Peptide LL-37 Research Articles

Antimicrobial peptide LL37 and regulatory T cell associated with late-onset sepsis in very preterm infants

Stem cell therapy may prevent late-onset sepsis (LOS) via antimicrobial peptide LL37 secretion and regulatory Tcell (Treg) regulation. The early prediction of LOS is still a challenge. This study evaluated whether immunological state of LL37 or Tregs precedes LOS. We firstly analyzed the LL37 level, Treg proportion, and LOS incidence in very preterm infants treated with autologous cord blood mononuclear cells (ACBMNCs) in our previous trial. Then, we constructed a prediction model and built validation cohort. We found ACBMNC intervention reduced the incidence of LOS from 27.3% to 6.9% (p= 0.021). LL37 and Treg abundances were higher in the ACBMNCs group. The nomogram demonstrated that early-life Treg and LL37 characteristics were closely associated with LOS (area under the curve, AUC 0.936), with implications for early prediction and timely clinical management. This composite model was also helpful to evaluate the beneficial effect of ACBMNCs intervention on LOS, thus promoting translational research.

Hybrid antimicrobial coating based on conjugate of hyaluronic acid with peptide LL-37 for PEO-modified titanium implants

A conjugate of hyaluronic acid and antimicrobial peptide LL-37 was synthesized for the first time. The hybrid compound was tested as an antimicrobial organic coating for titanium samples with an inorganic sublayer obtained by plasma electrolytic oxidation (PEO) of the surface. As a result of in vitro studies, the antibacterial effect of the hybrid molecule within the inorganic PEO coating was established, which consists of a significant (p 0.05) suppression of the ability of Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium and Escherichia coli to form biofilms. The presented approach can be utilized for the subsequent design and development of non-fouling antimicrobial coatings to decrease the risk of infectious diseases caused by bacteria when using implants.

Development of a defibrinated human blood hemolysis assay for rapid testing of hemolytic activity compared to computational prediction

Cytotoxicity studies determining hemolytic properties of antimicrobial peptides or other drugs are an important step in the development of novel therapeutics for clinical use. Hemolysis is an affordable, accessible, and rapid method for initial assessment of cellular toxicity for all drugs under development. However, variability in species of red blood cells and protocols used may result in significant differences in results. AMPs generally possess higher selectivity for bacterial cells but can have toxicity against host cells at high concentrations. Knowing the hemolytic activity of the peptides we are developing contributes to our understanding of their potential toxicity. Computational approaches for predicting hemolytic activity of AMPs exist and were tested head-to-head with our experimental results. ResultsStarting with an observation of high hemolytic activity of LL-37 peptide against human red blood cells that were collected in EDTA, we explored alternative approaches to develop a more robust, accurate and simple hemolysis assay using defibrinated human blood. We found significant differences between the sensitivity of defibrinated red blood cells and EDTA treated red blood cells. SignificanceAccurately determining the hemolytic activity using human red blood cells will allow for a more robust calculation of the therapeutic index of our potential antimicrobial compounds, a critical measure in their pre-clinical development. ConclusionWe introduce a standardized, more accurate protocol for assessing hemolytic activity using defibrinated human red blood cells. This approach, facilitated by the increased commercial availability of de-identified human blood and defibrination methods, offers a robust tool for evaluating toxicity of emerging drug compounds, especially AMPs.

Hybrid Antimicrobial Coating Based on Conjugate of Hyaluronic Acid with LL-37 Peptide for PEO-Modified Titanium Implants

Hybrid Antimicrobial Coating Based on Conjugate of Hyaluronic Acid with LL-37 Peptide for PEO-Modified Titanium Implants

Susceptibility of Legionella gormanii Membrane-Derived Phospholipids to the Peptide Action of Antimicrobial LL-37-Langmuir Monolayer Studies.

