Human LL-37 Research Articles

Role of Cathelicidins in Atherosclerosis and Associated Cardiovascular Diseases

Cathelicidins (human LL-37 and rat CRAMP) are multifunctional peptides involved in various cardiovascular conditions. This review integrates the recent findings about the functional involvement of LL-37/CRAMP across atherosclerosis, acute coronary syndrome, myocardial infarction, heart failure, diabetic cardiomyopathy, and platelet aggregation/thrombosis. In atherosclerosis, LL-37 interacts with scavenger receptors to modulate lipid metabolism and binds with mitochondrial DNA and lipoproteins. In acute coronary syndrome, LL-37 influences T cell responses and mitigates calcification within atherosclerotic plaques. During myocardial infarction and ischaemia/reperfusion injury, LL-37/CRAMP exhibits dual roles: protecting against myocardial damage through the AKT and ERK1/2 signalling pathways, while exacerbating inflammation via TLR4 and NLRP3 inflammasome activation. In heart failure, LL-37/CRAMP attenuates hypertrophy and fibrosis via NF-κB inhibition and the activation of the IGFR1/PI3K/AKT and TLR9/AMPK pathways. Moreover, in diabetic cardiomyopathy, these peptides alleviate oxidative stress and fibrosis by inhibiting TGFβ/Smad and AMPK/mTOR signalling and provide anti-inflammatory effects by reducing NF-κB nuclear translocation and NLRP3 inflammasome formation. LL-37/CRAMP also modulates platelet aggregation and thrombosis through the FPR2 and GPVI receptors, impacting apoptosis, autophagy, and other critical cellular processes. This comprehensive overview underscores LL-37/CRAMP as a promising therapeutic target in cardiovascular diseases, necessitating further elucidation of its intricate signalling networks and biological effects for clinical translation.

Cathelicidin boosts the antifungal activity of neutrophils and improves prognosis during Aspergillus fumigatus keratitis.

Aspergillus fumigatus (A. fumigatus) is one of the common pathogens of fungal keratitis. Fungal growth and invasion cause excessive inflammation and corneal damage, leading to severe vision loss. Neutrophils are the primary infiltrating cells critical for fungal clearance. Cathelicidin [LL-37 in humans and cathelicidin-related antimicrobial peptide (CRAMP) in mice], a natural antimicrobial peptide, can directly inhibit the growth of many pathogens and regulate immune responses. However, the role of cathelicidin and its effect on neutrophils in A. fumigatus keratitis remain unclear. By establishing A. fumigatus keratitis mouse models, we found that cathelicidin was increased in A. fumigatus keratitis. It could reduce fungal loads, lower clinical scores, and improve corneal transparency. Restriction of CRAMP on fungal proliferation was largely counteracted in CD18-/- mice, in which neutrophils cannot migrate into infected sites. When WT neutrophils were transferred into CD18-/- mice, corneal fungal loads were distinctly reduced, indicating that neutrophils are vital for CRAMP-mediated resistance. Furthermore, cathelicidin promoted neutrophils to phagocytose and degrade conidia both in vitro and in vivo. CXC chemokine receptor 2 (CXCR2) was reported to be a functional receptor of LL-37 on neutrophils. CXCR2 antagonist SB225002 or phospholipase C (PLC) inhibitor U73122 weakened LL-37-induced phagocytosis. Meanwhile, LL-37 induced PLC γ phosphorylation, which was attenuated by SB225002. SB225002 or the autophagy inhibitors Bafilomycin-A1 and 3-Methyladenine weakened LL-37-induced degradation of conidia. Transmission electron microscopy (TEM) observed that LL-37 increased autophagosomes in Aspergillus-infected neutrophils. Consistently, LL-37 elevated autophagy-associated protein expressions (Beclin-1 and LC3-II), but this effect was weakened by SB225002. Collectively, cathelicidin reduces fungal loads and improves the prognosis of A. fumigatus keratitis. Both in vitro and in vivo, cathelicidin promotes neutrophils to phagocytose and degrade conidia. LL-37/CXCR2 activates PLC γ to amplify neutrophils' phagocytosis and induces autophagy to eliminate intracellular conidia.

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.

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.

