RESUMO
α-Synuclein (a-syn) oligomers and fibrils are behind neurodegeneration in Parkinson's disease (PD), but therapeutically targeting them is challenging. Amphipathic and cationic helical peptides inhibit amyloid formation and suppress neurotoxicity by selectively binding the solvent-accessible regions in these toxic species. Can endogenous peptides, like LL-37, constitute a new therapeutic paradigm in PD?
Assuntos
Doença de Parkinson , Amiloide , Humanos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismoRESUMO
LL37 alone and in complex with self-DNA triggers inflammatory responses in myeloid cells and plays a crucial role in the development of systemic autoimmune diseases, like psoriasis and systemic lupus erythematosus. We demonstrated that LL37/self-DNA complexes induce long-term metabolic and epigenetic changes in monocytes, enhancing their responsiveness to subsequent stimuli. Monocytes trained with LL37/self-DNA complexes and those derived from psoriatic patients exhibited heightened glycolytic and oxidative phosphorylation rates, elevated release of proinflammatory cytokines, and affected naïve CD4+ T cells. Additionally, KDM6A/B, a demethylase of lysine 27 on histone 3, was upregulated in psoriatic monocytes and monocytes treated with LL37/self-DNA complexes. Inhibition of KDM6A/B reversed the trained immune phenotype by reducing proinflammatory cytokine production, metabolic activity, and the induction of IL-17-producing T cells by LL37/self-DNA-treated monocytes. Our findings highlight the role of LL37/self-DNA-induced innate immune memory in psoriasis pathogenesis, uncovering its impact on monocyte and T cell dynamics.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Catelicidinas , DNA , Monócitos , Psoríase , Humanos , Monócitos/imunologia , Monócitos/metabolismo , Psoríase/imunologia , DNA/imunologia , DNA/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Histona Desmetilases/metabolismo , Histona Desmetilases/genética , Linfócitos T CD4-Positivos/imunologia , Reprogramação Celular/imunologia , Citocinas/metabolismo , Citocinas/imunologia , Imunidade Inata , Masculino , Epigênese Genética , Feminino , Memória Imunológica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Interleucina-17/metabolismo , Interleucina-17/imunologia , Células CultivadasRESUMO
The intracellular Ca2+ concentration is mainly controlled by Ca2+ channels. These channels form complexes with K+ channels, which function to amplify Ca2+ flux. In cancer cells, voltage-gated/voltage-dependent Ca2+ channels and non-voltage-gated/voltage-independent Ca2+ channels have been reported to interact with K+ channels such as Ca2+-activated K+ channels and voltage-gated K+ channels. These channels are activated by an increase in cytosolic Ca2+ concentration or by membrane depolarization, which induces membrane hyperpolarization, increasing the driving force for Ca2+ flux. These complexes, composed of K+ and Ca2+ channels, are regulated by several molecules including lipids (ether lipids and cholesterol), proteins (e.g. STIM), receptors (e.g. S1R/SIGMAR1), and peptides (e.g. LL-37) and can be targeted by monoclonal antibodies, making them novel targets for cancer research.
Assuntos
Neoplasias , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Humanos , Lipídeos , Neoplasias/tratamento farmacológico , Potássio/metabolismo , Canais de Potássio/metabolismoRESUMO
Rosacea, a prevalent chronic facial inflammatory condition, afflicts millions worldwide. Its multifaceted pathogenesis poses challenges for effective treatment. Tranilast (TR), an analog of a tryptophan metabolite, has demonstrated anti-inflammatory and anti-fibrotic properties across various diseases. Yet, its potential in rosacea treatment remains understudied. Here, we induced rosacea-like symptoms in mice via prolonged LL-37 injections and administered TR intervention. Our findings reveal that TR mitigated skin lesions, reduced skin thickness, and suppressed inflammatory cell infiltration within the dermis of LL-37 mice. Notably, TR downregulated the expression of rosacea-associated inflammatory cytokines (TNF-α, IL-6, IL-1ß, and IL-18) and the antimicrobial peptide CAMP, while also inhibiting NLRP3 inflammasome activation and the TLR4 signaling pathway. Furthermore, TR attenuated LL-37-induced fibrosis and hindered the transforming growth factor-ß1 (TGF-ß1)/Smad2/3 pathway. In summary, our study underscores TR's therapeutic potential in rosacea by mitigating both skin inflammation and fibrosis, thereby offering a promising treatment avenue for this condition.
