide KR-20: Implications for Trichomoniasis.

María G Ramírez-Ledesma, Eva E Ávila, Nayeli Alva-Murillo

Trichomoniasis is the most prevalent non-viral sexually transmitted infection worldwide and is caused by Trichomonas vaginalis. The development of resistance against the standard treatment, metronidazole, highlights the need for alternative therapeutic approaches. The role of innate immune cells is crucial for understanding trichomoniasis; however, the contribution of monocytes remains poorly characterized. We previously reported that the antimicrobial peptides LL-37 and its derivative KR-20 are trichomonacidal. In other systems, LL-37 displays immunomodulatory effects. Nevertheless, whether these peptides modulate monocyte responses in the presence of T. vaginalis remains unknown, which was the aim of this study. U937 monocytes were co-incubated with LL-37 or KR-20 (3 h), with or without parasite. Monocyte metabolic activity, nitric oxide production, and relative expression of innate immune genes were assessed. LL-37 decreased monocyte metabolic activity and upregulated TNF-α expression (10 and 5 μM, respectively) in parasite-challenged monocytes. Meanwhile, KR-20 (2.5-10 μM) preserved metabolic activity, bound microbial components (LPS), reduced parasite-induced nitric oxide production, and downregulated the expression of IL-8, TNF-α, IL-1β, and COX-2 in infected monocytes. This work provides initial evidence that KR-20 modulates innate immune response in monocytes during T. vaginalis infection, suggesting its potential-yet to be fully validated-as an immunomodulatory candidate for trichomoniasis.

Identification and characterization of novel antimicrobial peptides from Camelus dromedarius: a combined bioinformatics and experimental study.

Wafa Al-Mamari, Yasmin Elhag, Samir Al Bulushi +5 more

There is an urgent need for new antimicrobial agents to address the emerging antimicrobial resistance and the lack of novel antibiotics on the market. Antimicrobial peptides (AMPs) have gained significant interest as potential antibiotics over the past 30 years due to their broad activity against bacteria. So far, the presence, characteristics, and function of AMPs in camel immunity remain to be explored. Therefore, this study aims to identify and functionally characterize AMPs in Camelus Dromedarius using in-silico and experimental approaches. In-silico identification and prediction of cathelicidin peptides properties were conducted using Blastp, Conserved Domain, Signal P-5.0, Peptide Cutter-Expasy, and the Antimicrobial Sequence Scanning System (AMPA) database. Physicochemical and biological properties were characterized using bioinformatics analysis tools. The experimental assays of synthetic AMPs were performed using circular dichroism (CD) spectroscopy, colony-forming assay, sytox green uptake assay, transmission and scanning electron microscopy, and hemolysis assay. Three cathelicidin peptides were identified from Camelus Dromedarius which were designated as CdPMAP-23, Cdprotegrin-3 (CdPG-3), and Cdcathelin-like (CdCATH). CdPG-3 and CdCATH demonstrated significant antibacterial effects against all tested Gram-negative and Gram-positive strains, including Escherichia. coli (Multidrug resistant) and Methicillin-Resistant Staphylococcus aureus (ATCC 700699). These two peptides caused significant membrane leakage and damage to Escherichia. coli (ATCC 25922), with CdPMAP-23 showing a lesser effect. Lower concentrations of CdPMAP-23, CdPG-3 and CdCATH exhibited low to moderate lytic activity against red blood cells in humans, camels, and chickens. This study identified novel AMPs from dromedary camels with potential therapeutic value against multidrug-resistant strains. The results show that AMPs are present in dromedary camels, setting out a foundation for further studies on the unique features of their innate immune system.

Advances in the pathogenesis of rosacea.

Hui Wang, Cheng Zhou

Rosacea is a chronic inflammatory cutaneous disorder predominantly affecting the centrofacial region, whose pathogenesis is complex and not yet fully understood. In this review we summarized the latest significant advances in the pathogenesis of rosacea in recent years. In genomic studies, the application of bioinformatics techniques such as whole-genome sequencing has identified novel susceptibility genes and linked multiple pathogenic mechanisms. Neurovascular dysfunction resulting from abnormal neuropeptides expression and dysregulated amino acid metabolism constitutes an important pathogenic factor in rosacea. The TLR2/LL-37/mTORC1 signaling axis, as a core regulatory pathway in innate immunity has been elucidated in detail. In addition, the dysbiosis of skin and gut microbiota, together with the impairment of skin barrier function, is also closely associated with the onset and progression of this disease. The deeper understanding of the pathogenesis of rosacea will benefit the development of new drugs and promote individualized diagnosis and treatment.

Regulation of virulence factors of Pseudomonas aeruginosa by Scutellaria baicalensis, Prunella vulgaris and antimicrobial peptide LL-37.