Antimicrobial Peptide Recognition Motif of the Substrate Binding Protein SapA from Nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with respiratory diseases, including otitis media and exacerbations of chronic obstructive pulmonary disease. NTHi exhibits resistance to killing by host antimicrobial peptides (AMPs) mediated by SapA, the substrate binding protein of the sensitivity to antimicrobial peptides (Sap) transporter. However, the specific mechanisms by which SapA selectively binds various AMPs such as defensins and cathelicidin are unknown. In this study, we report mutational analyses of both defensin AMPs and the SapA binding pocket to define the specificity of AMP recognition. Bactericidal assays revealed that NTHi lacking SapA are more susceptible to human beta defensins and LL-37, while remaining highly resistant to a human alpha defensin. In contrast to homologues, our research underscores the distinct specificity of NTHi SapA, which selectively recognizes and binds to peptides containing the charged-hydrophobic motif PKE and RRY. These findings provide valuable insight into the divergence of SapA among bacterial species and NTHi SapA's ability to selectively interact with specific AMPs to mediate resistance.

Design and In-silico Screening of Short Antimicrobial Peptides (AMPs) as Anti-Tubercular Agents Targeting INHA

Introduction: Tuberculosis has been studied as a potentially serious bacterial infection affecting the lung caused by Mycobacterium tuberculosis. In addition to its severe impact on health, resistance to existing drugs has also been seen as a rising concern in the space of medicinal solutions. Therapeutic peptides have the potential to complement existing drug designs to provide effective outcomes against Mycobacterium tuberculosis-resistant strains. Methods: This study illustrated a computational approach to design and test peptides against NADHdependent enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis. A human antimicrobial peptide LL-37 was used as a template, and a further 6 peptides were designed, and their binding and interactions against NADH-dependent enoyl-acyl carrier protein reductase were examined. Further, toxicity, immunogenicity, and a broad spectrum of physicochemical properties were calculated to evaluate the therapeutic and safety profile of these peptides. Results: These peptides were structurally modelled and docked with the protein to determine their binding poses and affinity. The molecular interaction of LL-37 with protein was treated as a reference to evaluate the effectiveness of designed peptides. Solvent accessible surface area (SASA) and ΔG binding free energy of docked complexes assisted in the ranking of these peptides. Eventually, peptides P1: LLGDFFRKSKEK, P3: LLFGDRFLLKEK and P7: LLGDFFRLLKEK were selected for 100 ns molecular dynamic simulation as they showed predicted dissociation constants of 8.7×10-4 M, 3.3×10-4 M and 1.2×10-4 M, respectively. These peptides showed direct hydrogen bond formation with ILE21 and LYS165, which are critical active site residues of the protein. The structural variation pattern collected from the MD simulation suggested a strong and stable binding of P3 and P1 with the protein with RMSD 4-5 Å with the starting conformation under the non-fluctuating state. These two peptides showed relatively similar binding results compared with the control peptide LL-37. Comprehensive structural analysis was performed for the middle structures of the most populated cluster generated from 100 ns MD simulation trajectory. Conclusion: Later, MMPBSA binding energies of these structures were computed, where the average binding free energies of P1, P3, and P7 peptides were -146.93 kcal/mole, -161.16 kcal/mole, and - 151.44 kcal/mole, respectively. These energies suggested that P3 is strongly bound to the active site of NADH-dependent enoyl-acyl carrier protein reductase. Overall, this study proposed the application of these peptides as a possible therapeutic solution to inhibit the growth of Mycobacterium tuberculosis.

The MprF homolog LysX synthesizes lysyl-diacylglycerol contributing to antibiotic resistance and virulence.

