RESUMO
Rattusin, an α-defensin-related antimicrobial peptide isolated from the small intestine of rats, has been previously characterized through NMR spectroscopy to elucidate its three-dimensional structure, revealing a C2 homodimeric scaffold stabilized by five disulfide bonds. This study aimed to identify the functional region of rattusin by designing and synthesizing various short analogs, subsequently leading to the development of novel peptide-based antibiotics. The analogs, designated as F1, F2, F3, and F4, were constructed based on the three-dimensional configuration of rattusin, among which F2 is the shortest peptide and exhibited superior antimicrobial efficacy compared to the wild-type peptide. The central cysteine residue of F2 prompted an investigation into its potential to form a dimer at neutral pH, which is critical for its antimicrobial function. This activity was abolished upon the substitution of the cysteine residue with serine, indicating the necessity of dimerization for antimicrobial action. Further, we synthesized ß-hairpin-like analogs, both parallel and antiparallel, based on the dimeric structure of F2, which maintained comparable antimicrobial potency. In contrast to rattusin, which acts by disrupting bacterial membranes, the F2 dimer binds directly to DNA, as evidenced by fluorescence assays and DNA retardation experiments. Importantly, F2 exhibited negligible cytotoxicity up to 515 µg/mL, assessed via hemolysis and MTT assays, underscoring its potential as a lead compound for novel peptide-based antibiotic development.
Assuntos
alfa-Defensinas , Animais , alfa-Defensinas/química , alfa-Defensinas/farmacologia , alfa-Defensinas/síntese química , Testes de Sensibilidade Microbiana , Ratos , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/síntese química , Multimerização Proteica/efeitos dos fármacos , DNA/metabolismo , DNA/química , Hemólise/efeitos dos fármacos , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Sequência de AminoácidosRESUMO
The antibacterial and antiviral functions of human defensin 5 lay the foundation for its role as a core host protective component. In addition, HD5 also has the function of inhibiting tumor proliferation and immune regulation. However, everything has two sides; cytotoxic and proinflammatory properties may exist, while HD5 performs physiological functions. Accordingly, the modification and engineering of HD5 are particularly important. Therefore, this review summarizes the role of HD5 in various aspects of host defense, as well as modification of HD5 to ameliorate the biological activity, with a view to promoting the clinical use of HD5.
Assuntos
alfa-Defensinas , Humanos , alfa-Defensinas/química , alfa-Defensinas/metabolismo , alfa-Defensinas/farmacologia , AntibacterianosRESUMO
Fecal-oral pathogens encounter constitutively expressed enteric alpha-defensins in the intestine during replication and transmission. Alpha-defensins can be potently antiviral and antibacterial; however, their primary sequences, the number of isoforms, and their activity against specific microorganisms often vary greatly between species, reflecting adaptation to species-specific pathogens. Therefore, alpha-defensins might influence not only microbial evolution and tissue tropism within a host but also species tropism and zoonotic potential. To investigate these concepts, we generated a panel of enteric and myeloid alpha-defensins from humans, rhesus macaques, and mice and tested their activity against group A rotaviruses, an important enteric viral pathogen of humans and animals. Rotaviral adaptation to the rhesus macaque correlated with resistance to rhesus enteric, but not myeloid, alpha-defensins and sensitivity to human alpha-defensins. While mouse rotaviral infection was increased in the presence of mouse enteric alpha-defensins, two prominent genotypes of human rotaviruses were differentially sensitive to human enteric alpha-defensins. Furthermore, the effects of cross-species alpha-defensins on human and mouse rotaviruses did not follow an obvious pattern. Thus, exposure to alpha-defensins may have shaped the evolution of some, but not all, rotaviruses. We then used a genetic approach to identify the viral attachment and penetration protein, VP4, as a determinant of alpha-defensin sensitivity. Our results provide a foundation for future studies of the VP4-dependent mechanism of defensin neutralization, highlight the species-specific activities of alpha-defensins, and focus future efforts on a broader range of rotaviruses that differ in VP4 to uncover the potential for enteric alpha-defensins to influence species tropism. IMPORTANCE Rotavirus is a leading cause of severe diarrhea in young children. Like other fecal-oral pathogens, rotaviruses encounter abundant, constitutively expressed defensins in the small intestine. These peptides are a vital part of the vertebrate innate immune system. By investigating the impact that defensins from multiple species have on the infectivity of different strains of rotavirus, we show that some rotaviral infections can be inhibited by defensins. We also found that some, but not all, rotaviruses may have evolved resistance to defensins in the intestine of their host species, and some even appropriate defensins to increase their infectivity. Because rotaviruses infect a broad range of animals and rotaviral infections are highly prevalent in children, identifying immune defenses against infection and how they vary across species and among viral genotypes is important for our understanding of the evolution, transmission, and zoonotic potential of these viruses as well as the improvement of vaccines.