Qian Xiao, Kaiwen Du, Li Luo +6 more

Introduction. The increasing resistance and the pathogen's complex multi-drug resistance mechanisms made the selection of effective antimicrobial treatments more challenging for Pseudomonas aeruginosa (P. aeruginosa). The study aimed to explore the effect of Scutellaria baicalensis, Prunella vulgaris and antimicrobial peptide LL-37 on the virulence factors of P. aeruginosa.Hypothesis. Previous studies have shown that extracts from traditional Chinese medicines, Scutellaria baicalensis and Prunella vulgaris, can also enhance the effects of antibiotics and reduce antibiotic resistance in P. aeruginosa. Antimicrobial peptide LL-37 shows the potential as a new-generation candidate for treating multi-drug-resistant bacteria, which has advantages over traditional antibiotics, whilst the combination role between Scutellaria baicalensis, Prunella vulgaris and LL-37 in P. aeruginosa remains unknown.Aim. We explored whether the combined use of Scutellaria baicalensis, Prunella vulgaris and LL-37 can exert antibacterial effects through the quorum sensing (QS) system.Methodology. The minimal inhibitory concentrations of Scutellaria baicalensis, Prunella vulgaris and LL-37 were determined for PAO1 and PA-ΔlasI/rhlI using micro broth dilution. The antibacterial activity of Scutellaria baicalensis combined with LL-37 and Prunella vulgaris combined with LL-37 was also assessed. The growth abilities of PAO1 were analysed after being treated with Scutellaria baicalensis, Prunella vulgaris and LL-37, respectively. Elastase secretion was measured using Congo red-elastic proteinase assays. And the expressions of QS genes (lasI, rhlR) were analysed by real-time PCR.Results. Single or combined treatments of Scutellaria baicalensis and LL-37 and Prunella vulgaris and LL-37 would significantly reduce elastase secretion. There were no significant differences in proliferation between the groups at any timepoint. All treatments downregulated lasI and rhlR gene expressions.Conclusion. Scutellaria baicalensis, Prunella vulgaris and antimicrobial peptide LL-37 all down-regulate the QS system-related genes of P. aeruginosa, inhibiting the secretion of virulence factors and reducing bacterial toxicity.

The Ly6ghigh Neutrophil Subset Dictates Breast Cancer Lung Metastasis via CD8+ T Cell Death.

Rui Wang, Xiaoqi Liu, Yixuan Hou +10 more

Background: Lung metastasis is a leading cause of breast cancer (BC)-related mortality, driven by the immunosuppressive traits of the metastatic tumor microenvironment. However, the mechanisms underlying cell-cell crosstalk in shaping immune evasion within the metastatic niche remain poorly defined. Neutrophil extracellular traps (NETs) and their associated proteins, such as cathelicidin, have emerged as key mediators of metastatic regulation in cancer. Here, we aimed to decipher the interaction between a neutrophil subset characterized by high expression of lymphocyte antigen 6 complex locus g (Ly6ghigh) and cluster of differentiation 8-positive T lymphocytes (CD8+ T cells), mediated via cathelicidin embedded in NETs, as well as their synergistic mechanism and cooperative role in promoting lung metastasis of BC. Methods: We characterized neutrophil heterogeneity and functional dynamics by performing single-cell RNA sequencing and flow cytometry on lung tissues derived from murine models of BC lung metastasis. We utilized cathelicidin-related antimicrobial peptide (Cramp) knockout mice to dissect the role of cathelicidin in NETs. The spatial colocalization of apoptotic CD8+ T cells and NETs was analyzed using multiplex immunofluorescence, and the molecular interactions were probed by protein binding assays. Results: Neutrophils in the lung metastatic niche were classified into 2 subsets based on the Ly6g expression: Ly6ghigh and Ly6glow neutrophils. Ly6glow neutrophils, which were recruited in the macrometastatic stage, exhibited myeloid-derived suppressor cell-like characteristics. Notably, Ly6ghigh neutrophils induced CD8+ T cell apoptosis through NET formation, with apoptotic CD8+ T cells spatially clustered within NET-rich areas. Mechanistically, NET-derived cathelicidin (Cramp in mice) directly bound to mitochondrial adenine nucleotide translocator 1 (Ant1) in CD8+ T cells, triggering conformational changes and complex formation with voltage-dependent anion channel 1 (Vdac1). These events resulted in the opening of the mitochondrial permeability transition pore and loss of mitochondrial membrane potential. Conclusions: Our study demonstrates that Ly6ghigh neutrophils play a critical role in immunosuppression and immune evasion through NET-induced apoptosis of CD8+ T cells. These findings underscore the importance of NETs and cathelicidin in BC lung metastasis, suggesting their potential as therapeutic targets in restoring antitumor immunity and in preventing metastatic progression.

Natural and Synthetic Peptides as Alternatives to Antibiotics in Intestinal Infections-A Review.

Lala Stepanyan, Monika Israyelyan, Alessandro Gori +7 more

Antimicrobial peptides (AMPs), evolutionarily conserved components of innate immunity characterized by their broad-spectrum efficacy and minimal resistance development, are increasingly recognized as promising therapeutic candidates. This review aims to integrate current knowledge concerning natural and synthetic antimicrobial peptides and their therapeutic effectiveness in addressing gastrointestinal infections.

Structural insights, immunomodulatory functions, and therapeutic potential of host defense peptides in avoiding antimicrobial resistance.