Lysyl-diacylglycerol (Lys-DAG) was identified three decades ago in Mycobacterium phlei, but the biosynthetic pathway and function of this aminoacylated lipid have since remained uncharacterized. Combining genetic methods, mass spectrometry, and biochemical approaches, we show that the multiple peptide resistance factor (MprF) homolog LysX from Corynebacterium pseudotuberculosis and two mycobacterial species is responsible for Lys-DAG synthesis. LysX is conserved in most Actinobacteria and was previously implicated in the synthesis of another modified lipid, lysyl-phosphatidylglycerol (Lys-PG), in Mycobacterium tuberculosis. Although we detected low levels of Lys-PG in the membrane of C. pseudotuberculosis, our data suggest that Lys-PG is not directly synthesized by LysX and may require an additional downstream pathway, which is as yet undefined. Our results show that LysX in C. pseudotuberculosis is a major factor of resistance against a variety of positively charged antibacterial agents, including cationic antimicrobial peptides (e.g., human peptide LL-37 and polymyxin B) and aminoglycosides (e.g., gentamycin and apramycin). Deletion of lysX caused an increase in cellular membrane permeability without dissipation of the membrane potential, suggesting that loss of the protein does not result in mechanical damage to the cell membrane. Furthermore, lysX-deficient cells exhibited an attenuated virulence phenotype in a Galleria mellonella infection model, supporting a role for LysX during infection. Altogether, Lys-DAG represents a novel molecular determinant for antimicrobial resistance and virulence that may be widespread in Actinobacteria and points to a richer landscape than previously realized of lipid components contributing to overall membrane physiology in this important bacterial phylum. IMPORTANCE In the past two decades, tRNA-dependent modification of membrane phosphatidylglycerol has been implicated in altering the biochemical properties of the cell surface, thereby enhancing the antimicrobial resistance and virulence of various bacterial pathogens. Here, we show that in several Actinobacteria, the multifunctional protein LysX attaches lysine to diacylglycerol instead of phosphatidylglycerol. We found that lysyl-diacylglycerol (Lys-DAG) confers high levels of resistance against various cationic antimicrobial peptides and aminoglycosides and also enhances virulence. Our data show that Lys-DAG is a lipid commonly found in important actinobacterial pathogens, including Mycobacterium and Corynebacterium species.

Recombinant Lactococcus lactis Expressing Human LL-37 Prevents Deaths from Viral Infections in Piglets and Chicken.

Novel antibiotic substitutes are increasingly in demand in the animal husbandry industry. An oral recombinant Lactococcus lactis (L. lactis) expressing human LL-37 (oral LL-37) was developed and its safety and antiviral effectiveness in vivo was tested. In addition to impairing liposome integrity, LL-37 polypeptide from recombinant L. lactis could prevent the host cell infection by a variety of viruses, including recombinant SARS, SARS-CoV-2, Ebola virus, and vesicular stomatitis virus G. Subchronic toxicity studies performed on Sprague-Dawley rats showed that no cumulative toxicity was found during short-term intervention. Oral LL-37 treatment after the onset of fever could reduce mortality in piglets infected with porcine reproductive and respiratory syndrome virus. Moreover, body weight gain of piglets receiving treatment was progressively restored, and nucleic acid positive rebound was not undetected after discontinuation. Oral LL-37 consistently increased the lifespan of chickens infected with Newcastle viruses. These findings suggested a potential use of recombinantly modified microorganisms in veterinary medicine.

Pathways of hLL-3717-29 Aggregation Give Insight into the Mechanism of α-Amyloid Formation.

α-amyloids present a novel self-assembly principle that can be utilized to prepare functional biomaterials. Evidence of α-amyloid formation in the active core of the human LL-37 protein (comprising residues 17 to 29) was associated with this peptide's membranolytic property. Though mechanistic pathways of β-amyloid formation are known, such studies are scarce in α-amyloids. Modern computational techniques allow such mechanistic studies in molecular detail. Here, we propose aggregation pathways in hLL-3717-29 through molecular dynamics simulations. We first identified oligomers among peptides based on a distance criterion. The distribution of oligomers was then used to build Markov state models from which pathways were obtained using the framework of transition path theory. We checked the structural stability of the peptides during oligomerization, which is crucial from their functional point of view. We also investigated the key residues that participate in oligomer formation, the interactions between them, and the effect of residue mutations on the binding free energy of the peptides. Our findings suggest that larger oligomers are produced from the association of smaller and intermediate oligomers. The peptides retain their helical structure during aggregation with transient occurrences of 3-10 helix and turns. Hydrophobic interactions are vital in the aggregation of these peptides with Ile24 playing a crucial role. Mutation of this residue to alanine decreases the peptides' binding free energy, resulting in reduced aggregation tendency.