LL-37 is the only member of the cathelicidin-type host defense peptide family in humans. It exhibits broad-spectrum bactericidal activity, which represents a distinctive advantage for future therapeutic targets. The presence of choline in the growth medium for bacteria changes the composition and physicochemical properties of their membranes, which affects LL-37's activity as an antimicrobial agent. In this study, the effect of the LL-37 peptide on the phospholipid monolayers at the liquid-air interface imitating the membranes of Legionella gormanii bacteria was determined. The Langmuir monolayer technique was employed to prepare model membranes composed of individual classes of phospholipids-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL)-isolated from L. gormanii bacteria supplemented or non-supplemented with exogenous choline. Compression isotherms were obtained for the monolayers with or without the addition of the peptide to the subphase. Then, penetration tests were carried out for the phospholipid monolayers compressed to a surface pressure of 30 mN/m, followed by the insertion of the peptide into the subphase. Changes in the mean molecular area were observed over time. Our findings demonstrate the diversified effect of LL-37 on the phospholipid monolayers, depending on the bacteria growth conditions. The substantial changes in membrane properties due to its interactions with LL-37 enable us to propose a feasible mechanism of peptide action at a molecular level. This can be associated with the stable incorporation of the peptide inside the monolayer or with the disruption of the membrane leading to the removal (desorption) of molecules into the subphase. Understanding the role of antimicrobial peptides is crucial for the design and development of new strategies and routes for combating resistance to conventional antibiotics.

Rapid evolution of an adaptive multicellular morphology of Candida auris during systemic infection

Candida auris has become a serious threat to public health. The mechanisms of how this fungal pathogen adapts to the mammalian host are poorly understood. Here we report the rapid evolution of an adaptive C. auris multicellular aggregative morphology in the murine host during systemic infection. C. auris aggregative cells accumulate in the brain and exhibit obvious advantages over the single-celled yeast-form cells during systemic infection. Genetic mutations, specifically de novo point mutations in genes associated with cell division or budding processes, underlie the rapid evolution of this aggregative phenotype. Most mutated C. auris genes are associated with the regulation of cell wall integrity, cytokinesis, cytoskeletal properties, and cellular polarization. Moreover, the multicellular aggregates are notably more recalcitrant to the host antimicrobial peptides LL-37 and PACAP relative to the single-celled yeast-form cells. Overall, to survive in the host, C. auris can rapidly evolve a multicellular aggregative morphology via genetic mutations.

Enhancement of innate immunity in gingival epithelial cells by vitamin D and HDAC inhibitors.

The human host defense peptide LL-37 is a component of the innate immune defense mechanisms of the oral cavity against colonization by microbes associated with periodontal disease. We have previously shown that the active form of vitamin D, 1,25(OH)2D3, can induce the expression of LL-37 in gingival epithelial cells (GEC), and prevent the invasion and growth of periopathogenic bacteria in these cells. Further, experimental vitamin D deficiency resulted in increased gingival inflammation and alveolar bone loss. Epidemiological studies have shown associations between vitamin D deficiency and periodontal disease in humans, suggesting application of vitamin D could be a useful therapeutic approach. Further, since we have shown the local activation of vitamin D by enzymes expressed in the GEC, we hypothesized that we could observe this enhancement with the stable, and inexpensive inactive form of vitamin D, which could be further increased with epigenetic regulators. We treated 3-dimensional primary cultures of GEC topically with the inactive form of vitamin D, in the presence and absence of selected histone deacetylase (HDAC) inhibitors. LL-37 mRNA levels were quantified by quantitative RT-PCR, and inhibition of invasion of bacteria was measured by fluorescence microscopy. Vitamin D treatment led to an induction of LL-37 mRNA levels, as well as an inhibition of pro-inflammatory cytokine secretion. This effect was further enhanced by HDAC inhibitors, most strongly when the HDAC inhibitor, phenyl butyrate (PBA) was combined with Vitamin D3. This was observed both in solution and in a prototype gel formulation using sodium butyrate. Finally, this combination treatment led to an increase in the antimicrobial activity against infection by Porphyromonas gingivalis and Filifactor alocis, bacteria associated with periodontal lesions, as well as herpes simplex virus, which has also been shown to be associated with periodontal lesions. Our results demonstrate that a combination of inactive vitamin D and sodium butyrate could be developed as a safe treatment for periodontal disease.

