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1.
Int J Mol Sci ; 22(4)2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33546369

RESUMO

Antimicrobial peptides (AMPs) possess great potential for combating drug-resistant bacteria. Thanatin is a pathogen-inducible single-disulfide-bond-containing ß-hairpin AMP which was first isolated from the insect Podisus maculiventris. The 21-residue-long thanatin displays broad-spectrum activity against both Gram-negative and Gram-positive bacteria as well as against various species of fungi. Remarkably, thanatin was found to be highly potent in inhibiting the growth of bacteria and fungi at considerably low concentrations. Although thanatin was isolated around 25 years ago, only recently has there been a pronounced interest in understanding its mode of action and activity against drug-resistant bacteria. In this review, multiple modes of action of thanatin in killing bacteria and in vivo activity, therapeutic potential are discussed. This promising AMP requires further research for the development of novel molecules for the treatment of infections caused by drug resistant pathogens.


Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias/efeitos dos fármacos , Fungos/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros
2.
Phys Chem Chem Phys ; 23(2): 1475-1488, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33399594

RESUMO

A promising alternative to classical antibiotics are antimicrobial peptides and their synthetic mimics (smAMPs) that supposedly act directly on membranes. For a more successful design of smAMPs, we need to know how the type of interaction with the membrane determines the type of membrane perturbation. How this, in turn, transfers into selectivity and microbial killing activity is largely unknown. Here, we characterize the action of two smAMPs: MM:CO (a copolymer of hydrophobic cyclooctyl subunits and charged ß-monomethyl-α-aminomethyl subunits) and the highly charged poly-NM (a homopolymer of α-aminomethyl subunits). By thorough characterization of vesicle leakage experiments, we elucidate complex membrane perturbation behavior in zwitterionic or negatively charged vesicles. Vesicle leakage data does not entirely agree with the growth inhibition of microbes. Our ensemble of advanced membrane permeabilization approaches clarifies these discrepancies. Long cumulative leakage kinetics show that the two smAMPs act either by transient leakage or by rare stochastic leakage events that occur at charge neutralization in the sample. We determine the strengths of individual leakage events induced by the smAMPs in membranes of various compositions. These strengths indicate changes in leakage mechanism over time and concentration range. Thus, our sophisticated analysis of vesicle leakage experiments reveals a fine-tuned flexibility in membrane permeabilization mechanisms. These details are indispensable in judging and designing membrane-active compounds.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Lipossomas Unilamelares/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Fluoresceínas/química , Glicerofosfatos/química , Interações Hidrofóbicas e Hidrofílicas , Permeabilidade/efeitos dos fármacos , Ligação Proteica , Eletricidade Estática , Lipossomas Unilamelares/química
3.
Arch Biochem Biophys ; 699: 108751, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33421380

RESUMO

Antimicrobial and cell-penetrating peptides have been the object of extensive studies for more than 60 years. Initially these two families were studied separately, and more recently parallels have been drawn. These studies have given rise to numerous methodological developments both in terms of observation techniques and membrane models. This review presents some of the most recent original and innovative developments in this field, namely droplet interface bilayers (DIBs), new fluorescence approaches, force measurements, and photolabelling.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Peptídeos Penetradores de Células/química , Corantes Fluorescentes/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microscopia de Fluorescência/métodos , Marcadores de Fotoafinidade/química , Espectrometria de Fluorescência/métodos
4.
Int J Mol Sci ; 22(2)2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33451135

