RESUMO
θ-Defensins, the only cyclic peptides of animal origin, have been isolated from the leukocytes of rhesus macaques and baboons. Their biogenesis is unusual because each peptide is an 18-residue chimera formed by the head-to-tail splicing of nonapeptides derived from two separate precursors. θ-Defensins have multiple arginines and a ladder-like tridisulfide array spanning their two antiparallel ß-strands. Human θ-defensin genes contain a premature stop codon that prevents effective translation of the needed precursors; consequently, these peptides are not present in human leukocytes. Synthetic θ-defensins with sequences that correspond to those encoded within the human pseudogenes are called retrocyclins. Retrocyclin-1 inhibits the cellular entry of HIV-1, HSV, and influenza A virus. The rhesus θ-defensin RTD-1 protects mice from an experimental severe acute respiratory syndrome coronavirus infection, and retrocyclin-1 protects mice from infection by Bacillus anthracis spores. The small size, unique structure, and multiple host defense activities of θ-defensins make them intriguing potential therapeutic agents.
Assuntos
Defensinas/química , Peptídeos Cíclicos/química , Defensinas/farmacologia , Defensinas/fisiologia , Humanos , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/fisiologiaRESUMO
Defensins are antimicrobial peptides that contribute broadly to innate immunity, including protection of mucosal tissues. Human α-defensin (HD) 6 is highly expressed by secretory Paneth cells of the small intestine. However, in contrast to the other defensins, it lacks appreciable bactericidal activity. Nevertheless, we report here that HD6 affords protection against invasion by enteric bacterial pathogens in vitro and in vivo. After stochastic binding to bacterial surface proteins, HD6 undergoes ordered self-assembly to form fibrils and nanonets that surround and entangle bacteria. This self-assembly mechanism occurs in vivo, requires histidine-27, and is consistent with x-ray crystallography data. These findings support a key role for HD6 in protecting the small intestine against invasion by diverse enteric pathogens and may explain the conservation of HD6 throughout Hominidae evolution.
Assuntos
Imunidade Inata , Imunidade nas Mucosas , Intestino Delgado/imunologia , alfa-Defensinas/química , alfa-Defensinas/metabolismo , Adesinas Bacterianas/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/ultraestrutura , Intestino Delgado/microbiologia , Intestino Delgado/ultraestrutura , Substâncias Macromoleculares/química , Substâncias Macromoleculares/imunologia , Substâncias Macromoleculares/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Modelos Moleculares , Nanoestruturas , Celulas de Paneth/imunologia , Celulas de Paneth/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/ultraestrutura , Yersinia enterocolitica/imunologia , Yersinia enterocolitica/patogenicidade , alfa-Defensinas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
θ-Defensins are cyclic octadecapeptides found in nonhuman primates whose broad antiviral spectrum includes HIV-1, HSV-1, severe acute respiratory syndrome coronavirus, and influenza A virus (IAV). We previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This study describes two families of peptides, hapivirins and diprovirins, whose design was inspired by retrocyclins. The goal was to develop smaller partially cyclic peptides that retain the antiviral activity of retrocyclins, while being easier to synthesize. The novel peptides also allowed for systemic substitution of key residues to evaluate the role of charge or hydrophobicity on antiviral activity. Seventy-two hapivirin or diprovirin peptides are described in this work, including several whose anti-IAV activity equals or exceeds that of normal α- or θ-defensins. Some of these also had strong antibacterial and antifungal activity. These new peptides were active against H3N2 and H1N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum t(1/2) in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection, and, when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogs provides an approach for creating novel antiviral agents for IAV infections.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Defensinas/imunologia , Defensinas/farmacologia , Vírus da Influenza A/imunologia , Sequência de Aminoácidos , Animais , Antivirais/imunologia , Linhagem Celular , Técnicas de Química Sintética , Cromatografia Líquida de Alta Pressão , Defensinas/síntese química , Cães , Humanos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Monócitos/virologia , Neutrófilos/virologia , Peptídeos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Human myeloid α-defensins called HNPs play multiple roles in innate host defense. The Trp-26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association. To gain additional insights into the functional significance of dimerization, we compared wild type HNP1 to dimerization-impaired, N-methylated HNP1 monomers and to disulfide-tethered obligate HNP1 dimers. The structural effects of these modifications were confirmed by x-ray crystallographic analyses. Like the previously studied W26A mutation, N-methylation of Ile-20 dramatically reduced the ability of HNP1 to kill Staphylococcus aureus, inhibit LF, and bind gp120. Importantly, this modification had minimal effect on the ability of HNP1 to kill Escherichia coli. The W26A and MeIle-20 mutations impaired defensin activity synergistically. N-terminal covalent tethering rescued the ability of W26A-HNP1 to inhibit LF but failed to restore its defective killing of S. aureus. Surface plasmon resonance studies revealed that Trp-26 mediated the association of monomers and canonical dimers of HNP1 to immobilized HNP1, LF, and gp120, and also indicated a possible mode of tetramerization of HNP1 mediated by Ile-20 and Leu-25. This study demonstrates that dimerization contributes to some but not all of the many and varied activities of HNP1.