Assuntos
Infecções por Rotavirus , Rotavirus , alfa-Defensinas , Animais , Humanos , Intestino Delgado/imunologia , Intestino Delgado/virologia , Macaca mulatta , Camundongos , Rotavirus/efeitos dos fármacos , Rotavirus/genética , Infecções por Rotavirus/fisiopatologia , Infecções por Rotavirus/virologia , Proteínas Estruturais Virais/metabolismo , alfa-Defensinas/genética , alfa-Defensinas/metabolismo , alfa-Defensinas/farmacologiaRESUMO
Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.
Assuntos
alfa-Defensinas , Sequência de Aminoácidos , Animais , Bactérias , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Precursores de Proteínas , alfa-Defensinas/química , alfa-Defensinas/farmacologia , alfa-Defensinas/fisiologiaRESUMO
The inflammatory response to SARS/CoV-2 (COVID-19) infection may contribute to the risk of thromboembolic complications. α-Defensins, antimicrobial peptides released from activated neutrophils, are anti-fibrinolytic and prothrombotic in vitro and in mouse models. In this prospective study of 176 patients with COVID-19 infection, we found that plasma levels of α-defensins were elevated, tracked with disease progression/mortality or resolution and with plasma levels of interleukin-6 (IL-6) and D-dimers. Immunohistochemistry revealed intense deposition of α-defensins in lung vasculature and thrombi. IL-6 stimulated the release of α-defensins from neutrophils, thereby accelerating coagulation and inhibiting fibrinolysis in human blood, imitating the coagulation pattern in COVID-19 patients. The procoagulant effect of IL-6 was inhibited by colchicine, which blocks neutrophil degranulation. These studies describe a link between inflammation and the risk of thromboembolism, and they identify a potential new approach to mitigate this risk in patients with COVID-19 and potentially in other inflammatory prothrombotic conditions.
Assuntos
COVID-19/metabolismo , Inflamação/metabolismo , Tromboembolia/prevenção & controle , alfa-Defensinas/sangue , Adulto , Idoso , Animais , Coagulação Sanguínea/efeitos dos fármacos , COVID-19/complicações , COVID-19/diagnóstico , COVID-19/virologia , Estudos de Casos e Controles , Colchicina/farmacologia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Humanos , Inflamação/complicações , Interleucina-6/sangue , Interleucina-6/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Animais , Neutrófilos/efeitos dos fármacos , Estudos Prospectivos , Fatores de Risco , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Tromboembolia/etiologia , Trombose/etiologia , Trombose/metabolismo , Moduladores de Tubulina/farmacologia , alfa-Defensinas/farmacologiaRESUMO
The escalating predicament of multidrug resistant cancer cells and associated side effects of conventional chemotherapy necessitates the exploration of alternative anticancer therapies. The present study evaluated anticancer therapeutic potential of human defensin 5 (HD-5) against colon cancer. The in vivo anticancer efficacy of HD-5 against 1,2-dimethylhydrazine (DMH) induced colon cancer was elucidated in terms of tumor biostatistics, number of aberrant crypt foci (ACF), in situ apoptosis assay,changes in morphological as well as histological architecture of colon(s). The direct interaction of peptide was investigated by incubating peptide with normal and/or cancerous colonocytes followed by phase contrast, Hoechst 3342 and AO/PI staining as well as confocal microscopy. Changes in membrane dynamics were evaluated by MC 540 and N-NBD-PE staining. In vivo decrease(s) in tumor parameters, number of aberrant crypt foci along with marked increase in the rate of apoptosis was observed.H&E staining revealed neutrophils infiltration and restoration of normal architecture in treated colon(s) which was consistent with scanning electron microscopic observations. Furthermore, non-membranolytic mechanism was found to be acquired by peptide as it could traverse cell membrane gaining access to nucleus and cytoplasm thereby disintegrating cellular architecture. MC 540 and NBD-PE staining revealed that peptide could bind to cancerous cells by taking advantage of altered fluidity levels. Our results indicated that HD-5 exhibited strong cancer cell killing and does not affect normal host cells. The peptide can be exploited as promising option to combat developing menace of colon cancer and/or can at least be used as an adjunct to present day chemotherapies.