RESUMO

Antimicrobial peptides (AMPs) are intensively studied in terms of alternative drugs. Sub5 is a synthetic 12-mer AMP with substitutions of five amino acids of bactenecin 2A (Bac2A), a linear-ized bactenecin variant of bovine. Sub5 is highly effective against fungi with an ability to trans-locate cell membrane, but its targets are unknown. Systematic analysis of Sub5 targets will facil-itate our understanding on its mechanism of action. In this study, we used high-throughput Saccharomyces cerevisiae proteome microarrays to explore the potential protein targets of Sub5. The screening results showed 128 potential protein targets of Sub5. Bioinformatics analysis of protein targets of Sub5 revealed significant gene ontology (GO) enrichment in actin related pro-cess of "actin filament-based process", "actin filament organization", "actin cortical patch or-ganization", regulation of "actin filament bundle assembly". Moreover, the other enriched cat-egories in GO enrichment mostly contained actin associate proteins. In total, 11 actin-associated proteins were identified in the protein targets of Sub5. Protein family (PFAM) enrichment anal-ysis shows protein domain enriched in actin binding, i.e., "Cytoskeletal-regulatory complex EF hand (helix E-loop-helix F motif)". Being consistent with GO analysis, Search Tool for the Re-trieval of Interacting Genes/Proteins (STRING) analysis of the protein targets of Sub5 showed ac-tin network with involvement of 15 protein targets. Along with actin-network, STRING analysis showed protein-protein interaction network in ribonucleoprotein, transcription and translation, chromosome, histone, and ubiquitin related, DNA repair, and chaperone. Multiple Expression motifs for Motif Elicitation (MEME) suite provided a consensus binding motif of [ED][ED]EEE[ED][ED][ED][ED][ED], in total of 75 protein targets of Sub5. This motif was present in 9 out of 15 actin-related proteins identified among protein targets of Sub5.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteoma , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Proteínas de Transporte , Biologia Computacional/métodos , Ontologia Genética , Anotação de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos
5.
J Dairy Sci ; 104(2): 2106-2122, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33358157

RESUMO

Timely and objective diagnosis and classification of mastitis is crucial to ensure adequate management and therapeutic decisions. Analyzing specific biomarkers in milk could be advantageous compared with subjective or semiquantitative criteria, such as palpation of the udder in clinical mastitis cases or evaluation of somatic cell count using cow side tests (e.g., California Mastitis Test) in subclinical mastitis quarters. The objective of this study was to investigate the diagnostic value of 3 biomarkers; cathelicidin, milk amyloid A, and haptoglobin for the diagnosis of subclinical and clinical mastitis. Furthermore, the suitability of these biomarkers to differentiate between mild, moderate, and severe clinical mastitis and the influence of different pathogens on biomarker levels was tested. A total of 67 healthy cows, 119 cows with subclinical mastitis, and 212 cows with clinical mastitis were enrolled in the study. Although cathelicidin, haptoglobin, and milk amyloid A were measured in all samples from healthy cows and those with subclinical mastitis, haptoglobin, and cathelicidin results were only available from 121 out of 212 cows with clinical mastitis. Milk amyloid A was measured in all samples. In cows with clinical mastitis, the mastitic quarter and a second healthy quarter serving as a healthy in-cow control quarter were sampled. It was possible to differentiate between healthy quarters, quarters with subclinical mastitis, and quarters with clinical mastitis using all 3 biomarkers. Concerning cathelicidin, thresholds were 0.000 [sensitivity (Se) = 0.83, specificity (Sp) = 0.97] and 0.053 (Se = 0.98, Sp = 0.99) for normalized optical density at 450 nm (NOD450) for differentiating between healthy quarters and quarters with subclinical or clinical mastitis, respectively. Thresholds of 1.28 µg/mL (Se = 0.65, Sp = 0.76) and 1.81 µg/mL (Se = 0.77, Sp = 0.83) for milk amyloid A and 3.65 µg/mL (Se = 0.92, Sp = 0.94) and 5.40 µg/mL mL (Se = 0.96, Sp = 0.99) for haptoglobin were calculated, respectively. Healthy in-cow control quarters from cows with CM showed elevated milk amyloid A and haptoglobin levels compared with healthy quarters from healthy cows. Only the level of milk amyloid A was higher in severe clinical mastitis cases compared with mild ones. In contrast to clinical mastitis, cathelicidin and haptoglobin in subclinical mastitis quarters were significantly influenced by different bacteriological results. The measurement of cathelicidin, milk amyloid A, and haptoglobin in milk proved to be a reliable method to detect quarters with subclinical or clinical mastitis.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Haptoglobinas/metabolismo , Mastite Bovina/diagnóstico , Proteína Amiloide A Sérica/metabolismo , Animais , Biomarcadores/metabolismo , Bovinos , Contagem de Células/veterinária , Feminino , Glândulas Mamárias Animais , Leite/citologia
6.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33372152

RESUMO

Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models of Staphylococcus aureus or Candida albicans infection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.