Assuntos
alfa-Defensinas/química , alfa-Defensinas/imunologia , Cristalografia por Raios X , Dimerização , Escherichia coli/fisiologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Humanos , Imunidade Inata , Conformação Molecular , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , alfa-Defensinas/genéticaRESUMO
Defensins are small, multifunctional cationic peptides. They typically contain six conserved cysteines whose three intramolecular disulfides stabilize a largely ß-sheet structure. This review of human α-defensins begins by describing their evolution, including their likely relationship to the Big Defensins of invertebrates, and their kinship to the ß-defensin peptides of many if not all vertebrates, and the θ-defensins found in certain non-human primates. We provide a short history of the search for leukocyte-derived microbicidal molecules, emphasizing the roles played by luck (good), preconceived notions (mostly bad), and proper timing (essential). The antimicrobial, antiviral, antitoxic, and binding properties of human α-defensins are summarized. The structural features of α-defensins are described extensively and their functional contributions are assessed. The properties of HD6, an enigmatic Paneth cell α-defensin, are contrasted with those of the four myeloid α-defensins (HNP1-4) and of HD5, the other α-defensin of human Paneth cells. The review ends with a decalogue that may assist researchers or students interested in α-defensins and related aspects of neutrophil function.
Assuntos
Anti-Infecciosos/imunologia , Imunidade Inata , Celulas de Paneth/imunologia , alfa-Defensinas/imunologia , Animais , Anti-Infecciosos/química , Evolução Biológica , Biologia Computacional , Humanos , Imunidade nas Mucosas , Neutrófilos/imunologia , Conformação Proteica , alfa-Defensinas/química , alfa-Defensinas/genéticaAssuntos
Dissulfetos/química , Dissulfetos/metabolismo , beta-Defensinas/química , beta-Defensinas/imunologia , Animais , Anti-Infecciosos/química , Anti-Infecciosos/imunologia , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/imunologia , Colo/imunologia , Colo/metabolismo , Colo/microbiologia , Simulação por Computador , Humanos , Imunidade Inata , Modelos Moleculares , Oxirredução , Conformação Proteica , beta-Defensinas/metabolismo , beta-Defensinas/farmacologiaRESUMO
Lactobacillus iners is a common constituent of the human vaginal microbiota. This species was only recently characterized due to its fastidious growth requirements and has been hypothesized to play a role in the pathogenesis of bacterial vaginosis. Here we present the identification and molecular characterization of a protein toxin produced by L. iners. The L. iners genome encodes an open reading frame with significant primary sequence similarity to intermedilysin (ILY; 69.2% similarity) and vaginolysin (VLY; 68.4% similarity), the cholesterol-dependent cytolysins from Streptococcus intermedius and Gardnerella vaginalis, respectively. Clinical isolates of L. iners produce this protein, inerolysin (INY), during growth in vitro, as assessed by Western analysis. INY is a pore-forming toxin that is activated by reducing agents and inhibited by excess cholesterol. It is active across a pH range of 4.5 to 6.0 but is inactive at pH 7.4. At sublytic concentrations, INY activates p38 mitogen-activated protein kinase and allows entry of fluorescent phalloidin into the cytoplasm of epithelial cells. Unlike VLY and ILY, which are human specific, INY is active against cells from a broad range of species. INY represents a new target for studies directed at understanding the role of L. iners in states of health and disease at the vaginal mucosal surface.