Assuntos
Neoplasias do Colo/prevenção & controle , Fragmentos de Peptídeos/farmacologia , alfa-Defensinas/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinógenos/toxicidade , Colo/efeitos dos fármacos , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dimetilidrazinas/toxicidade , Feminino , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
AIMS: The α-defensins or human neutrophil peptides (HNP 1-3) that exist in azurophilic granules are found to have anticancer activity. The pattern of disulfide bonds in α-defensins is crucial for the functional properties. Therefore, synthesis using the chemical and recombinant approaches is a challenging. A safe source for the production of α-defensins can be the use of leukoreduction filters in blood banks that contain large quantities of neutrophils and are discarded after use. The aim of this study was to purify α-defensins from neutrophils trapped in leukofilters and to investigate its anticancer activity. MATERIALS AND METHODS: Immunoprecipitation was performed to purify α-defensins and the presence of protein was confirmed by Western Blot. The Jurkat T-cell line was incubated with different concentrations (5, 10 and 15⯵g/ml) of purified HNP1-3 for 16â¯h. Cell viability was measured using a WST-1 assay and apoptosis was analyzed for Annexin V/PI markers. Caspase-3/7 activity was determined using fluorescence assay. The effects of purified α-defensins were compared to commercial HNP 1-3. KEY FINDINGS: Purified HNP 1-3 decreased the viability at 10 and 15⯵g/ml and commercial HNP 1-3 at 15⯵g/ml concentrations. Following to the purified HNP1-3 treatment, the percentage of Annexin V positive population and caspase-3 activity were significantly increased compared to control (pâ¯=â¯0.000 and pâ¯=â¯0.001, respectively) and commercial HNP1-3 (pâ¯=â¯0.034 and pâ¯=â¯0.018, respectively). SIGNIFICANCE: Results indicated the anticancer activity of HNP1-3 which can be used as future chemotherapeutic drugs. Furthermore, leukofilters can be considered as economic source for purifying these peptides.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neutrófilos/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , alfa-Defensinas/farmacologia , Anti-Infecciosos/farmacologia , Grânulos Citoplasmáticos/metabolismo , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Células Tumorais CultivadasRESUMO
Active hemostatic agents can play a crucial role in saving patients' lives during surgery. Active hemostats have several advantages including utilization of natural blood coagulation and biocompatibility. Among them, although human neutrophil peptide-1 (HNP-1) has been previously reported with the hemostatic mechanism, which part of HNP-1 facilitates the hemostatic activity is not known. Here, a partial peptide (HNP-F) promoting hemostasis, originating from HNP-1, has been newly identified by the blood coagulation ability test. HNP-F shows the best hemostatic effect between the anterior half and posterior half of peptides. Moreover, microscopic images show platelet aggregation and an increase in the concentration of platelet factor 4, and the scanning electron microscope image of platelets support platelet activation by HNP-F. Thromboelastography indicates decreased clotting time and increased physical properties of blood clotting. Mouse liver experiments demonstrate improved hemostatic effect by treatment of peptide solution. Cell viability and hemolysis assays confirm the HNP-F's biosafety. It is hypothesized that the surface charge and structure of HNP-F could be favorable to interact with fibrinogen or thrombospondin-1. Collectively, because HNP-F as an active peptide hemostat has many advantages, it could be expected to become a potent hemostatic biomaterial, additive or pharmaceutical candidate for various hemostatic applications.
Assuntos
Hemostasia/efeitos dos fármacos , alfa-Defensinas , Animais , Sobrevivência Celular/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Tromboelastografia , alfa-Defensinas/química , alfa-Defensinas/genética , alfa-Defensinas/farmacologiaRESUMO
Surgical site infections (SSI) represent a serious health problem that occur after invasive surgery, thus new antimicrobial biomaterials able to prevent SSI are needed. Silks are natural biopolymers with excellent biocompatibility, low immunogenicity and controllable biodegradability. Spider silk-based materials can be bioengineered and functionalized with specific peptides, such as antimicrobial peptides, creating innovative polymers. Herein, we explored new drug-free multifunctional silk films with antimicrobial properties, specifically tailored to hamper microbial infections. Different spider silk domains derived from the dragline sequence of the spider Nephila clavipes (6mer and 15mer, 27 and 41 kDa proteins, respectively) were fused with the two antimicrobial peptides, Hepcidin (Hep) and Human Neutrophil peptide 1 (HNP1). The self-assembly features of the spider silk domains (ß-sheets) were maintained after functionalization. The bioengineered 6mer-HNP1 protein demonstrated inhibitory effects against microbial pathogens. Silk-based films with 6mer-HNP1 and different contents of silk fibroin (SF) significantly reduced bacterial adhesion and biofilm formation, whereas higher bacterial counts were found on the films prepared with 6mer or SF alone. The silk-based films showed no cytotoxic effects on human foreskin fibroblasts. The positive cellular response, together with structural and antimicrobial properties, highlight the potential of these multifunctional silk-based films as new materials for preventing SSI.