Cathelicidin (LL-37) causes expression of inflammatory factors in coronary artery endothelial cells of Kawasaki disease by activating TLR4-NF-κB-NLRP3 signaling.

Kawasaki disease (KD) is a type of vasculitis with an unidentified etiology. Cathelicidin (LL-37) may be involved in the development of the KD process; therefore, further research to investigate the molecular mechanism of LL-37 involvement in KD is warranted. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, NLRP3, and LL-37 in the sera of healthy subjects, children with KD, and children with pneumonia. Subsequently, human recombinant LL-37 or/and toll-like receptors 4 (TLR4)-specific inhibitor TAK-242 stimulated human coronary artery endothelial cells (HCAECs), CCK-8 was used to detect cell proliferation, flow cytometry to detect apoptosis, transmission electron microscopy to observe cytoskeletal changes, Transwell to measure cell migration ability, ELISA to detect inflammatory factor levels, Western blot analysis to analyze protein levels of toll-like receptors 4 (TLR4) and NF-κB p-65, and quantitative real-time polymerase chain reaction (qRT-PCR) to determine LL-37, NLRP3 mRNA levels. In this study, we found that the level of LL-37 was highly expressed in the serum of children with KD, and after LL-37 stimulation, apoptosis was significantly increased in HCAECs, and the expression levels of TLR4, NLRP3 and inflammatory factors in cells were significantly enhanced. Intervention with the TLR4-specific inhibitor TAK-242 significantly alleviated the LL-37 effects on cellular inflammation, TLR4, NLRP3 promotion effect. Our data suggest that LL-37 induces an inflammatory response in KD coronary endothelial cells via TLR4-NF-κB-NLRP3, providing a potential target for the treatment of KD.

Bactericidal Activity to Escherichia coli: Different Modes of Action of Two 24-Mer Peptides SAAP-148 and OP-145, Both Derived from Human Cathelicidine LL-37.

OP-145 and SAAP-148, two 24-mer antimicrobial peptides derived from human cathelicidin LL-37, exhibit killing efficacy against both Gram-positive and Gram-negative bacteria at comparable peptide concentrations. However, when it comes to the killing activity against Escherichia coli, the extent of membrane permeabilization does not align with the observed bactericidal activity. This is the case in living bacteria as well as in model membranes mimicking the E. coli cytoplasmic membrane (CM). In order to understand the killing activity of both peptides on a molecular basis, here we studied their mode of action, employing a combination of microbiological and biophysical techniques including differential scanning calorimetry (DSC), zeta potential measurements, and spectroscopic analyses. Various membrane dyes were utilized to monitor the impact of the peptides on bacterial and model membranes. Our findings unveiled distinct binding patterns of the peptides to the bacterial surface and differential permeabilization of the E. coli CM, depending on the smooth or rough/deep-rough lipopolysaccharide (LPS) phenotypes of E. coli strains. Interestingly, the antimicrobial activity and membrane depolarization were not significantly different in the different LPS phenotypes investigated, suggesting a general mechanism that is independent of LPS. Although the peptides exhibited limited permeabilization of E. coli membranes, DSC studies conducted on a mixture of synthetic phosphatidylglycerol/phosphatidylethanolamine/cardiolipin, which mimics the CM of Gram-negative bacteria, clearly demonstrated disruption of lipid chain packing. From these experiments, we conclude that depolarization of the CM and alterations in lipid packing plays a crucial role in the peptides' bactericidal activity.

A Synthetic Cyclized Antimicrobial Peptide with Potent Effects against Drug-Resistant Skin Pathogens.

Dermal infections requiring treatment are usually treated with conventional antibiotics, but the rise of bacterial resistance to first-line antibiotics warrants alternative therapeutics. Here, we report that a backbone-cyclized antimicrobial peptide, CD4-PP, designed from the human host defense peptide LL-37, has strong direct antibacterial effects on antibiotic sensitive as well as resistant-type strains and clinical isolates of common skin pathogens in the low (<2) μM range. In addition, it influences innate immunity in keratinocytes, and treatment with CD4-PP is able to clear bacterial infections in infected keratinocytes. Additionally, CD4-PP treatment significantly reduces the wound area in a lawn of keratinocytes infected with MRSA. In conclusion, CD4-PP has the potential to serve as a future drug treating wounds infected with antibiotic-resistant bacteria.