RESUMO
The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3-4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 µM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Catelicidinas , Osteoblastos , Vitamina D , Humanos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Vitamina D/farmacologia , Vitamina D/metabolismo , Vitamina D/análogos & derivados , Células THP-1 , Complexo de Endopeptidases do Proteassoma/metabolismo , Sobrevivência Celular/efeitos dos fármacosRESUMO
Tumor immunotherapies targeting PD-(L)1 exhibit anti-tumor efficacy in only 10-30% of patients with various cancers. Literature has demonstrated that a "hot tumor" which contains high T lymphocytes in the tumor microenvironment exhibits a better response to immunotherapies than a "cold tumor." This study aimed to investigate whether tumor-intrinsic IFNα and CXCL10 determine the recruitment and activation of CD8+ T cells to become "hot tumor." In this study, we found that CXCL10 overexpressed in a variety of tumors including lung, colon, and liver tumors with a correlation with PD-L1. High PD-L1 and CXCL10 are associated with better survival rates in tumor patients receiving immunotherapies. IFNs-downstream transcriptional factor IRF-1 and STAT1 were correlated with PD-L1 and CXCL10 expression. We demonstrated that IRF-1 and STAT1 were both bound with the promoters of PD-L1 and CXCL10, sharing the same signaling pathway and determining IFNs-mediated PD-L1 and CXCL10 expression. In addition, IFNα significantly increased activation marker IFNγ in PBMCs, promoting M1 type monocyte differentiation, CD4+ T, and CD8+ T cell activation. Particularly, we found that CD8+ T lymphocytes abundantly expressed CXCR3, a receptor of CXCL10, by flow cytometry, indicating that tumor-intrinsic CXCL10 potentially recruited CD8+ T in tumor microenvironment. To demonstrate the hypothesis, immunotherapy-sensitive CT26 and immunotherapy-resistant LL/2 were used and we found that CT26 cells exhibited higher IFNα, IFNγ, CXCL10, and PD-L1 levels compared to LL/2, leading to higher IFNγ expression in mouse splenocytes. Moreover, we found that CD8+ T cells were recruited by CXCL10 in vitro, whereas SCH546738, an inhibitor of CXCR3, inhibited T cell migration and splenocytes-mediated anti-tumor effect. We then confirmed that CT26-derived tumor was sensitive to αPD-L1 immunotherapy and LL/2-tumor was resistant, whereas αPD-L1 significantly increased T lymphocyte activation marker CD107a in CT26-derived BALB/c mice. In conclusion, this study revealed that CXCL10 expression is correlated with PD-L1 in tumors, sharing the same signaling pathway and associating with better immunotherapeutic efficacy. Further evidence in the syngeneic tumor models demonstrated that immunotherapy-sensitive CT26 intrinsically exhibited higher IFNα and CXCL10 compared to immunotherapy-resistant LL/2 to recruit and activate CD8+ T cells in the tumor microenvironment, exhibiting "hot tumor" characteristic of sensitizing αPD-L1 immunotherapies.