Human Host Defense Peptide LL-37 Suppresses TNFα-Mediated Matrix Metalloproteinases MMP9 and MMP13 in Human Bronchial Epithelial Cells

Introduction: TNFα-inducible matrix metalloproteinases play a critical role in the process of airway remodeling in respiratory inflammatory disease including asthma. The cationic host defense peptide LL-37 is elevated in the lungs during airway inflammation. However, the impact of LL-37 on TNFα-driven processes is not well understood. Here, we examined the effect of LL-37 on TNFα-mediated responses in human bronchial epithelial cells (HBECs). Methods: We used a slow off-rate modified aptamer-based proteomics approach to define the HBEC proteome altered in response to TNFα. Abundance of selected protein candidates and signaling intermediates was examined using immunoassays, ELISA and Western blots, and mRNA abundance was examined by qRT-PCR. Results: Proteomics analysis revealed that 124 proteins were significantly altered, 12 proteins were enhanced by ≥2-fold compared to unstimulated cells, in response to TNFα. MMP9 was the topmost increased protein in response to TNFα, enhanced by ∼10-fold, and MMP13 was increased by ∼3-fold, compared to unstimulated cells. Furthermore, we demonstrated that LL-37 significantly suppressed TNFα-mediated MMP9 and MMP13 in HBEC. Mechanistic data revealed that TNFα-mediated MMP9 and MMP13 production is controlled by SRC kinase and that LL-37 enhances related upstream negative regulators, namely, phospho-AKT (T308) and TNFα-mediated TNFAIP3 or A20. Conclusions: The findings of this study suggest that LL-37 may play a role in intervening in the process of airway remodeling in chronic inflammatory respiratory disease such as asthma.

Enterobacterales Biofilm-Specific Genes and Antimicrobial and Anti-Inflammatory Biomarkers in the Blood of Patients with Ischemic Heart Disease.

Ischemic heart disease (IHD) is the most prevalent type of cardiovascular disease. The main cause of IHD is atherosclerosis, which is a multifactorial inflammatory disease of blood vessels. Studies show that bacteria might have a significant impact on the pathogenesis of atherosclerosis and plaque rupture. This study aimed to evaluate the complexity of interactions between bacteria and the human body concerning metabolites and bacterial genes in patients with ischemic heart disease. Bacterial 16S rDNA and wcaF, papC, and sdhC genes were detected in whole blood using a real-time PCR methodology. An enzyme-linked immunosorbent assay was used to measure the concentration of the LL-37 protein. An analysis of ARA in blood plasma was performed. Bacterial 16S rDNA was detected in 31% of the study patients, and the genes wcaF and sdhC in 20%. Enterobacterales genes were detected more frequently in patients younger than 65 years than in patients aged 65 years and older (p = 0.018) and in patients with type 2 diabetes (p = 0.048). Concentrations of the human antimicrobial peptide LL-37 and 12S-HETE concentrations were determined to be higher if patients had 16S rDNA and biofilm-specific genes. The results of this study enhance the understanding that Enterobacterales bacteria may participate in the pathogenesis of atherosclerosis and IHD. Bacterial DNA and host metabolites in higher concentrations appear to be detected.

Complexation of fungal extracellular nucleic acids by host LL-37 peptide shapes neutrophil response to Candida albicans biofilm.