Assuntos
Anti-Infecciosos/química , Materiais Biocompatíveis/química , Fibroínas/química , Hepcidinas/biossíntese , Proteínas Recombinantes de Fusão/química , alfa-Defensinas/biossíntese , Animais , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Linhagem Celular , Sobrevivência Celular , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroínas/biossíntese , Fibroínas/genética , Fibroínas/farmacologia , Expressão Gênica , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Hepcidinas/genética , Hepcidinas/farmacologia , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , Polimerização , Conformação Proteica em Folha beta , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Aranhas/fisiologia , Infecção da Ferida Cirúrgica/prevenção & controle , Suturas/microbiologia , alfa-Defensinas/genética , alfa-Defensinas/farmacologiaRESUMO
AIMS: Microglia-mediated neuroinflammation plays an important role in focal ischemic stroke, a disorder with no effective therapeutic agents. Since microglial polarization to the M2 phenotype and reduction of oxidative stress are mediated through AMP-activated protein kinase (AMPK) and nuclear factor erythroid 2-related factor 2 (Nrf2) activation, we assessed the dual therapeutic effect of AMPK and Nrf2 activation by a novel neuroprotectant HP-1c in the treatment of ischemic stroke. RESULTS: We developed a novel class of hybrids (HP-1a-HP-1f) of telmisartan and 2-(1-hydroxypentyl)-benzoate (HPBA) as a ring-opening derivative of NBP. The most promising hybrid, HP-1c, exhibited more potent anti-inflammatory and neuroprotective effects in vitro and reduced brain infarct volume and improved neurological deficits in a rat model of transient focal cerebral ischemia when compared with telmisartan alone, NBP alone, or a combination of telmisartan and NBP. HP-1c had a therapeutic window of up to 24 h, ameliorated ischemic cerebral injury in permanent focal cerebral ischemia, and improved motor function. The beneficial effects of HP-1c in ischemic stroke were associated with microglial polarization to the M2 phenotype and reduced oxidative stress. HP-1c also shifted the M1/M2 polarization in a mouse neuroinflammatory model. The anti-inflammatory and anti-oxidative effects of HP-1c were associated with AMPK-Nrf2 pathway activation for neuroprotection. We showed that HP-1c penetrates the brain, has a plasma half-life of around 3.93 h, and has no toxicity in mice. Innovation and Conclusion: Our study results suggest that HP-1c, with dual AMPK- and Nrf2-activating properties, may have potential in further studies as a novel therapy for ischemic stroke. Antioxid. Redox Signal. 28, 141-163.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Encefalite/etiologia , Encefalite/metabolismo , Microglia/metabolismo , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/metabolismo , Acidente Vascular Cerebral/complicações , Animais , Antioxidantes , Isquemia Encefálica , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Infarto Cerebral , Modelos Animais de Doenças , Encefalite/imunologia , Feminino , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microglia/imunologia , Microglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , PPAR gama/metabolismo , Ratos , alfa-Defensinas/química , alfa-Defensinas/farmacologiaRESUMO
The increasing incidence of multidrug-resistant Acinetobacter baumannii (MDRAb) infections worldwide has necessitated the development of novel antibiotics. Human defensin 5 (HD5) is an endogenous peptide with a complex architecture and antibacterial activity against MDRAb In the present study, we attempted to simplify the structure of HD5 by removing disulfide bonds. We found that the Cys2-4 bond was most indispensable for HD5 to inactivate MDRAb, although the antibacterial activity of the derivative was significantly attenuated. We then replaced the noncationic and nonhydrophobic residues with electropositive Arg to increase the antibacterial activity of HD5 derivative that contains a Cys2-4 bond, obtaining another derivative termed HD5d5. The in vitro antibacterial assay and irradiation-wound-infection animal experiment both showed that HD5d5 was much more effective than HD5 at eliminating MDRAb Further investigations revealed that HD5d5 efficiently bound to outer membrane lipid A and penetrated membranes, leading to bacterial collapse and peptide translocation. Compared to HD5, more HD5d5 molecules were located in the cytoplasm of MDRAb, and HD5d5 was more efficient at reducing the activities of superoxide dismutase and catalase, causing the accumulation of reactive oxygen species that are detrimental to microbes. In addition, HD5 failed to suppress the pathogenic outer membrane protein A of Acinetobacter baumannii (AbOmpA) at concentrations up to 50 µg/ml, whereas HD5d5 strongly bound to AbOmpA and exhibited a dramatic toxin-neutralizing ability, thus expanding the repertoire of drugs that is available to treat MDRAb infections.
Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica , Infecção dos Ferimentos/tratamento farmacológico , alfa-Defensinas/farmacologia , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/mortalidade , Infecções por Acinetobacter/patologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/crescimento & desenvolvimento , Acinetobacter baumannii/metabolismo , Animais , Antibacterianos/síntese química , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catalase/antagonistas & inibidores , Catalase/genética , Catalase/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Lipídeo A/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Engenharia de Proteínas/métodos , Isoformas de Proteínas/síntese química , Isoformas de Proteínas/farmacologia , Transporte Proteico , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Análise de Sobrevida , Irradiação Corporal Total , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/mortalidade , Infecção dos Ferimentos/patologia , alfa-Defensinas/síntese químicaRESUMO
α, ß, and θ defensins are effectors of the innate immune system with potent antibacterial, antiviral, and antifungal activity. Defensins have direct antiviral activity in cell culture, with varied mechanisms for individual viruses, although some common themes have emerged. In addition, defensins have potent immunomodulatory activity that can alter innate and adaptive immune responses to viral infection. In some cases, there is evidence for paradoxical escape from defensin neutralization or enhancement of viral infection. The direct and indirect activities of defensins have led to their development as therapeutics and vaccine components. The major area of investigation that continues to lag is the connection between the effects of defensins in cell culture models and viral pathogenesis in vivo. Model systems to study defensin biology, including more physiologic models designed to bridge this gap, are also discussed.
Assuntos
Defensinas/metabolismo , Viroses/imunologia , alfa-Defensinas/metabolismo , beta-Defensinas/metabolismo , Adenoviridae/efeitos dos fármacos , Adenoviridae/patogenicidade , Animais , Antivirais/farmacologia , Defensinas/genética , Defensinas/farmacologia , Defensinas/uso terapêutico , HIV/efeitos dos fármacos , HIV/patogenicidade , Herpesviridae/efeitos dos fármacos , Herpesviridae/patogenicidade , Humanos , Imunidade Inata , Imunomodulação , Camundongos , Papillomaviridae/efeitos dos fármacos , Papillomaviridae/patogenicidade , Viroses/tratamento farmacológico , alfa-Defensinas/genética , alfa-Defensinas/farmacologia , alfa-Defensinas/uso terapêutico , beta-Defensinas/genética , beta-Defensinas/farmacologia , beta-Defensinas/uso terapêuticoRESUMO
Human neutrophil peptides (HNPs) not only have antimicrobial properties, but also exert multiple immunomodulatory effects depending on the concentration used. We have previously demonstrated that the intraperitoneal administration of high-dose HNP-1 (100 µg/day) aggravates murine dextran sulfate sodium (DSS)-induced colitis, suggesting a potential pro-inflammatory role for HNPs at high concentrations. However, the role of low physiological concentrations of HNPs in the intestinal tract remains largely unknown. The aim of this study was to examine the effects of low concentrations of HNPs on intestinal inflammation. We first examined the effects of the mild transgenic overexpression of HNP-1 in DSS-induced colitis. HNP-1 transgenic mice have plasma HNP-1 levels similar to the physiological concentrations in human plasma. Compared to wild-type mice treated with DSS, HNP-1 transgenic mice treated with DSS had significantly lower clinical and histological scores, and lower colonic mRNA levels of pro-inflammatory cytokines, including interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. We then injected low-dose HNP-1 (5 µg/day) or phosphate-buffered saline (PBS) intraperitoneally into C57BL/6N and BALB/c mice administered DSS. The HNP-1-treated mice exhibited significantly milder colitis with reduced expression levels of pro-inflammatory cytokines compared with the PBS-treated mice. Finally, we examined the in vitro effects of HNP-1 on the expression of cytokines associated with macrophage activation. Low physiological concentrations of HNP-1 did not significantly affect the expression levels of IL-1ß, TNF-α, IL-6 or IL-10 in colonic lamina propria mononuclear cells activated with heat-killed Escherichia coli, suggesting that the anti-inflammatory effects of HNP-1 on murine colitis may not be exerted by direct action on intestinal macrophages. Collectively, our data demonstrated a biphasic dose-dependent effect of HNP-1 on DSS-induced colitis: an amelioration at low concentrations and an aggravation at high concentrations. Low concentrations of HNPs may contribute to the maintenance of intestinal homeostasis.