Assuntos
Colesterol/metabolismo , Citotoxinas/metabolismo , Lactobacillus/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citotoxinas/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Lactobacillus/efeitos dos fármacos , Lactobacillus/genética , Mutação , Estresse Fisiológico , Raios UltravioletaRESUMO
Listeria monocytogenes is a facultative intracellular pathogen that infects a large diversity of host cells, including macrophages. To avoid the phagosome microbicidal environment, L. monocytogenes secretes a pore-forming toxin (listeriolysin O, LLO) that releases the bacterium into the cytoplasm. We hypothesized that the α-defensins (HNPs) and/or humanized θ-defensin (RC-1) peptides produced by human and non-human primate neutrophils, respectively, cooperate with macrophages to control L. monocytogenes infection. Our results establish that HNP-1 and RC-1 enable macrophages to control L. monocytogenes intracellular growth by inhibiting phagosomal escape, as a consequence, bacteria remain trapped in a LAMP-1-positive phagosome. Importantly, HNP-1 interaction with macrophages and RC-1 interaction with bacteria are required to prevent macrophage infection. In accordance with these results, RC-1 is a more potent anti-listerial peptide than HNP-1 and HNP-1 is acquired by macrophages and trafficked to the phagocytosed bacteria. Finally, HNP-1 and RC-1 antimicrobial activity is complemented by their ability to prevent LLO function through two mechanisms, blocking LLO-dependent perforation of macrophage membranes and the release of LLO from the bacteria. In conclusion, at the site of infection the cooperation between antimicrobial peptides, such as HNP-1, and macrophages likely plays a critical role in the innate immune defence against L. monocytogenes.
Assuntos
Defensinas/imunologia , Listeria monocytogenes/crescimento & desenvolvimento , Macrófagos/imunologia , Macrófagos/microbiologia , alfa-Defensinas/imunologia , Animais , Anti-Infecciosos/imunologia , Toxinas Bacterianas/metabolismo , Células Cultivadas , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Macrófagos/citologia , Camundongos , Fagossomos/imunologia , Fagossomos/microbiologiaRESUMO
BACKGROUND: RC-101 is a congener of the antiretroviral peptide retrocyclin, which we and others have reported is active against clinical HIV-1 isolates from all major clades, does not hemagglutinate, and is non-toxic and non-inflammatory in cervicovaginal cell culture. Herein, film-formulated RC-101 was assessed for its antiviral activity in vitro, safety in vivo, retention in the cervix and vagina, and ability to remain active against HIV-1 and SHIV after intravaginal application in macaques. METHODOLOGY/PRINCIPAL FINDINGS: RC-101 was formulated as a quick-dissolving film (2000 µg/film), retained complete activity in vitro as compared to unformulated peptide, and was applied intravaginally in six pigtailed macaques daily for four days. At one and four days following the final application, the presence of RC-101 was assessed in peripheral blood, cervicovaginal lavage, cytobrushed cervicovaginal cells, and biopsied cervical and vaginal tissues by quantitative western blots. One day following the last film application, cervical biopsies from RC-101-exposed and placebo-controlled macaques were collected and were subjected to challenge with RT-SHIV in an ex vivo organ culture model. RC-101 peptide was detected primarily in the cytobrush and biopsied cervical and vaginal tissues, with little to no peptide detected in lavage samples, suggesting that the peptide was associated with the cervicovaginal epithelia. RC-101 remained in the tissues and cytobrush samples up to four days post-application, yet was not detected in any sera or plasma samples. RC-101, extracted from cytobrushes obtained one day post-application, remained active against HIV-1 BaL. Importantly, cervical biopsies from RC-101-treated animals reduced RT-SHIV replication in ex vivo organ culture as compared to placebo-treated animals. CONCLUSIONS/SIGNIFICANCE: Formulated RC-101 was stable in vivo and was retained in the mucosa. The presence of antivirally active RC-101 after five days in vivo suggests that RC-101 would be an important molecule to develop further as a topical microbicide to prevent HIV-1 transmission.