Assuntos
Quimiocina CXCL10 , Imunoterapia , Interferon-alfa , Microambiente Tumoral , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/imunologia , Microambiente Tumoral/imunologia , Animais , Camundongos , Humanos , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Ativação Linfocitária/imunologia , Linhagem Celular Tumoral , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Feminino , Fator de Transcrição STAT1/metabolismoRESUMO
Photocatalytic nanoparticles offer antimicrobial effects under illumination due to the formation of reactive oxygen species (ROS), capable of degrading bacterial membranes. ROS may, however, also degrade human cell membranes and trigger toxicity. Since antimicrobial peptides (AMPs) may display excellent selectivity between human cells and bacteria, these may offer opportunities to effectively "target" nanoparticles to bacterial membranes for increased selectivity. Investigating this, photocatalytic TiO2 nanoparticles (NPs) are coated with the AMP LL-37, and ROS generation is found by C11-BODIPY to be essentially unaffected after AMP coating. Furthermore, peptide-coated TiO2 NPs retain their positive ζ-potential also after 1-2 h of UV illumination, showing peptide degradation to be sufficiently limited to allow peptide-mediated targeting. In line with this, quartz crystal microbalance measurements show peptide coating to promote membrane binding of TiO2 NPs, particularly so for bacteria-like anionic and cholesterol-void membranes. As a result, membrane degradation during illumination is strongly promoted for such membranes, but not so for mammalian-like membranes. The mechanisms of these effects are elucidated by neutron reflectometry. Analogously, LL-37 coating promoted membrane rupture by TiO2 NPs for Gram-negative and Gram-positive bacteria, but not for human monocytes. These findings demonstrate that AMP coating may selectively boost the antimicrobial effects of photocatalytic NPs.
Assuntos
Membrana Celular , Nanopartículas , Titânio , Titânio/química , Titânio/farmacologia , Humanos , Catálise , Nanopartículas/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Técnicas de Microbalança de Cristal de QuartzoRESUMO
The autoantigens LL37 and ADAMTSL5 contribute to induce pathogenetic T-cells responses in a subset of psoriatic patients. Whether the presence of LL37-and/or ADAMTS5-reactive T-cells influences the clinical response to treatment is still unknown. The aim of the study is to evaluate the clinical responses to the anti-IL-23 risankizumab in LL37 and/or ADAMTSL5-reactive patients in comparison with non-reactive ones and to assess whether genetics (HLA-Cw06.02) or BMI influences the response to treatment. Patients were screened at baseline for the presence of circulating LL37 or/and ADAMTSL5-reactive T-cells and were treated as per protocol with risankizumab. Effectiveness data (PASI scores) were collected at weeks 4, 16, 28, 40 and 52. Data were also analyzed based on HLA-Cw06.02 status and BMI. The overall response to treatment of patients with autoreactivity to LL37 or ADAMTSL5 did not differ compared to the non-reactive cohort as measured as PASI75/90/100 at different time points; however, subjects that had autoreactive T-cells to both LL37 and ADAMTS5 demonstrated suboptimal response to treatment starting at week16. HLA-Cw06:02+ patients demonstrated faster response to risankizumab at week 4 compared to HLA-Cw06:02-. Additionally, the response to treatment was influenced by the BMI with slower responses seen in overweight and obese patients at week 4 and week16. In conclusion, while the presence of either LL37-and ADAMTS5-reactive circulating T-cells do not influence the clinical response to risankizumab, the presence of the double reactivity to both LL37 and ADAMTS5 decreases the clinical responses. Moreover, we evidenced that HLA-Cw06+ respond faster to IL-23 inhibition and that BMI, associated to autoreactivity, can influence the speed in response.