The EGCG and α-Mangosteen Stimulate SHED-IL10 and SHED-LL37 Metabolite Concentration.

Stem cells of human exfoliated deciduous teeth (SHED) metabolites are secreted molecules from SHED, namely cytokines, chemokines, and growth factors. The metabolite can be used in various regenerative therapy based on cell-free immunomodulatory potential effects, like interleukin 10 (IL-10) and LL37. This molecule can stimulate with epigallocatechin gallate (EGCG) and α-mangosteen and has been proven to have anti-inflammatory and antibacterial effects. This study aimed to identify the effect of EGCG and α-mangosteen to SHED metabolite, called SHED-IL10 and SHED-LL37, from six passages to obtain the optimum stimulation and able to use as periodontitis regeneration treatment. The six different passages of SHED were prepared in Dulbecco's modified Eagle medium and added with EGCG 80% (10 μM), EGCG 95% (10 μM), or α-mangosteen (10 μM). After a 24 hours incubation, each passage was measured with the metabolite concentration, SHED-IL10 and SHED-LL37, with human IL-10 and LL37 using enzyme-linked immunosorbent assay. Each different concentration was then analyzed statistically. The addition of EGCG 95% is able to stimulate the SHED-IL10 optimum concentration in passage 1 (p < 0.01). But, in the different conditions, the addition of EGCG 80%, EGCG 95%, and α-mangosteen was able to stimulate the SHED-LL37 optimum concentration in passage 2 (p < 0.001). The addition of EGCG and α-mangosteen can stimulate the SHED-IL10 and SHED-LL37 concentrations. These two metabolites are promising as regenerative therapy through anti-inflammatory and antibacterial properties.

Synergy between Human Peptide LL-37 and Polymyxin B against Planktonic and Biofilm Cells of Escherichia coli and Pseudomonas aeruginosa.

The rise in antimicrobial resistant bacteria is limiting the number of effective treatments for bacterial infections. Escherichia coli and Pseudomonas aeruginosa are two of the pathogens with the highest prevalence of resistance, and with the greatest need for new antimicrobial agents. Combinations of antimicrobial peptides (AMPs) and antibiotics that display synergistic effects have been shown to be an effective strategy in the development of novel therapeutic agents. In this study, we investigated the synergy between the AMP LL-37 and various classes of antibiotics against E. coli and P. aeruginosa strains. Of the six antibiotics tested (ampicillin, tetracycline, ciprofloxacin, gentamicin, aztreonam, and polymyxin B (PMB)), LL-37 displayed the strongest synergy against E. coli MG1655 and P. aeruginosa PAO1 laboratory strains when combined with PMB. Given the strong synergy, the PMB + LL-37 combination was chosen for further examination where it demonstrated synergy against multidrug-resistant and clinical E. coli isolates. Synergy of PMB + LL-37 towards clinical isolates of P. aeruginosa varied and showed synergistic, additive, or indifferent effects. The PMB + LL-37 combination treatment showed significant prevention of biofilm formation as well as eradication of pre-grown E. coli and P. aeruginosa biofilms. Using the Galleria mellonella wax worm model, we showed that the PMB + LL-37 combination treatment retained its antibacterial capacities in vivo. Flow analyses were performed to characterize the mode of action. The results of the present study provide proof of principle for the synergistic response between LL-37 and PMB and give novel insights into a promising new antimicrobial combination against gram-negative planktonic and biofilm cells.

Transforming Cross-Linked Cyclic Dimers of KR-12 into Stable and Potent Antimicrobial Drug Leads.