Assuntos
Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Sequência de Aminoácidos/genética , Animais , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Encéfalo/imunologia , Encéfalo/metabolismo , Morte Celular/efeitos dos fármacos , Simulação por Computador , Bases de Dados Genéticas , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neuropeptídeos/metabolismo , Filogenia , Transdução de Sinais/fisiologia
7.
J Insect Sci ; 20(6)2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33347588

RESUMO

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.


Assuntos
Moscas Domésticas/imunologia , Proteínas de Insetos/isolamento & purificação , Intestinos/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Perfilação da Expressão Gênica , Genes de Insetos , Moscas Domésticas/genética , Moscas Domésticas/metabolismo , Imunidade Inata/fisiologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/imunologia , Larva/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA
8.
PLoS One ; 15(12): e0238675, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33370283

RESUMO

Host defense peptides (HDPs) are an important first line of defense with antimicrobial and immunomodulatory properties. Selection for increased body weight is hypothesized to be related to reduced immune response. We studied the relationships among body weight, age, and the HDP expression patterns in intestine and immune organs. We used chickens with marked differences of body sizes. The non-selected Daweishan mini chickens showed the highest indexes of immune organs and the lowest concentrations of the plasma immune parameters C3, C4, IgA, and IgY, while the commercial Avian broiler showed the opposite results. The Daweishan mini chickens showed the highest mRNA expressions of HDP genes in small intestine followed by the semi-selected Wuding chickens. Compared with local breeds, broiler chickens showed higher mRNA expression of HDP genes in spleen, thymus, and bursa. Body weight and HDP expression levels were negatively correlated in the intestine and positively in the immune organs. Our results indicated that the HDP immune regulatory roles in small intestine acted as first line of defense in innate immunity in local breeds, and as an adaptive immunity in broiler chickens. Selection was associated with different expression expressions of HDP genes in breed-, age-, and organ-specific manners.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Aviárias/genética , Peso Corporal/genética , Galinhas/genética , Expressão Gênica , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Aviárias/metabolismo , Galinhas/metabolismo , Perfilação da Expressão Gênica , Imunidade Inata , Intestino Delgado/metabolismo , Baço/metabolismo , Distribuição Tecidual
9.
Science ; 370(6514)2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060333

RESUMO

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and a source of nutrients for intracellular pathogens. We demonstrate that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated by danger signals. In response to lipopolysaccharide (LPS), multiple host defense proteins, including interferon-inducible guanosine triphosphatases and the antimicrobial cathelicidin, assemble into complex clusters on LDs. LPS additionally promotes the physical and functional uncoupling of LDs from mitochondria, reducing fatty acid metabolism while increasing LD-bacterial contacts. Thus, LDs actively participate in mammalian innate immunity at two levels: They are both cell-autonomous organelles that organize and use immune proteins to kill intracellular pathogens as well as central players in the local and systemic metabolic adaptation to infection.


Assuntos
Bactérias/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Gotículas Lipídicas/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Ácidos Graxos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/imunologia
10.
Mol Immunol ; 126: 14-24, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32739720

RESUMO

Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) form a subfamily of the large superfamily of G-protein-coupled receptors. LGRs can be divided into three groups. LGR2 from Drosophila melanogaster is involved in cuticle tanning (melanization and sclerotization). In this study, one LGR2 (MnLGR2) was identified from Macrobrachium nipponense. MnLGR2 has an open reading frame of 4515 bp encoding a protein with 1504 amino acids. MnLGR2 is comprised of a 7-transmembrane domain, 12 leucine-rich repeats, and 5 low-complexity regions. The highest expression level of MnLGR2 was observed in gills. The expression levels of MnLGR2 in gills and stomach could be regulated by bacterial challenge. Knockdown of MnLGR2 upregulated the expression of anti-microbial peptide (AMP) genes. Further study indicated that inhibition of AMP expression by MnLGR2 was through inhibition of relish-mediated AMP expression. In addition to the negative regulation of AMP expression, MnLGR2 participated in positive regulation of phenol oxidase (PO) activity and expression of proPO activating pathway-related genes (proPO-activating factor and proPO-activating enzymes). Therefore, MnLGR2 plays an important role in prawn innate immunity.