Assuntos
Psoríase , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Resultado do Tratamento , Psoríase/tratamento farmacológico , Psoríase/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Autoantígenos/imunologia , Proteína ADAMTS5/metabolismo , Anticorpos Monoclonais/uso terapêutico , Interleucina-23 , Índice de Massa Corporal , Autoimunidade , Proteínas ADAMTS , Antígenos HLA-CRESUMO
Innate immunity is vital for animal homeostasis and survival. First-line immuno-defense for fish larvae involves mucus enriched with leukolectin (LL) secreted by dermal lectocytes. Later during the critical transition from yolk-nutrition to feeding, additional larval immuno-protection in zebrafish (zF) is provided by macrophages containing LL (lectophages). This work investigated new LL-expression in embryos and in blood, structures of fish leukocytic LL and LL-genes, and LL-presence in chicken leukocytes. In zF-embryos, lectophages appear â¼10 hpf, while later, cells co-expressing myeloperoxidase- and LL-mRNA were detected (â¼19 hpf). Furthermore, protein-extracts of Atlantic salmon (Ssal) leukocytes contained LL-proteins, compartmentalized in the cytosol. Cloning and sequencing revealed 94 % nt-sequence identity between variants of Ssal-leukolectins. Highly conserved LLs allowed production of epitope-specific anti-LL IgGs. Immuno-fluorescence-analysis demonstrated that most Ssal-bloodcells were LL-negative, but both some large cells with protrusions and some small, rounded cells did express LL. Immunoperoxidase-staining method confirmed LL-expression in some Ssal-leukocytes, identified as macrophages, PMN-leukocytes, thrombocytes and dendritic cells. However, closer examination revealed a dichotomy of these cell-categories into either LL-positive, or LL-negative variants. In situ hybridization demonstrated profuse LL-expression in Ssal head kidney interstitial tissue, while LL-transcripts were absent in large kidney tubules. Both hematopoietic (non-pigmented) marrow cells and melano-macrophages expressed LL-mRNA, implying that leukolectins provide lifelong innate immuno-protection. PCR-amplification using Ssal-leukocytic DNA as template, and direct sequencing yielded a leukocytic ll-gene. Some cells in salmon, cod, halibut, oikopleura and zebrafish embryos express LL-proteins and/or LL-mRNA, and LL-mRNA is detected in salmon, cod and chicken leukocytes. However, current genomes for these species lack recognizable LL-loci except the Ssal_v3.1 Genome-assembly. The data demonstrate an unexpected dichotomy of some leukocyte lineages into LL-positive or LL-negative cell-variants. Such dichotomies suggest exploring differential impacts from the duplicated leukocyte-lineages in health and disease.
Assuntos
Imunidade Inata , Leucócitos , Salmo salar , Peixe-Zebra , Animais , Imunidade Inata/genética , Peixe-Zebra/imunologia , Peixe-Zebra/genética , Leucócitos/imunologia , Salmo salar/imunologia , Salmo salar/genética , Galinhas/imunologia , Galinhas/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologiaRESUMO
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.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Catelicidinas/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Bactérias , Staphylococcus aureus , Escherichia coli/genética , Proteínas de Transporte/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
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.
Assuntos
Anti-Infecciosos , Legionella , Legionellaceae , Humanos , Fosfolipídeos , Peptídeos Catiônicos Antimicrobianos/farmacologia , ColinaRESUMO
Antimicrobial peptide LL37 is a promising antibacterial candidate due to its potent antimicrobial activity with no known bacterial resistance. However, intrinsically LL37 is susceptible to degradation in wound fluids limits its effectiveness. Bacterial toxins which are released after cell lysis are found to hinder wound healing. To address these challenges, encapsulating LL37 in microspheres (MS) and loading the MS onto activated carbon (AC)-chitosan (CS) hydrogel. This advanced wound dressing not only protects LL37 from degradation but also targets bacterial toxins, aiding in the healing of chronic wound infections. First, LL37 MS and LL37-AC-CS hydrogel were prepared and characterised in terms of physicochemical properties, drug release, and peptide-polymer compatibility. Antibacterial and antibiofilm activity, bacterial toxin elimination, cell migration, and cell cytotoxicity activities were investigated. LL37-AC-CS hydrogel was effective against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. LL37-AC-CS hydrogel bound more endotoxin than AC with CS hydrogel alone. The hydrogel also induced cell migration after 72 h and showed no cytotoxicity towards NHDF after 72 h of treatment. In conclusion, the LL37-AC-CS hydrogel was shown to be a stable, non-toxic advanced wound dressing method with enhanced antimicrobial and antitoxin activity, and it can potentially be applied to chronic wound infections to accelerate wound healing.