Assuntos
Anti-Inflamatórios/farmacologia , Colite/etiologia , Colite/patologia , alfa-Defensinas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Colite/tratamento farmacológico , Colite/metabolismo , Citocinas/metabolismo , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Expressão Gênica , Predisposição Genética para Doença , Humanos , Mediadores da Inflamação , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Transgênicos , alfa-Defensinas/sangue , alfa-Defensinas/genéticaRESUMO
Neutrophils are the first and most numerous cells to arrive at the site of an inflammatory insult and are among the first to die. We previously reported that alpha defensins, released from apoptotic human neutrophils, augmented the antimicrobial capacity of macrophages while also inhibiting the biosynthesis of proinflammatory cytokines. In vivo, alpha defensin administration protected mice from inflammation, induced by thioglychollate-induced peritonitis or following infection withSalmonella entericaserovar Typhimurium. We have now dissected the antiinflammatory mechanism of action of the most abundant neutrophil alpha defensin, Human Neutrophil Peptide 1 (HNP1). Herein we show that HNP1 enters macrophages and inhibits protein translation without inducing the unfolded-protein response or affecting mRNA stability. In a cell-free in vitro translation system, HNP1 powerfully inhibited both cap-dependent and cap-independent mRNA translation while maintaining mRNA polysomal association. This is, to our knowledge, the first demonstration of a peptide released from one cell type (neutrophils) directly regulating mRNA translation in another (macrophages). By preventing protein translation, HNP1 functions as a "molecular brake" on macrophage-driven inflammation, ensuring both pathogen clearance and the resolution of inflammation with minimal bystander tissue damage.
Assuntos
Macrófagos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Infecções por Salmonella/metabolismo , Salmonella typhimurium , alfa-Defensinas/metabolismo , Animais , Humanos , Macrófagos/patologia , Camundongos , Infecções por Salmonella/patologia , alfa-Defensinas/farmacologiaRESUMO
Oxidized human defensin 5 (HD5OX), a Paneth cell-secreted antibacterial peptide with three characteristic disulfide bonds, protects the host from invasion by morbigenous microbes in the small intestine. HD5OX can be reduced by thioredoxin (Trx) in vitro, while the biochemical properties of the reduced linear peptide, HD5RED, remain unclear. Here, we first confirm that HD5RED does exist in vivo. Furthermore, we reveal that the recruitment of HD5RED to the outer membrane of Gram-negative bacteria and to the anionic lipid A is lower than that of HD5OX, and HD5RED is less efficient in penetrating bacterial outer and inner membranes and inducing membrane depolarization, which confers an attenuated antibacterial activity to HD5RED. However, due to its higher structural flexibility, the binding of HD5RED to bacterial lipopolysaccharide (LPS) is markedly stronger than that of HD5OX. Consequently, HD5RED is more effective in suppressing the production of the pro-inflammatory cytokine TNF-α in LPS-stimulated macrophages by blocking the interaction between LPS and LPS-binding protein, thus suggesting that HD5RED might act as a scavenger to neutralize LPS in the gut. This study provides insights into the antibacterial and immunoregulatory effects of HD5RED and expands the known repertoire of the enteric defensins.