Candida albicans remains the predominant cause of fungal infections, where adhered microbial cells form biofilms - densely packed communities. The central feature of C. albicans biofilms is the production of an extracellular matrix (ECM) consisting of polymers and extracellular nucleic acids (eDNA, eRNA), which significantly impedes the infiltration of host cells. Neutrophils, as crucial players in the innate host defense, employ several mechanisms to eradicate the fungal infection, including NETosis, endocytosis, or the release of granules containing, among others, antimicrobial peptides (AMPs). The main representative of these is the positively charged peptide LL-37 formed from an inactive precursor (hCAP18). In addition to its antimicrobial functions, this peptide possesses a propensity to interact with negatively charged molecules, including nucleic acids. Our in vitro studies have demonstrated that LL-37 contacting with C. albicans nucleic acids, isolated from biofilm, are complexed by the peptide and its shorter derivatives, as confirmed by electrophoretic mobility shift assays. We indicated that the generation of the complexes induces discernible alterations in the neutrophil response to fungal nucleic acids compared to the effects of unconjugated molecules. Our analyses involving fluorescence microscopy, flow cytometry, and Western blotting revealed that stimulation of neutrophils with DNA:LL-37 or RNA:LL-37 complexes hamper the activation of pro-apoptotic caspases 3 and 7 and fosters increased activation of anti-apoptotic pathways mediated by the Mcl-1 protein. Furthermore, the formation of complexes elicits a dual effect on neutrophil immune response. Firstly, they facilitate increased nucleic acid uptake, as evidenced by microscopic observations, and enhance the pro-inflammatory response, promoting IL-8 production. Secondly, the complexes detection suppresses the production of reactive oxygen species and attenuates NETosis activation. In conclusion, these findings may imply that the neutrophil immune response shifts toward mobilizing the immune system as a whole, rather than inactivating the pathogen locally. Our findings shed new light on the intricate interplay between the constituents of the C. albicans biofilm and the host's immune response and indicate possible reasons for the elimination of NETosis from the arsenal of the neutrophil response during contact with the fungal biofilm.

Cathelicidin peptide analogues inhibit EV71 infection through blocking viral entry and uncoating.

Given the serious neurological complications and deaths associated with enterovirus 71 (EV71) infection, there is an urgent need to develop effective antivirals against this viral infection. In this study, we demonstrated that two Cathelicidin-derived peptides, LL-18 and FF-18 were more potent against EV71 infection than the parent peptide LL-37, which is the mature and processed form of Cathelicidin. These peptides could directly bind to the EV71 virus particles, but not to coxsackievirus, indicative of their high specificity. The binding of peptides with the virus surface occupied the viral canyon region in a way that could block virus-receptor interactions and inhibit viral uncoating. In addition, these peptide analogues could also relieve the deleterious effect of EV71 infection in vivo. Therefore, Cathelicidin-derived peptides might be excellent candidates for further development of antivirals to treat EV71 infection.

A novel film spray containing curcumin inhibits SARS-CoV-2 and influenza virus infection and enhances mucosal immunity

BackgroundInfection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza virus is still a major worldwide health concern. Plants are a good source of bioactive compounds to be used as preventive measures for both inhibiting the virus binding and enhancing mucosal innate immunity. Curcumin has been shown to possess antiviral activity and modulate innate immunity. Therefore, the purpose of this study was to develop an oro-nasal film spray containing curcumin and determine its antiviral activity against SARS-CoV-2 and influenza virus infection, as well as its effects on mucosal innate immunity and inflammatory cytokines in vitro.MethodsThe antiviral activity of the film spray against SARS-CoV-2, influenza A/H1N1, A/H3N2, and influenza B was assessed in vitro by plaque reduction assay. Cytotoxicity of the film spray to oral keratinocytes and nasal epithelial cells was assessed by MTT assay, and cytotoxicity to Vero and MDCK cells was assessed by an MTS-based cytotoxicity assay. Oral and nasal innate immune markers in response to the film spray were determined by ELISA and by a commercial Milliplex Map Kit, respectively.ResultsOur data show that the film spray containing curcumin can inhibit both SARS-CoV-2 and influenza virus infections while maintaining cell viability. Results obtained among 4 viruses revealed that curcumin film spray demonstrated the highest inhibitory activity against SARS-CoV-2 with the lowest EC50 of 3.15 µg/ml and the highest SI value of 4.62, followed by influenza B (EC50 = 6.32 µg/ml, SI = 2.04), influenza A/H1N1 (EC50 = 7.24 µg/ml, SI = 1.78), and influenza A/H3N2 (EC50 > 12.5 µg/ml, SI < 1.03), respectively. Antimicrobial peptides LL-37 and HD-5, IL-6 and TNF-α produced by oral keratinocytes were significantly induced by the film spray, while hBD2 was significantly reduced.ConclusionFilm spray containing curcumin possesses multiple actions against SARS-CoV-2 infection by inhibiting ACE-2 binding in target cells and enhancing mucosal innate immunity. The film spray can also inhibit influenza virus infection. Therefore, the curcumin film spray may be effective in preventing the viral infection of both SARS-CoV-2 and influenza.