Assuntos
Antibacterianos , Bandagens , Quitosana , Escherichia coli , Hidrogéis , Microesferas , Pseudomonas aeruginosa , Staphylococcus aureus , Quitosana/química , Antibacterianos/farmacologia , Antibacterianos/administração & dosagem , Antibacterianos/química , Hidrogéis/química , Hidrogéis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Humanos , Pseudomonas aeruginosa/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/prevenção & controle , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Catelicidinas , Testes de Sensibilidade Microbiana/métodos , Toxinas Bacterianas , Liberação Controlada de Fármacos , Movimento Celular/efeitos dos fármacos , Carbono/química , Biofilmes/efeitos dos fármacosRESUMO
Trafficking of M-protein (Mprt) from the cytosol of Group A Streptococcus pyogenes (GAS) occurs via Sec translocase membrane channels that associate with Sortase A (SrtA), an enzyme that catalyzes cleavage of Mprt at the proximal C-terminal [-LPST355∗GEAA-] motif and subsequent transpeptidation of the Mprt-containing product to the cell wall (CW). These steps facilitate stable exposure of the N-terminus of Mprt to the extracellular milieu where it interacts with ligands. Previously, we found that inactivation of SrtA in GAS cells eliminated Mprt CW transpeptidation but effected little reduction in its cell surface exposure, indicating that the C-terminus of Mprt retained in the cytoplasmic membrane (CM) extends its N-terminus to the cell surface. Herein, we assessed the effects of mutating the Thr355 residue in the WT SrtA consensus sequence (LPST355∗GEAA-) in a specific Mprt, PAM. In vitro, we found that synthetic peptides with mutations (LPSX355GEAA) in the SrtA cleavage site displayed slower cleavage activities with rSrtA than the WT peptide. Aromatic residues at X had the lowest activities. Nonetheless, PAM/[Y355G] still transpeptidated the CW in vivo. However, when using isolated CMs from srtA-inactivated GAS cells, rapid cleavage of PAM/[LPSY355GEAA] occurred at E357∗ but transpeptidation did not take place. These results show that another CM-resident enzyme nonproductively cleaved PAM/[LPSYGE357∗AA]. However, SrtA associated with the translocon channel in vivo cleaved and transpeptidated PAM/[LPSX355∗GEAA] variants. These CM features allow diverse cleavage site variants to covalently attach to the CW despite the presence of other potent nonproductive CM proteases.
Assuntos
Aminoaciltransferases , Proteínas de Bactérias , Parede Celular , Streptococcus pyogenes , Aminoaciltransferases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Evolução Biológica , Parede Celular/metabolismo , Cisteína Endopeptidases , Mutação , Streptococcus pyogenes/classificação , Streptococcus pyogenes/enzimologiaRESUMO
Clostridioides difficile is a leading cause of antibiotic-associated diarrheal disease. C. difficile colonization, growth, and toxin production in the intestine is strongly associated with its ability to use amino acids to generate energy, but little is known about the impact of specific amino acids on C. difficile pathogenesis. The amino acid glycine is enriched in the dysbiotic gut and is suspected to contribute to C. difficile infection. We hypothesized that the use of glycine as an energy source contributes to colonization of the intestine and pathogenesis of C. difficile. To test this hypothesis, we deleted the glycine reductase (GR) genes grdAB, rendering C. difficile unable to ferment glycine, and investigated the impact on growth and pathogenesis. Our data show that the grd pathway promotes growth, toxin production, and sporulation. Glycine fermentation also had a significant impact on toxin production and pathogenesis of C. difficile in the hamster model of disease. Furthermore, we determined that the grd locus is regulated by host cathelicidin (LL-37) and the cathelicidin-responsive regulator, ClnR, indicating that the host peptide signals to control glycine catabolism. The induction of glycine fermentation by LL-37 demonstrates a direct link between the host immune response and the bacterial reactions of toxin production and spore formation.