Assuntos
Proteínas de Artrópodes/metabolismo , Palaemonidae/fisiologia , Proteínas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Mucosa Gástrica/metabolismo , Técnicas de Silenciamento de Genes , Brânquias/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade Inata , Muda/fisiologia , Monofenol Mono-Oxigenase/metabolismo , Palaemonidae/microbiologia , Proteínas/genética , Receptores Acoplados a Proteínas-G/genética , Staphylococcus aureus/imunologia , Fatores de Transcrição , Regulação para Cima/imunologia , Vibrio parahaemolyticus/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia
11.
Mol Immunol ; 126: 65-72, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32768860

RESUMO

The insect gut participates in initial local immune responses by producing reactive oxygen and nitrogen species as well as anti-microbial peptides to resist pathogenic invasions. Nitric oxide (NO), a signaling and an immune effector molecule synthesized by the enzyme NO synthase (NOS), mediates an early step of the signal transduction pathway. In this study, we evaluated NO levels after Nosema pernyi infection in Antheraea pernyi gut. NOS activity was higher in the microsporidia-infected gut of A. pernyi than in that of control. Three NOS-related genes were cloned, and their spatio-temporal expression patterns were evaluated. ApNOS2 was expressed quickly in the midgut after N. pernyi infection. Sodium nitroprusside, dihydrate (SNP), or Nω-L-nitro-arginine methyl ester, hydrochloride (L-NAME), altered the NO content in A. pernyi midgut. Anti-microbial peptides (AMPs) in the groups exposed to N. pernyi plus SNP and N. pernyi plus L-NAME exhibited higher and lower expression, respectively, relative to the control. These results indicate that microsporidia infection triggers short-term activation of NO and NOS genes in the A. pernyi gut that is downregulated after 24 h. Notably, infection rates can be influenced by a NOS inhibitor. Furthermore, NO can be induced by pathogens. Similarly, NO content in the A. pernyi gut also influences AMPs in humoral immunity and some immune-related genes. Our results suggest that nitric oxide plays a vital role in A. pernyi gut immunity.


Assuntos
Trato Gastrointestinal/imunologia , Microsporidiose/veterinária , Mariposas/imunologia , Óxido Nítrico/metabolismo , Nosema/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/antagonistas & inibidores , Proteínas de Artrópodes/metabolismo , Regulação para Baixo , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Imunidade Humoral/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Microsporidiose/imunologia , Mariposas/enzimologia , Mariposas/microbiologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Análise Espaço-Temporal
12.
Proc Natl Acad Sci U S A ; 117(32): 19446-19454, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723829

RESUMO

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibiotic-resistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-state 15N NMR data. While horine was potent primarily against gram-positive pathogens, verine showed broad-spectrum antimicrobial activity. Both peptides protected greater than 80% mice from infection-caused deaths. Moreover, horine and verine also displayed significant systemic efficacy in different murine models comparable to conventional antibiotics. In addition, they could eliminate resistant pathogens and preformed biofilms. Significantly, the peptides showed no nephrotoxicity to mice after intraperitoneal or intravenous administration for 1 wk. Our study underscores the significance of horine and verine in fighting drug-resistant pathogens.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/metabolismo , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Infecções Bacterianas/tratamento farmacológico , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Membrana Celular/metabolismo , Bases de Dados de Proteínas , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Resultado do Tratamento
13.
Life Sci ; 257: 118111, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32682918