Assuntos
Antivirais/farmacologia , Peptídeos/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Administração Intravaginal , Animais , Antivirais/administração & dosagem , Colo do Útero/efeitos dos fármacos , Colo do Útero/virologia , Colposcopia , Relação Dose-Resposta a Droga , Feminino , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Células HeLa , Humanos , Macaca nemestrina , Peptídeos/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Fatores de Tempo , Vagina/efeitos dos fármacos , Vagina/virologiaRESUMO
BACKGROUND: Many gram-positive bacteria produce pore-forming exotoxins that contain a highly conserved, 12-residue domain (ECTGLAWEWWRT) that binds cholesterol. This domain is usually flanked N-terminally by arginine and C-terminally by valine. We used this 14-residue sequence as a template to create a small library of peptides that bind cholesterol and other lipids. METHODOLOGY/RESULTS: Several of these peptides manifested anti-inflammatory properties in a predictive in vitro monocyte chemotactic assay, and some also diminished the pro-inflammatory effects of low-density lipoprotein in apoE-deficient mice. The most potent analog, Oxpholipin-11D (OxP-11D), contained D-amino acids exclusively and was identical to the 14-residue design template except that diphenylalanine replaced cysteine-3. In surface plasmon resonance binding studies, OxP-11D bound oxidized (phospho)lipids and sterols in much the same manner as D-4F, a widely studied cardioprotective apoA-I-mimetic peptide with anti-inflammatory properties. In contrast to D-4F, which adopts a stable alpha-helical structure in solution, the OxP-11D structure was flexible and contained multiple turn-like features. CONCLUSION: Given the substantial evidence that oxidized phospholipids are pro-inflammatory in vivo, OxP-11D and other Oxpholipins may have therapeutic potential.
Assuntos
Anti-Inflamatórios/síntese química , Colesterol/metabolismo , Exotoxinas/química , Peptídeos/farmacologia , Fosfolipídeos/metabolismo , Anti-Inflamatórios/metabolismo , Proteínas de Bactérias/química , Desenho de Fármacos , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/metabolismo , Ligação ProteicaRESUMO
We performed a comprehensive alanine scan of human alpha-defensin HNP1 and tested the ability of the resulting analogs to kill Staphylococcus aureus, inhibit anthrax lethal factor, and bind human immunodeficiency virus-1 gp120. By far, the most deleterious mutation for all of these functions was W26A. The activities lost by W26A-HNP1 were restored progressively by replacing W26 with non-coded, straight-chain aliphatic amino acids of increasing chain length. The hydrophobicity of residue 26 also correlated with the ability of the analogs to bind immobilized wild type HNP1 and to undergo further self-association. Thus, the hydrophobicity of residue 26 is not only a key determinant of the direct interactions of HNP1 with target molecules, but it also governs the ability of this peptide to form dimers and more complex quaternary structures at micromolar concentrations. Although all defensin peptides are cationic, their amphipathicity is at least as important as their positive charge in enabling them to participate in innate host defense.