Is it possible to enhance structural stability and biological activity of KR-12, a truncated antimicrobial peptide derived from the human host defense peptide LL-37? Based on the mapping of essential residues in KR-12, we have designed backbone-cyclized dimers, cross-linked via a disulfide bond to improve peptide stability, while at the same time improving on-target activity. Circular dichroism showed that each of the dimers adopts a primarily alpha-helical conformation (55% helical content) when bound to lyso-phosphatidylglycerol micelles, indicating that the helical propensity of the parent peptide is maintained in the new cross-linked cyclic form. Compared to KR-12, one of the cross-linked dimers showed 16-fold more potent antimicrobial activity against human pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans and 8-fold increased activity against Escherichia coli. Furthermore, these peptides retained antimicrobial activity at physiologically relevant conditions, including in the presence of salts and in human serum, and with selective Gram-negative antibacterial activity in rich growth media. In addition to giving further insight into the structure-activity relationship of KR-12, the current work demonstrates that by combining peptide stabilization strategies (dimerization, backbone cyclization, and cross-linking via a disulfide bond), KR-12 can be engineered into a potent antimicrobial peptide drug lead with potential utility in a therapeutic context.

The Human Host Defense Peptide LL-37 Overexpressed in Lung Cell Lines by Methanolic Extract of Valeriana officinalis

The Human Host Defense Peptide LL-37 Overexpressed in Lung Cell Lines by Methanolic Extract of Valeriana officinalis

Peptide Stability Is Important but Not a General Requirement for Antimicrobial and Antibiofilm Activity In Vitro and In Vivo.

Peptide stability to proteases has been a major requirement for developing peptide therapeutics. This study investigates the effects of peptide stability on antimicrobial and antibiofilm activity under various conditions. For this purpose, two human cathelicidin-derived peptides differing in stability to proteases were utilized. While GF-17, a peptide derived from the major antimicrobial region of human LL-37, can be rapidly cleaved by proteases, the engineered peptide 17BIPHE2 is resistant to multiple proteases. In the standard antimicrobial susceptibility, killing kinetics, and membrane permeabilization assays conducted in vitro using planktonic bacteria, these two peptides displayed similar potency. The two peptides were also similarly active against methicillin-resistant Staphylococcus aureus (MRSA) USA300 prior to biofilm formation. However, 17BIPHE2 was superior to GF-17 in disrupting preformed biofilms probably due to both enhanced stability and slightly higher DNA binding capacity. In a wax moth model, 17BIPHE2 better protected insects from MRSA infection-caused death than GF-17, consistent with the slower degradation of 17BIPHE2 than GF-17. Here, peptide antimicrobial activity was found to be critical for in vivo efficacy. When incorporated in the nanofiber/microneedle delivery device, GF-17 and 17BIPHE2 displayed a similar effect in eliminating MRSA in murine chronic wounds, underscoring the advantage of nanofibers in protecting the peptide from degradation. Since nanoformulation can ease the requirement of peptide stability, it opens the door to a direct use of natural peptides or their cocktails for antimicrobial treatment, accelerating the search of effective antibiofilm peptides to treat chronic wounds.

Structural Basis of Peptide-Based Antimicrobial Inhibition of a Resistance-Nodulation-Cell Division Multidrug Efflux Pump.

ABSTRACTBacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters.IMPORTANCE RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a cationic antibiotic-sensitizing peptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.

Metal-Ion Interactions with Dodecapeptide Fragments of Human Cationic Antimicrobial Protein LL-37 [hCAP(134-170)

Isothermal titration calorimetry, circular dichroism (CD) techniques, and in silico analysis were used to determine potential metal binding sites in human cationic antimicrobial protein (hCAP) corresponding to overlapping the dodecapeptide sequences of hCAP(134–170) referred to as LL-37. The correct antibacterial action of LL-37 is closely related to its established unique structure. Disturbances in the LL-37 structure (e.g., unwanted presence of metal ions) lead to a radical change in its biological functions. Five fragments of the LL-37 [hCAP(134–170)], namely, hCAP(134–145) (A1), hCAP(140–151) (A2), hCAP(146–157) (A3), hCAP(152–163) (A4), and hCAP(159–170) (A5), were taken into account and their affinity to Mn(II) and Zn(II) ions was rigorously assessed. We prove that only three of the investigated peptides (A1, A2, and A5) are capable of forming thermodynamically stable complexes with metal ions. Additionally, based on density functional theory (DFT) calculations, we propose the most likely coordination modes of metal(II) to peptides as well as discuss the chemical nature of the interactions. Finally, we present the structural features of the strongest binding peptide, hCAP(159–170), responsible for the metal binding. The presented results provide important structural and thermodynamic information to understand the influence of some metal ions on the activity of hCAP(134–170).