RESUMO

BACKGROUND AND AIMS: Bacterial translocation (BT) is strongly associated with disease progression and poor outcome in cirrhotic patients. The role of Pregnane X receptor (PXR) in regulating bacterial translocation in cirrhosis is unknown. We previously showed that Ginkgolide-A (GA), a natural PXR ligand, attenuated BT in cirrhotic mice by abrogating inflammation along the gut-liver-axis, and by protecting small intestinal tight junctions (TJ). Here, we aimed to investigate the effect of GA in activating PXR and associated antimicrobial peptides (AMPs) in regulating BT in experimental cirrhosis. METHODS: Male Swiss albino mice were administered CCl4 (0.5 mL/kg body-weight, i.p twice a week) for 12 consecutive weeks. After the 12th week, mice were randomized and administered with GA (100-mg/kg body-weight, oral) every-day for 2 weeks. At termination, blood, gut and liver tissues were collected for molecular studies. RESULTS: GA treatment to cirrhotic mice significantly increased the expression of small intestinal PXR and Regenerating family member 3 alpha (Reg3A), which were otherwise reduced in CCl4 cirrhotic mice. Moreover, compared to naive mice a significantly reduced Lactobacillus, and increased Bacteroides and Enterococcus 16s rRNA levels were observed in the small intestine and liver of cirrhotic mice. Treatment with GA to cirrhotic mice significantly reduced intestinal overgrowth and translocation of Enterococcus and Bacteroides to the liver. Furthermore, GA treatment significantly attenuated intestinal permeability and BT marker soluble-CD14 (sCD14), which were increased in CCl4 cirrhotic mice. CONCLUSION: The study showed for the first time that, GA treatment to cirrhotic rodents attenuates BT, by improving PXR and Reg3A expression.


Assuntos
Translocação Bacteriana/efeitos dos fármacos , Ginkgolídeos/farmacologia , Lactonas/farmacologia , Cirrose Hepática Experimental/metabolismo , Proteínas Associadas a Pancreatite/metabolismo , Receptor de Pregnano X/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Western Blotting , Células Hep G2 , Humanos , Cirrose Hepática Experimental/complicações , Masculino , Camundongos , Simulação de Acoplamento Molecular , Receptor de Pregnano X/efeitos dos fármacos
14.
Arch Oral Biol ; 117: 104827, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32673820

RESUMO

OBJECTIVE: The aim of the study was to assess whether or not Vitamin D deficiency was associated with the GCF and gingival tissue antimicrobial peptides (AMP), namely, human beta defensin-2 (hBD-2) and cathelicidin (LL-37) level in chronic periodontitis (CP) and gingivitis patients. DESIGN: A total of 80 volunteers were included in this study. Forty was classified as Vitamin D deficient (25(OH)D <20 ng/mL), and 40 Vitamin D sufficient patients (25(OH)D ≥ 20 ng/mL). Of these, 20 of them were affected by gingivitis and 20 by CP. Following sampling, the hBD-2 and LL-37 concentration in gingival tissues and GCF were determined by the ELISA method. RESULTS: The hBD-2 and LL-37 levels were higher in periodontitis compared to gingivitis patients within Vitamin D sufficient and deficient groups. The AMP levels of GCF and gingival tissue in the vitamin D deficient group was lower compared to sufficient serum 25(OH)D within gingivitis and CP groups. Additionally, a non-parametric regression model known as the generalized additive model was used to identify the contribution of diagnosis, Vitamin D status, and other potential clinical variables on the local levels of AMPs. Regression analysis showed that the periodontal disease status, serum vitamin D concentration were independent predictors for elevated GCF AMP levels. Finally, a positive correlation between GCF and tissue levels of both hBD-2 (r = 0.82; <0.0001) and LL-37 (r = 0.65; <0.0001) was detected. CONCLUSION: This study shows that serum 25(OH)D deficiency is associated with decreased hBD-2 and LL-37 expression of GCF and gingival tissues in both gingivitis and CP patients.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Periodontite Crônica , Gengivite , Vitamina D/sangue , beta-Defensinas/metabolismo , Líquido do Sulco Gengival , Humanos
15.
Appl Environ Microbiol ; 86(19)2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32709730