Assuntos
Lipopolissacarídeos/farmacologia , alfa-Defensinas/metabolismo , Proteínas de Fase Aguda/metabolismo , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Líquidos Corporais/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Escherichia coli/efeitos dos fármacos , Humanos , Íleo/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Oxirredução , Ligação Proteica , Salmonella typhimurium/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , alfa-Defensinas/química , alfa-Defensinas/farmacologiaRESUMO
OBJECTIVES: The present study aimed to investigate the effect of HNP-1 on the matrix metalloproteinase (MMP)-2, -8 and -9 secretions of two oral squamous cell carcinoma (OSCC) cell lines (UT-SCC-43A and UT-SCC-43B). DESIGN: In all experiments, the two OSCC cell lines were incubated with graded concentrations (0, 1, 5, and 10 µg/ml) of HNP-1 for 24 and 48 h. Cell viability was measured using a colorimetric proliferation test and cell death was analyzed with a colorimetric cytotoxicity detection kit. Enzyme activity of MMP-2 and MMP-9 was detected by using gelatin zymography, and molecular weight forms of MMP-8 were determined by Western-blot and a densitometric quantitation method. RESULTS: Both cell lines showed a significant increase in LDH toxicity at 24h (UT-SCC-43A: p=0.005 & UT-SCC-43B: p=0.014). Reduced gelatinolytic activities of proMMP-2 were detected in UT-SCC-43B cell line after 24 and 48 h of incubation with HNP-1 (1 µg/ml: p<0.001, 5 µg/ml: p<0.001, and 10 µg/ml: p=0.0225). MMP-8 levels of both cell lines decreased at 200-250 kDa after 24h of incubation, while after 48 h only UT-SCC-43B decreased at 45-50 kDa. CONCLUSIONS: Our results indicate that HNP-1 suppresses the secretion of MMP-2, -8, and -9 in OSCC cell lines.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/enzimologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/enzimologia , Metaloproteinases da Matriz/metabolismo , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/enzimologia , alfa-Defensinas/farmacologia , Anti-Infecciosos/farmacologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Densitometria/métodos , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Gelatina/metabolismo , Gelatinases/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metaloproteinases da Matriz/biossíntese , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Streptococcus mitis is a colonizer of the oral cavity and the nasopharynx, and is closely related to Streptococcus pneumoniae. Both species occur in encapsulated and unencapsulated forms, but in S. mitis the role of the capsule in host interactions is mostly unknown. Therefore, the aim of this study was to examine how capsule expression in S. mitis can modulate interactions with the host with relevance for colonization. The S. mitis type strain, as well as two mutants of the type strain, an isogenic capsule deletion mutant, and a capsule switch mutant expressing the serotype 4 capsule of S. pneumoniae TIGR4, were used. Wild-type and capsule deletion strains of S. pneumoniae TIGR4 were included for comparison. We found that capsule production in S. mitis reduced adhesion to oral and lung epithelial cells. Further, exposure of oral epithelial cells to encapsulated S. mitis resulted in higher interleukin-6 and CXCL-8 transcription levels relative to the unencapsulated mutant. Capsule expression in S. mitis increased the sensitivity to human neutrophil peptide 1-3 but reduced the sensitivity to human ß-defensin-3 and cathelicidin. This was in contrast with S. pneumoniae in which capsule expression has been generally associated with increased sensitivity to human antimicrobial peptides (AMPs). Collectively, these findings indicate that capsule expression in S. mitis is important in modulating interactions with epithelial cells, and is associated with increased or reduced susceptibility to AMPs depending on the nature of the AMP.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cápsulas Bacterianas/efeitos dos fármacos , Cápsulas Bacterianas/metabolismo , Queratinócitos/microbiologia , Boca/citologia , Streptococcus mitis/citologia , Streptococcus mitis/efeitos dos fármacos , alfa-Defensinas/farmacologia , Aderência Bacteriana , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/imunologia , Catelicidinas/farmacologia , Linhagem Celular Tumoral , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Humanos , Interleucina-6/genética , Interleucina-8/genética , Queratinócitos/imunologia , Boca/imunologia , Boca/microbiologia , Mutação , Streptococcus mitis/genética , Streptococcus mitis/imunologia , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/fisiologia , beta-Defensinas/farmacologiaRESUMO
Human α-defensin 6 (HD6), unlike other mammalian defensins, does not exhibit bactericidal activity, particularly against aerobic bacteria. Monomeric HD6 has a tertiary structure similar to other α-defensins in the crystalline state. However, the physico-chemical reasons behind the lack of antibacterial activity of HD6 are yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD6 analogs. A linear analog of HD6, in which the distribution of arginine residues was similar to active α-defensins, shows broad-spectrum antimicrobial activity, indicating that atypical distribution of arginine residues contributes to the inactivity of HD6. Peptides spanning the N-terminal cationic segment were active against a wide range of organisms. Antimicrobial potency of these shorter analogs was further enhanced when myristic acid was conjugated at the N-terminus. Cytoplasmic localization of the analogs without fatty acylation was observed to be necessary for bacterial killing, while they exhibited fungicidal activity by permeabilizing Candida albicans membranes. Myristoylated analogs and the linear full-length arginine analog exhibited activity by permeabilizing bacterial and fungal membranes. Our study provides insights into the lack of bactericidal activity of HD6 against aerobic bacteria.