Assuntos
Clostridioides difficile , Cricetinae , Animais , Clostridioides difficile/metabolismo , Catelicidinas , Fermentação , Virulência , Aminoácidos/metabolismo , Glicina/metabolismo , Proteínas de Bactérias/genética , Esporos/metabolismoRESUMO
Rosacea is a chronic inflammatory skin disease whose late manifestations have not yet been clearly reported in animal models. The objective of this study is to describe the skin lesions and major histopathological changes in a rosacea-like phenotype in mice induced by prolonged LL-37 administration and furthermore, to assess the potential of long-term LL-37 administration in inducing irreversible rosacea-like skin lesion models. Balb/c mice were continuously injected intradermally with LL-37 every 12 h to induce a rosacea-like phenotype. After LL-37 injections were administered for 20 consecutive days, the area of rosacea-like lesions gradually expanded in the first 13 days, then entered a stable phase. Haematoxylin and eosin (H&E) and Van Gieson's staining showed a high degree of inflammatory cell aggregation, thickening of the epidermis and dermis, and collagen deposition in large quantities. The results of immunofluorescence staining and Western blotting showed that the expression of α-SMA, TNF-α, vimentin, and COL1 in the skin of mice was significantly upregulated. Short-term LL-37 administration induced rosacea-like lesions that only featured the aggregation of inflammatory factors and thickening of the epidermis, whereas no collagen hyperplasia was observed, and a full recovery was noticed. However, rosacea-like skin lesions induced by long-term LL-37 administration did not completely recover. Our study compares rosacea-like lesions induced by short-term versus long-term LL-37 administration, and the results suggest that irreversible rosacea-like lesions can be induced by long-term LL-37 administration.
RESUMO
Oligomerization of antimicrobial peptides (AMPs) is critical in their effects on pathogens. LL-37 and its truncated fragments are widely investigated regarding their structures, antimicrobial activities, and application, such as developing new antibiotics. Due to the small size and weak intermolecular interactions of LL-37 fragments, it is still elusive to establish the relationship between oligomeric states and antimicrobial activities. Here, an α-hemolysin nanopore, mass spectrometry (MS), and molecular dynamic (MD) simulations are used to characterize the oligomeric states of two LL-37 fragments. Nanopore studies provide evidence of trapping events related to the oligomer formation and provide further details on their stabilities, which are confirmed by MS and MD simulations. Furthermore, simulation results reveal the molecular basis of oligomer dynamics and states of LL-37 fragments. This work provides unique insights into the relationship between the oligomer dynamics of AMPs and their antimicrobial activities at the single-molecule level. The study demonstrates how integrating methods allows deciphering single molecule level understanding from nanopore sensing approaches.
Assuntos
Anti-Infecciosos , Nanoporos , Proteínas Hemolisinas/química , Simulação de Dinâmica MolecularRESUMO
We have previously demonstrated spermicidal activity of LL-37 antimicrobial peptide on mouse/human sperm and its contraceptive effects in female mice. With its microbicidal action against Neisseria gonorrhoeae, LL-37 warrants development into a multipurpose prevention technology (MPT) agent for administering into the female reproductive tract (FRT). However, it is important to verify that multiple administrations of LL-37 do not lead to damage of FRT tissues and/or irreversible loss of fecundity. Herein, we transcervically injected LL-37 (36 µM-10× spermicidal dose) into female mice in estrus in three consecutive estrous cycles. A set of mice were sacrificed for histological assessment of the vagina/cervix/uterus 24 h after the last injection, while the second set were artificially inseminated with sperm from fertile males 1 week afterwards, and then monitored for pregnancy. Mice injected with PBS in parallel were regarded as negative controls, whereas those injected with vaginal contraceptive foam (VCF, available over the counter), containing 12.5% nonoxynol-9, served as positive controls for vaginal epithelium disruption. We demonstrated that the vagina/cervix/uterus remained normal in both LL-37-injected and PBS-injected mice, which also showed 100% resumption of fecundity. In contrast, VCF-injected mice showed histological abnormalities in the vagina/cervix/uterus and only 50% of them resumed fecundity. Similarly, LL-37 multiply administered intravaginally caused no damage to FRT tissues. While our results indicate the safety of multiple treatments of LL-37 in the mouse model, similar studies have to be conducted in non-human primates and then humans. Regardless, our study provides an experimental model for studying in vivo safety of other vaginal MPT/spermicide candidates.