RESUMO

NisI confers immunity against nisin, with high substrate specificity to prevent a suicidal effect in nisin-producing Lactococcus lactis strains. However, the NisI maturation process as well as its influence on nisin resistance has not been characterized. Here, we report the roles of lipoprotein signal peptidase II (Lsp) and prolipoprotein diacylglyceryl transferase (Lgt) in NisI maturation and nisin resistance of L. lactis F44. We found that the resistance of nisin of an Lsp-deficient mutant remarkably decreased, while no significant differences in growth were observed. We demonstrated that Lsp could cleave signal peptide of NisI precursor in vitro Moreover, diacylglyceryl modification of NisI catalyzed by Lgt played a decisive role in attachment of NisI on the cell envelope, while it exhibited no effects on cleavage of the signal peptides of NisI precursor. The dissociation constant (KD ) for the interaction between nisin and NisI exhibited a 2.8-fold increase compared with that between nisin and pre-NisI with signal peptide by surface plasmon resonance (SPR) analysis, providing evidence that Lsp-catalyzed signal peptide cleavage was critical for the immune activity of NisI. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.IMPORTANCE Nisin, a safe and natural antimicrobial peptide, has a long and impressive history as a food preservative and is also considered a novel candidate to alleviate the increasingly serious threat of antibiotic resistance. Nisin is produced by certain L. lactis strains. The nisin immunity protein NisI, a membrane-bound lipoprotein, is expressed by nisin producers to avoid suicidal action. Here, we report the roles of Lsp and Lgt in NisI maturation and nisin resistance of L. lactis F44. The results verified the importance of Lsp to NisI-conferred immunity and Lgt to localization. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.


Assuntos
Proteínas de Bactérias/genética , Lactococcus lactis/genética , Lipoproteínas/genética , Proteínas de Membrana/genética , Nisina/genética , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Lactococcus lactis/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Nisina/metabolismo , Transferases/genética , Transferases/metabolismo
16.
Nat Commun ; 11(1): 2703, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483147

RESUMO

Mother's milk is the best choice for infants nutrition, however when it is not available or insufficient to satisfy the needs of the infant, formula is proposed as an effective substitute. Here, we report the results of a randomized controlled clinical trial (NCT03637894) designed to evaluate the effects of two different dietary regimens (standard formula and Lactobacillus paracasei CBA L74-fermented formula) versus breastfeeding (reference group) on immune defense mechanisms (primary endpoint: secretory IgA, antimicrobial peptides), the microbiota and its metabolome (secondary outcomes), in healthy full term infants according to the type of delivery (n = 13/group). We show that the fermented formula, safe and well tolerated, induces an increase in secretory IgA (but not in antimicrobial peptides) and reduces the diversity of the microbiota, similarly, but not as much as, breastmilk. Metabolome analysis allowed us to distinguish subjects based on their dietary regimen and mode of delivery. Together, these results suggest that a fermented formula favors the maturation of the immune system, microbiota and metabolome.


Assuntos
Dieta , Sistema Imunitário/fisiologia , Metaboloma/fisiologia , Microbiota/fisiologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Aleitamento Materno , Método Duplo-Cego , Fezes/microbiologia , Feminino , Fermentação , Humanos , Imunoglobulina A Secretora/metabolismo , Fórmulas Infantis , Recém-Nascido , Lactobacillus paracasei/metabolismo , Masculino , Leite Humano , beta-Defensinas/metabolismo
17.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 32(3): 330-335, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32385998