Gisele Regina Rodrigues, Michel Lopes Leite, Octavio Luiz Franco

Antimicrobial resistance (AMR) undermines the effectiveness of antibiotic treatment, and due to this, it now worldwide health concern. In general, antibiotics are used to control or combat bacteria, their widespread and irresponsible has contributed to the development bacterial resistance to some microbial drug. Due to this, treating patients identified with resistant bacteria is burdensome and, expensive, and success rates are low. Among the more successful tools are host defense peptides (HDPs), which represent a beneficial alternative due to their unique mechanisms of action and low toxicity toward host cells. HDPs are crucial innate immune system components, exhibiting antibacterial, antifungal, antiviral, and anti-inflammatory activities. Some peptides, such as human cathelicidin LL-37, exhibit direct pathogen-killing activity and the ability to modulate innate immune responses. HDPs also interact with various pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs), influencing downstream pathways essential for microbial clearance and inflammation regulation. These interactions damage the microbial membrane, stimulating cytokine production and immune cell recruitment. Furthermore, HDPs can modulate chemokine receptor signaling to coordinate leukocyte migration and enhance host protection. Despite these promising aspects, there are challenges to overcome, such as potential immunotoxicity, proteolytic instability, and low receptor specificity, which hinder clinical application. This review highlights the complex interaction between HDPs and immune receptors, which can be used to overcome AMR and inform next-generation anti-infective therapy development.

Increased cathelicidin LL-37 colonic expression is associated with tumor progression in colorectal cancer.

J Wlodarczyk, L Dziki, J Fichna

Colorectal cancer (CRC) remains a major global health challenge, with increasing incidence and limited treatment options. The antimicrobial peptide cathelicidin (LL-37) has been implicated in both tumorigenic and tumor-suppressive roles, but its precise function in CRC progression remains unclear. This study investigates the LL-37 expression in CRC and its association with key molecular pathways, including vitamin D signaling and G protein-coupled receptors (GPCRs). We analyzed LL-37 mRNA expression in 25 CRC tissue samples and matched healthy colonic mucosa using quantitative real-time PCR. Additionally, we assessed the expression of potential LL-37 target receptors, including formyl peptide receptor 2 (FPR2), toll-like receptors (TLR3, TLR4), CXC chemokine receptor 2 (CXCR2), and mas-related gene X2 (MrgX2). The correlation between LL-37 expression and clinicopathological factors, including tumor stage and nodal metastases, was also evaluated. LL-37 expression was significantly upregulated in CRC tissues compared to normal mucosa (p<0.001), with higher expression in advanced-stage CRC (AJCC stage III) and tumors with nodal metastases (p=0.006). Molecular analysis revealed significantly increased FPR2 expression and reduced TLR3 expression in CRC tissue, suggesting their involvement in tumor progression. Our findings suggest a role for LL-37 in CRC progression, potentially mediated through FPR2 activation and TLR3 suppression. The observed discrepancies in LL-37 function across studies highlight its complex, context-dependent role in tumor biology. Further research is needed to elucidate the mechanistic basis of LL-37 signaling and its potential as a therapeutic target in CRC.

Epithelial Heparan Sulfate Promotes Staphylococcus aureus Corneal Infection by Inhibiting Cathelicidins.

Kazutaka Hayashida, Jeffrey D Esko, Richard D Gallo +3 more

Cathelicidins are short cationic peptides with potent microbicidal activities and comprise an important arm of host innate immunity. Many cell types can produce cathelicidins, but they are mainly expressed by recruited immune cells and are induced in epithelial cells during infection. Although the mechanisms of bacterial killing by cathelicidins have been largely elucidated in vitro, those that regulate their activities in vivo are less well understood. Bacterial pathogens often co-opt host extracellular matrix (ECM) components and their functions to escape host defense; however, it is unclear whether such mechanisms exist against cathelicidins. Several studies have demonstrated that host heparan sulfate (HS) inhibits LL-37, the human cathelicidin, suggesting that bacteria might exploit HS to evade killing by cathelicidins. However, precisely how HS inhibits LL-37 and possibly other cathelicidins remains unknown, and the role of the HS-cathelicidin interaction in infectious disease has not been rigorously studied. Here, we found that deleting CRAMP, the murine cathelicidin, significantly increases the susceptibility of mice to Staphylococcus aureus corneal infection. We also determined that heparan compounds bind to CRAMP with low nanomolar affinity, the secondary structure of CRAMP is required for HS binding, and HS binding to CRAMP inhibits CRAMP binding to target bacterial cells. Furthermore, we found that heparan compounds inhibit the killing of S. aureus by cathelicidins derived from several mammalian species in a 2-O-sulfate-dependent manner. Additionally, we demonstrate for the first time that conditional deletion of HS2ST, the enzyme responsible for 2-O-sulfation of HS, in corneal epithelial cells significantly reduces the susceptibility of mice to corneal infection. Altogether, these data uncover an endogenous inhibition mechanism of cathelicidins where 2-O-sulfated epithelial HS tightly binds and neutralizes the antibacterial activity of cathelicidins.