LL-37_Renalexin hybrid peptide exhibits antimicrobial activity at lower MICs than its counterpart single peptides

An alarming global public health and economic peril has been the emergence of antibiotic resistance resulting from clinically relevant bacteria pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species constantly exhibiting intrinsic and extrinsic resistance mechanisms against last-resort antibiotics like gentamycin, ciprofloxacin, tetracycline, colistin, and standard ampicillin prescription in clinical practices. The discovery and applications of antimicrobial peptides (AMPs) with antibacterial properties have been considered and proven as alternative antimicrobial agents to antibiotics. In this study, we have designed, produced, and purified a recombinant novel multifunctional hybrid antimicrobial peptide LL-37_Renalexin for the first time via the application of newly designed flexible GS peptide linker coupled with the use of our previously characterized small metal-binding proteins SmbP and CusF3H+ as carrier proteins that allow for an enhanced bacterial expression, using BL21(DE3) and SHuffle T7(DE3) Escherichia coli strains, and purification of the hybrid peptide via immobilized metal affinity chromatography. The purified tag-free LL-37_Renalexin hybrid peptide exhibited above 85% reduction in bacteria colony-forming units and broad-spectrum antimicrobial effects against Staphylococcus aureus, Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), and Klebsiella pneumoniae bacteria clinical isolates at a lower minimum inhibition concentration level (10–33 μM) as compared to its counterpart single-AMPs LL-37 and Renalexin (50–100 μM).Key points• The hybrid antimicrobial peptide LL-37_Renalexin has been designed using a GS linker.• The peptide was expressed with the carrier proteins SmbP and CusF3H+.• The hybrid peptide shows antibacterial potency against clinical bacterial isolates.

Potential role of salivary vitamin D antimicrobial peptide LL-37 and interleukins in severity of dental caries: an exvivo study

IntroductionVitamin D performs various functions as a hormone by promoting calcium absorption but plays a major role in innate immunity,cell differentiation, cell maturation through its genomic effects via vitamin D receptor. The immune response also plays a major role in tooth surface and supporting structure destruction and playing a major factor in high caries formation. The inflammatory cytokines are released has proinflammatory cytokines and stimulate cells in disease process. Therefore, in the present study we have evaluated the association of salivary vitamin D, LL-37, interleukins 6 and 17A in various levels of severity of dental caries.MethodEthical approval was obtained (NU/CEC/2020/0339), 377 individuals reporting to department of conservative dentistry and endodontics, AB Shetty memorial institute of dental sciences were included based on inclusion criteria. The individuals were further divided into caries free(N = 105) and caries active(N = 272) based on their caries prevalence. The salivary were collected and evaluated for vitamin D, LL-37,IL-17A and IL-6.Results were statistically analysed with SPSS vs 22 (IBM Corp, USA). Normally distributed data were expressed as mean ± SD. Skewed data were expressed as median and interquartile range. To compare (mean) outcome measures between the two groups unpaired independent t-test was applied and for values in median IQR, Mann Whitney U test was used. All statistical analysis for P value were two-sided and significance was set to P ≤ 0.05.ResultsThe study showed that, the salivary vitamin D statistically decreased with increasing severity of caries which showed that vitamin D plays an important role in prevention of caries. Antimicrobial peptide LL-37 was higher in caries free group but was not statistically significant, salivary IL-6 level was higher in caries active group but intergroup comparison did not show significant difference. Salivary IL-17A did not show statistically significant between caries active and caries free group.ConclusionThe salivary levels of vitamin D may play a vital role in prevalence of dental caries and its severity which can be a underlying cause in presence of other etiological factors.

Pseudomonas aeruginosa two-component system CprRS regulates HigBA expression and bacterial cytotoxicity in response to LL-37 stress.