Assuntos
Anti-Infecciosos/farmacologia , Desenho de Fármacos , Peptídeos/farmacologia , alfa-Defensinas/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Antifúngicos/síntese química , Antifúngicos/química , Antifúngicos/farmacologia , Candida albicans/citologia , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Candida albicans/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Fenômenos Químicos , Dicroísmo Circular , Cistina/química , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Negativas/ultraestrutura , Humanos , Lipoilação , Potenciais da Membrana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Ácido Mirístico/química , Peptídeos/síntese química , Peptídeos/química , Estrutura Terciária de Proteína , Staphylococcus aureus/citologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/ultraestrutura , alfa-Defensinas/químicaRESUMO
θ-Defensins are pleiotropic, macrocyclic peptides that are expressed uniquely in Old World monkeys. The peptides are potent, broad-spectrum microbicides that also modulate inflammatory responses in vitro and in animal models of viral infection and polymicrobial sepsis. θ-Defensins suppress proinflammatory cytokine secretion by leukocytes stimulated with diverse Toll-like receptor (TLR) ligands. Studies were performed to delineate anti-inflammatory mechanisms of rhesus θ-defensin 1 (RTD-1), the most abundant θ-defensin isoform in macaque granulocytes. RTD-1 reduced the secretion of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-8 in lipopolysaccharide (LPS)-stimulated human blood monocytes and THP-1 macrophages, and this was accompanied by inhibition of nuclear factor κB (NF-κB) activation and mitogen-activated protein kinase (MAPK) pathways. Peptide inhibition of NF-κB activation occurred following stimulation of extracellular (TLRs 1/2 and 4) and intracellular (TLR9) receptors. Although RTD-1 did not inhibit MAPK in unstimulated cells, it induced phosphorylation of Akt in otherwise untreated monocytes and THP-1 cells. In the latter, this occurred within 10 min of RTD-1 treatment and produced a sustained elevation of phosphorylated Akt (pAkt) for at least 4 h. pAkt is a negative regulator of MAPK and NF-κB activation. RTD-1 inhibited IκBα degradation and p38 MAPK phosphorylation, and stimulated Akt phosphorylation in LPS-treated human primary monocytes and THP-1 macrophages. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) blocked RTD-1-stimulated Akt phosphorylation and reversed the suppression of NF-κB activation by the peptide. These studies indicate that the anti-inflammatory properties of θ-defensins are mediated by activation of the PI3K/Akt pathway and suppression of proinflammatory signals in immune-stimulated cells.
Assuntos
Citocinas/imunologia , Regulação da Expressão Gênica/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/imunologia , Monócitos/imunologia , NF-kappa B/imunologia , Peptídeos Cíclicos/imunologia , alfa-Defensinas/imunologia , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/citologia , Masculino , Monócitos/citologia , Peptídeos Cíclicos/farmacologia , Fosfatidilinositol 3-Quinases/imunologia , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , alfa-Defensinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Human α-defensin 5 (HD5) exhibits broad spectrum antimicrobial activity and plays an important role in mucosal immunity of the small intestine. Although there have been several studies, the structural requirements for activity and mechanism of bacterial killing is yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD5 and linear analogs. Cysteine deletions attenuated the antibacterial activity considerably. Candidacidal activity was affected to a lesser extent. Fatty acid conjugated linear analogs showed antimicrobial activity comparable activity to HD5. Effective surface charge neutralization of bacteria was observed for HD5 as compared to the non-fatty acylated linear analogs. Our results show that HD5 and non-fatty acylated linear analogs enter the bacterial cytoplasm without causing damage to the bacterial inner membrane. Although fatty acylated peptides exhibited antimicrobial activity comparable to HD5, their mechanism of action involved permeabilization of the Escherichia coli inner membrane. HD5 and analogs had the ability to bind plasmid DNA. HD5 had greater binding affinity to plasmid DNA as compared to the analogs. The three dimensional structure of HD5 favors greater interaction with the bacterial cell surface and also with DNA. Antibacterial activity of HD5 involves entry into bacterial cytoplasm and binding to DNA which would result in shut down of the bacterial metabolism leading to cell death. We show how a moderately active linear peptide derived from the α-defensin HD5 can be engineered to enhance antimicrobial activity almost comparable to the native peptide.