Assuntos
Peptídeos Antimicrobianos , Espermicidas , Gravidez , Masculino , Feminino , Humanos , Camundongos , Animais , Sêmen , Espermicidas/farmacologia , Nonoxinol/farmacologia , EspermatozoidesRESUMO
Recombinant LL-37 Lactococcus lactis (Oral LL-37) was designed to prevent progression of COVID-19 by targeting virus envelope, however, effectiveness and safety of Oral LL-37 in clinical application was unclear. A total of 238 adult inpatients, open-labelled, randomized, placebo-controlled, single-center study was conducted to investigate the primary end points, including negative conversion time (NCT) of SARS-CoV-2 RNA and adverse events (AEs). As early as intervened on 6th day of case confirmed, Oral LL-37 could significantly shorten NCT (LL-37 9.80 ± 2.67 vs. placebo 14.04 ± 5.89, p < 0.01). For Oral LL-37, as early as treated in 6 days, the adjusted hazard ratio (HR) for a primary event of nucleic acid negative outcome was 6.27-fold higher than 7-day-later (HR: 6.276, 95% confidence interval [CI]: 3.631-10.848, p < 0.0001), and the adjusted HR of Oral LL-37 within 6 days is higher than placebo (HR: 2.427 95% CI: 1.239-4.751, p = 0.0097). No severe AEs were observed during hospitalization and follow-up investigation. This study shows that early intervention of Oral LL-37 incredibly reduces NCT implying a potential for clearance of Omicron BA.5.1.3 without evident safety concerns.
Assuntos
COVID-19 , SARS-CoV-2 , Adulto , Humanos , COVID-19/prevenção & controle , RNA Viral , Hospitalização , Pacientes InternadosRESUMO
Human papillomavirus (HPV) type 16 is the most common sexually transmitted virus related to cervical cancer. Among different types of advanced novel therapies, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-mediated gene editing holds great promise for cancer treatment. In this research, optimal gRNA sequences targeting HPV16 E5, E6, E7, and p97 promoter for CRISPR/Cas9-mediated genome editing were designed by in silico prediction. After cloning, delivery of the recombinant vectors into C3, TC1 and HeLa tumor cells was evaluated by Lipofectamine 2000, and LL-37 antimicrobial peptide. Then, the levels of cell cycle proteins (p21, p53, and Rb) were investigated after treatment by western blot analysis. Finally, C57BL/6 mice were inoculated with C3 tumor cells, and treated with recombinant vectors and cisplatin. Based on the tumor size reduction and IHC results, the E6 + E7-treated group with a high percentage of cleaved caspase-3 positive cells (45.75%) and low mitotic index of 2-3 was determined as the best treatment among other groups. Moreover, the potential of LL-37 peptide to overcome the CRISPR/Cas9 delivery challenge was shown for the first time. Overall, our study suggests that the CRISPR/Cas9-mediated gene editing of pre-existing tumors is effective, specific and nontoxic, and the outlook for precise gene therapy in cancer patients is very bright.
Assuntos
Sistemas CRISPR-Cas , Papillomavirus Humano 16 , Camundongos , Animais , Humanos , Papillomavirus Humano 16/genética , Peptídeos Antimicrobianos , Camundongos Endogâmicos C57BL , OncogenesRESUMO
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.