Pseudomonas aeruginosa is a highly pathogenic bacterium known for its ability to sense and coordinate the production of virulence factors in response to host immune responses. However, the regulatory mechanisms underlying this process have remained largely elusive. In this study, we investigate the two-component system CprRS in P. aeruginosa and unveil the crucial role of the sensor protein CprS in sensing the human host defense peptide LL-37, thereby modulating bacterial virulence. We demonstrate that CprS acts as a phosphatase in the presence of LL-37, leading to the phosphorylation and activation of the response regulator CprR. The results prove that CprR directly recognizes a specific sequence within the promoter region of the HigBA toxin-antitoxin system, resulting in enhanced expression of the toxin HigB. Importantly, LL-37-induced HigB expression promotes the production of type III secretion system effectors, leading to reduced expression of proinflammatory cytokines and increased cytotoxicity towards macrophages. Moreover, mutations in cprS or cprR significantly impair bacterial survival in both macrophage and insect infection models. This study uncovers the regulatory mechanism of the CprRS system, enabling P. aeruginosa to detect and respond to human innate immune responses while maintaining a balanced virulence gene expression profile. Additionally, this study provides new evidence and insights into the complex regulatory system of T3SS in P. aeruginosa within the host environment, contributing to a better understanding of host-microbe communication and the development of novel strategies to combat bacterial infections.

Development of a 3D multifunctional collagen scaffold impregnated with peptide LL-37 for vascularised bone tissue regeneration

Bone is a highly dynamic connective tissue that provides structural support, locomotion and acts as a shield for many vital organs from damage. Bone inherits the ability to heal after non-severe injury. In case of severe bone abnormalities due to trauma, infections, genetic disorders and tumors, there is a demand for a scaffold that can enhance bone formation and regenerate the lost bone tissue. In this study, a 3D collagen scaffold (CS) was functionalized and assessed under in vitro and in vivo conditions. For this, a collagen scaffold coated with hydroxyapatite (Ap-CS) was developed and loaded with a peptide LL-37. The physico-chemical characterisation confirmed the hydroxyapatite coating on the outer and inner surfaces of Ap-CS. In vitro studies confirmed that LL-37 loaded Ap-CS promotes osteogenic differentiation of human osteosarcoma cells without showing significant cytotoxicity. The efficacy of the LL-37 loaded Ap-CS for bone regeneration was evaluated at 4 and 12 weeks post-implantation by histopathological and micro-CT analysis in rabbit femur defect model. The implanted LL-37 loaded Ap-CS facilitated the new bone formation at 4 weeks compared with Ap-CS without LL-37. The LL-37 loaded Ap-CS incorporating apatite and peptide LL-37 would be useful as a multifunctional scaffold for bone tissue engineering.

Heterologous expression of the novel dimeric antimicrobial peptide LIG in Pichia pastoris

The antimicrobial peptide (AMP) LI is a fusion product of antimicrobial peptide LL37 produced by human neutrophils and Indolicidin secreted by bovine neutrophils. LI retained the antimicrobial activity of the parental peptides and showed high cell selectivity. In this study, the flexible linker Gly-Ser-Gly (G-S-G) was used to ligate LI into dimeric LIG, and constructed the Pichia pastoris (P. pastoris) expression vector pPIC9K-6×His-3×FLAG-LIG. The total protein expression of P. pastoris GS115 reached the highest level (189.6 mg/L) after 96 h induction with 3 % methanol at the initial pH value of 7.0. Finally, 5.9 mg/L of recombinant LIG (rLIG) was obtained after enterokinase digestion and purification. The rLIG had high antimicrobial activity and low hemolytic activity. Compared with monomer LI, GSG linked dimeric LIG, which had no significant change in antimicrobial activity and had good salt ions stability. In this study, the dimeric antimicrobial peptide LIG was successfully expressed, which provided a new idea for the expression of AMPs in the P. pastoris expression system, and had important significance for the application of AMPs.

Discovery of novel antibacterial agent for the infected wound treatment: all-hydrocarbon stapling optimization of LL-37.