Assuntos
Multimerização Proteica , alfa-Defensinas/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunidade Inata/fisiologia , Mutação de Sentido Incorreto , Estrutura Quaternária de Proteína , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo , Relação Estrutura-Atividade , Triptofano/química , Triptofano/genética , Triptofano/imunologia , Triptofano/metabolismo , alfa-Defensinas/genética , alfa-Defensinas/imunologia , alfa-Defensinas/metabolismoRESUMO
Two cyclic theta-defensin peptides were isolated from leukocytes of the hamadryas baboon, Papio hamadryas, and purified to homogeneity by gel electrophoresis and reversed-phase high-performance liquid chromatography. Both peptides had high in vitro activity against Escherichia coli, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. Here, we report their de novo sequencing by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). This was accomplished by combining conventional enzymatic digestion with N-terminal derivatization by 2-sulfobenzoic acid cyclic anhydride (SACA) or 4-sulfophenylisothiocyanate (SPITC) to facilitate the interpretation of fragment ion spectra. In addition to the two cyclic theta-defensins (PhTDs) we also sequenced a novel Papio hamadryas alpha-defensin, PhD-4, which showed high sequence homology to rhesus alpha-defensin RMAD-1 and human alpha-defensin HNP-1.
Assuntos
Defensinas/química , Leucócitos/química , Papio hamadryas/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bactérias/efeitos dos fármacos , Benzenossulfonatos/química , Benzoatos/química , Candida albicans/efeitos dos fármacos , Simulação por Computador , Defensinas/genética , Defensinas/metabolismo , Defensinas/farmacologia , Isotiocianatos/química , Testes de Sensibilidade Microbiana , Papio hamadryas/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Homologia de Sequência de Aminoácidos , alfa-Defensinas/química , alfa-Defensinas/genéticaRESUMO
Antimicrobial peptides (AMPs), important effector molecules of the innate immune system, also provide templates for designing novel antibiotics. Protegrin, an especially potent AMP found in porcine leukocytes, was recently shown to form octameric transmembrane pores. We have employed a combination of experiments and models spanning length scales from the atomistic to the cellular level in order to elucidate the microbicidal mechanism of protegrin. Comparison of the modeling and experimental data suggests that approximately 10-100 protegrin pores are necessary to explain the observed rates of potassium leakage and Escherichia coli death in exponential-phase bacteria. The kinetics of viability loss suggest that bacterial death results largely from uncontrolled ion exchange processes and decay of transmembrane potential. However, ion exchange processes alone cannot account for the experimentally observed cell swelling and osmotic lysis-a redundant "overkill" mechanism most likely to occur in locales with high protegrin concentrations. Although our study is limited to protegrin and E. coli, the timeline of events described herein is likely shared by other AMPs that act primarily by permeabilizing microbial membranes. This work provides many of the missing links in describing antimicrobial action, as well as providing a quantitative connection between several previous experimental and simulation studies of protegrin.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/efeitos dos fármacos , Modelos Teóricos , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Íons/metabolismo , Potenciais da Membrana/fisiologia , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Antimicrobial host defense peptides (AHDPs) are effective against a wide range of microbes, including viruses. The arteriviral infection caused by porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating pandemic that causes the most economically significant disease of swine. We sought to determine if the expression of AHDPs was influenced by infection with PRRSV, and if porcine AHDPs have direct antiviral activity against PRRSV. Because pulmonary alveolar macrophages (PAMs) are primary targets of PRRSV infection, gene expression of porcine AHDPs was evaluated in lungs from fetal and 2-wk-old congenitally infected pigs. In PRRSV-positive lungs and PAMs, gene expression of most porcine AHDPs showed little upregulation. However, gene expression of porcine beta-defensin-1 (pBD-1), pBD-4, pBD-104, pBD-123, and pBD-125 were downregulated more than threefold in 2-wk-old congenitally infected pig lungs. Incubation of PRRSV with pBD-3 or PG-4 significantly inhibited viral infectivity in MARC-145 cells. Using nine protegrin or protegrin-derived peptides, we determined that a cyclic analog of PG-4 increased anti-PRRSV activity, and that substitution of phenylalanine with valine eliminated most PG-4 antiviral activity. In PAMs, pBD-3 and PG-4 at 5-40 microg/mL consistently suppressed PRRSV titers. Collectively, these findings suggest a potential role for some porcine AHDPs as innate antiviral effectors in PRRSV infection. Moreover, modulation of porcine innate immune mechanisms with AHDPs may be one means of limiting the impact of this costly pandemic viral disease.