Rationale: Antimicrobial peptide LL-37 has been recognized as a favorable alternative to antibiotics due to its broad antibacterial spectrum, low resistance development and diverse biological activities. However, its high manufactory cost, poor proteolytic stability, and unpredictable cytotoxicity seriously hindered its medical translation. Methods: To push the frontiers of its clinical application, all-hydrocarbon stapling strategy was exploited here for the structural modification of KR-12, the core and minimal fragment of LL-37. Results: Based on a library of KR-12 derivatives that designed and synthesized to be stapled at positions of either i, i+4 or i, i+7, structure to activity relationship was investigated. Among them, KR-12(Q5, D9) with the glutamine and aspartic acid residues stapled displayed increased helical content and positive charge. The reinforced α-helical conformation not only protected it from proteolytic hydrolysis but also improved its antibacterial efficacy via effective membrane perturbation and anti-inflammatory efficacy via compact LPS binding. Besides, the increased positive charge endowed it with an enhanced therapeutic index. On infected wound mouse model, it was demonstrated to eliminate bacteria and promote wound closure and regeneration effectively. Conclusion: Overall, the all-hydrocarbon stapling was proven to lay the foundation for the future development of antibacterial agents. KR-12(Q5, D9) could serve as a lead compound for the clinical treatment of bacterial infections.

Extracellular Traps in Patients Diagnosed With Bacterial Vaginosis, Trichomoniasis, Candidiasis, Noninfectious Vaginitis and Cytolytic Vaginosis.

Vaginal infections are a public health problem associated with serious health complications due to the exacerbated inflammation they generate. Vaginal inflammation may also occur in some noninfectious processes, such as noninfectious vaginitis and cytolytic vaginosis. Immune system cells respond to infections through various mechanisms, such as the formation of extracellular traps (ETs), which are DNA networks associated with effector proteins. Many pathogens induce ETs formation in vitro, as occurs in some natural infections. A recent report indicates that human vaginal infections in vivo generate ETs. Therefore, in this study, we aimed to identify ETs in samples from 40 donors who were diagnosed with infectious (i.e., bacterial vaginosis, candidiasis and trichomoniasis) and noninfectious (i.e., noninfectious vaginitis and cytolytic vaginosis) vaginal inflammation. We were able to observe ETs by identifying the LL-37 peptide, which is associated with DNA networks. In seven vaginal swabs from the control group (formed by 19 donors without vaginal infection symptoms), we detected at least one pathogen per sample and observed ETs; thus, these donors were considered asymptomatic. The remaining 12 donors were confirmed to be healthy, as their exudates did not present any tested pathogens, sign of inflammation or ETs. ETs in vaginal inflammatory processes can worsen inflammation but may also help control infection.

Antimicrobial Activity of Human C-Type Lectin Domain Family 3 Member A (CLEC3A)

C-type lectins (CTLs) are a group of proteins that play a crucial role in immunological functions. With the rise of antibiotic-resistant bacteria, CTLs have emerged as a potential alternative to traditional antibiotics and antimicrobial peptides (AMPs), the latter exhibiting limited application due to their low biostability. In this study, we used viable count assays to investigate the antimicrobial activity of the human C-type Lectin Domain Family 3 Member A (CLEC3A) and its two protein domains, CLEC3A Ex23 and CLEC3A Ex3, against gram-positive and gram-negative bacteria. Additionally, using immunoblot analysis, we assessed the biostability of CLEC3A and its protein domains in bacterial supernatant and murine serum. Our findings demonstrate that CLEC3A, CLEC3A Ex23, and CLEC3A Ex3 possess antimicrobial activity against gram-positive Staphyloccocus aureus and gram-negative Pseudomonas aeruginosa. CLEC3A is more effective against P. aeruginosa than the well-investigated antimicrobial peptide LL-37. Furthermore, CLEC3A and its domains have low sensitivity to bacterial and serum proteases, making them more advantageous for systemic application than most AMPs. In conclusion, our research has demonstrated that CLEC3A is not only a precursor of AMPs but also an antimicrobial protein itself, with favorable characteristics for therapeutic applications.