Assuntos
Síndrome Respiratória e Reprodutiva Suína , Inativação de Vírus/efeitos dos fármacos , beta-Defensinas/biossíntese , Animais , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Linhagem Celular , Chlorocebus aethiops , Feminino , Regulação da Expressão Gênica/imunologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , RNA/análise , RNA/biossíntese , RNA/genética , Suínos , Virulência , beta-Defensinas/imunologia , beta-Defensinas/farmacologiaRESUMO
Despite the small size and conserved tertiary structure of defensins, little is known at a molecular level about the basis of their functional versatility. For insight into the mechanism(s) of defensin function, we prepared enantiomeric pairs of four human defensins, HNP1, HNP4, HD5, and HBD2, and studied their killing of bacteria, inhibition of anthrax lethal factor, and binding to HIV-1 gp120. Unstructured HNP1, HD5, and HBD3 and several other human alpha- and beta-defensins were also examined. Crystallographic analysis showed a plane of symmetry that related (L)HNP1 and (D)HNP1 to each other. Either d-enantiomerization or linearization significantly impaired the ability of HNP1 and HD5 to kill Staphylococcus aureus but not Escherichia coli. In contrast, (L)HNP4 and (D)HNP4 were equally bactericidal against both bacteria. d-Enantiomers were generally weaker inhibitors or binders of lethal factor and gp120 than their respective native, all-l forms, although activity differences were modest, particularly for HNP4. A strong correlation existed among these different functions. Our data indicate: (a) that HNP1 and HD5 kill E. coli by a process that is mechanistically distinct from their actions that kill S. aureus and (b) that chiral molecular recognition is not a stringent prerequisite for other functions of these defensins, including their ability to inhibit lethal factor and bind gp120 of HIV-1.
Assuntos
alfa-Defensinas/química , Alanina/química , Aminobutiratos/química , Animais , Antígenos de Bactérias/química , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/química , Cristalografia por Raios X/métodos , Cisteína/química , Escherichia coli/metabolismo , Humanos , Cinética , Camundongos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/metabolismo , Estereoisomerismo , Ressonância de Plasmônio de SuperfícieRESUMO
Many pathogenic gram-positive bacteria release exotoxins that belong to the family of cholesterol-dependent cytolysins. Here, we report that human alpha-defensins HNP-1 to HNP-3 acted in a concentration-dependent manner to protect human red blood cells from the lytic effects of three of these exotoxins: anthrolysin O (ALO), listeriolysin O, and pneumolysin. HD-5 was very effective against listeriolysin O but less effective against the other toxins. Human alpha-defensins HNP-4 and HD-6 and human beta-defensin-1, -2, and -3 lacked protective ability. HNP-1 required intact disulfide bonds to prevent toxin-mediated hemolysis. A fully linearized analog, in which all six cysteines were replaced by aminobutyric acid (Abu) residues, showed greatly reduced binding and protection. A partially unfolded HNP-1 analog, in which only cysteines 9 and 29 were replaced by Abu residues, showed intact ALO binding but was 10-fold less potent in preventing hemolysis. Surface plasmon resonance assays revealed that HNP-1 to HNP-3 bound all three toxins at multiple sites and also that solution-phase HNP molecules could bind immobilized HNP molecules. Defensin concentrations that inhibited hemolysis by ALO and listeriolysin did not prevent these toxins from binding either to red blood cells or to cholesterol. Others have shown that HNP-1 to HNP-3 inhibit lethal toxin of Bacillus anthracis, toxin B of Clostridium difficile, diphtheria toxin, and exotoxin A of Pseudomonas aeruginosa; however, this is the first time these defensins have been shown to inhibit pore-forming toxins. An "ABCDE mechanism" that can account for the ability of HNP-1 to HNP-3 to inhibit so many different exotoxins is proposed.
Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Colesterol/farmacologia , Proteínas de Choque Térmico/toxicidade , Proteínas Hemolisinas/toxicidade , Hemólise/efeitos dos fármacos , Glicoproteínas de Membrana/toxicidade , Estreptolisinas/toxicidade , alfa-Defensinas/farmacologia , Sequência de Aminoácidos , Animais , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Coelhos , Soro/fisiologia , alfa-Defensinas/química , alfa-Defensinas/metabolismoRESUMO
Four of the six human alpha-defensins (human neutrophil peptides 1-3 and human alpha-defensin 5; HD5) have a lectin-like ability to bind glycosylated proteins. Using HD5 as a model, we applied surface plasmon resonance techniques to gain insights into this property. HD5 bound natural glycoproteins > neoglycoproteins based on BSA > nonglycosylated BSA >> free sugars. The affinity of HD5 for simple sugars covalently bound to BSA was orders of magnitude greater than its affinity for the same sugars in solution. The affinity of HD5 for protein-bound carbohydrates resulted from multivalent interactions which may also involve noncarbohydrate residues of the proteins. HD5 showed concentration-dependent self-association that began at submicromolar concentrations and proceeded to dimer and tetramer formation at concentrations below 5 microM. The (R9A, R28A) and (R13A, R32A) analogs of HD5 showed greatly reduced self-association as well as minimal binding to BSA and to BSA-affixed sugars. From this and other evidence, we conclude that the extensive binding of HD5 to (neo)glycoproteins results from multivalent nonspecific interactions of individual HD5 molecules with carbohydrate and noncarbohydrate moieties of the target molecule and that the primary binding events are magnified and enhanced by subsequent in situ assembly and oligomerization of HD5. Self-association and multivalent binding may play integral roles in the ability of HD5 to protect against infections caused by viruses and other infectious agents.
Assuntos
Antivirais/metabolismo , Metabolismo dos Carboidratos/imunologia , alfa-Defensinas/metabolismo , Acetilglucosamina/metabolismo , Linhagem Celular , Relação Dose-Resposta Imunológica , Glicosilação , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/imunologia , HIV-1/metabolismo , Hemaglutininas Virais/metabolismo , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/metabolismo , Humanos , Ligação Proteica/imunologia , Soroalbumina Bovina/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
We have reported that the alpha-defensins human neutrophil peptides (HNP)-1 and HNP-2 neutralize and aggregate influenza A virus (IAV) and promote uptake of IAV by neutrophils. These alpha-defensins were also shown to bind to surfactant protein (SP)-D and reduce its antiviral activity. In this study, we examined retrocyclin (RC)1 and RC2, humanized versions of the antiviral theta-defensins found in the leukocytes of certain nonhuman primates. RC1 was just as effective as HNP-1-3 in neutralizing IAV, and RC2 and RC101 (an analog of RC1) were more effective. In contrast, human beta-defensins (HBDs) showed less neutralizing activity. Human defensins 5 and 6 (mainly produced by intestinal Paneth cells) had viral neutralizing activity similar to HNP-1-3. Like HNP-1-3, RCs induced viral aggregation and promoted the uptake of IAV by neutrophils. We used surface plasmon resonance to evaluate binding of defensins to SP-D. HBDs, HD6, and HNP-4 bound minimally to SP-D. HNP-1-3 and RCs bound SP-D with high affinity; however, unlike HNP-1 and HNP-2, RCs did not inhibit SP-D antiviral activity. HBDs also did not inhibit antiviral activity of SP-D. Given their strong neutralizing activity and compatibility with SP-D, RCs may provide attractive prototypes for designing therapeutics that can prevent or treat respiratory infections caused by IAV.
Assuntos
Defensinas/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Proteína D Associada a Surfactante Pulmonar/imunologia , alfa-Defensinas/imunologia , beta-Defensinas/imunologia , Animais , Linhagem Celular , Galinhas , Cricetinae , Defensinas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Neutrófilos/imunologia , Neutrófilos/metabolismo , Ligação Proteica , Proteína D Associada a Surfactante Pulmonar/metabolismo , alfa-Defensinas/metabolismo , beta-Defensinas/metabolismoRESUMO
Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins-18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue "demidefensin" precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.