RESUMO

OBJECTIVE: To investigate the effect of heparin-binding protein (HBP) on the damage of vascular permeability in early burn. METHODS: (1) Clinical research: 12 patients with severe burns admitted to Suzhou Hospital of Nanjing Medical University from January 1st to August 30th in 2019 were enrolled. Eight patients with severe trauma admitted to the same hospital during the same period were also enrolled as controls to explain the specificity of burn injury. Whole blood samples were obtained within 0.5 hour after admission. The white blood cell count (WBC), absolute value and ratio of neutrophils, and serum HBP levels were measured. Serum samples of 12 patients with severe burn were collected within 9 days after admission, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of metabolism products of glycocalyx including syndecan-1 and hyaluronic acid (HA). The correlation between HBP and neutrophils ratio, syndecan-1 and HA were analyzed by linear correlation. (2) Basic research: a 30% total body surface area (TBSA) III degree angle burn model of Sprague-Dawley (SD) rat aged 6-8 weeks was prepared. In low molecular weight heparin (LMWH) intervention group (n = 5), 200 U/kg LMWH was injected subcutaneously immediately and every 2 hours after injury for 4 times in total; the burn group (n = 5) was given the same amount of normal saline. No intervention was given to the normal control group (n = 5). The peripheral venous blood was collected at 0, 2, 4, and 8 hours after injury, and the serum levels of HBP, syndecan-1 and HA were measured; the injury of glycocalyx on pulmonary vascular endothelial cells was observed under transmission electron microscope. RESULTS: (1) Clinical research results: the WBC, neutrophils absolute value and ratio, and HBP levels were increased in 12 patients with severe burn and 8 patients with severe trauma. There was no significant difference in the WBC, absolute value and ratio of neutrophils between severe burn and severe trauma patients [WBC (×109/L): 14.5±6.1 vs. 10.8±3.6, the absolute value of neutrophils (×109/L): 12.0±5.9 vs. 9.0±4.0, the ratio of neutrophils: 0.81±0.10 vs. 0.79±0.14, all P > 0.05], but the HBP levels in the burn patients were significantly higher than those in the trauma patients (µg/L: 192.92±61.73 vs. 51.17±23.05, P < 0.01). Twelve patients with severe burns had a sharp increase in serum syndecan-1 and HA levels after burns, which continued to maintain high levels and peaked at the 9th day [syndecan-1 (µg/L): 16.02±0.39, HA (µg/L): 106.83±4.90]. The analysis showed that HBP was positively correlated with neutrophils ratio, syndecan-1 and HA in severe burn patients at the 1st day after admission (r values were 0.805, 0.732 and 0.900, respectively, all P < 0.01). It indicated that the sharp increase of neutrophils after the burn released a lot of HBP, and the glycocalyx of the vascular endothelium was severely damaged. (2) Basic research results: the levels of serum HBP, syndecan-1 and HA in the burn group were increased sharply as compared with the normal control group, and continued to increase with time, reaching a peak at 8 hours after burn. In the LMWH intervention group, the serum levels of HBP, syndecan-1 and HA were significantly lower than those in the burn group, and the difference was still statistically significant after 8 hours [HBP (µg/L): 6.47±0.25 vs. 12.48±0.08, syndecan-1 (µg/L): 19.06±1.48 vs. 25.92±3.34, HA (µg/L): 35.76±2.10 vs. 54.91±2.64, all P < 0.01]. The results of transmission electron microscopy showed that in the normal control group, the glycocalyx pulmonary vascular endothelial cells was continuous, evenly distributed and dense. The glycocalyx on pulmonary vascular endothelial cells of rats were significantly damaged and shed 2 hours after burn in the burn group, and no glycocalyx was observed at 8 hours. In the LMWH intervention group, the glycocalyx on pulmonary vascular endothelial cells was damaged and the phenomenon of shedding was significantly relieved, and the glycocalyx could be observed 8 hours after the injury. CONCLUSIONS: The massive exudation of body fluids and the significant increase of vascular permeability in patients in early burns may be related to the destruction of the glycocalyx on endothelial cells by HBP released from increased neutrophils.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Permeabilidade Capilar , Heparina de Baixo Peso Molecular , Animais , Células Endoteliais , Humanos , Ratos , Ratos Sprague-Dawley
18.
Soft Matter ; 16(21): 5032-5043, 2020 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-32452495

RESUMO

Amphipathic peptides that partition into lipid bilayers affect the curvature elastic properties of their host. Some of these peptides are able to shift the Gaussian modulus to positive values, thus triggering an instability with respect to the formation of saddle curvatures. To characterize the generic aspects of the underlying mechanism, we employ a molecular lipid model that accounts for the interfacial tension between the polar and apolar regions of the membrane, for interactions between the lipid headgroups, and for the energy to stretch or compress the hydrocarbon chains. Peptides are modeled as cylinders that partition into the host membrane in a parallel orientation where they diminish the space available to the lipid headgroups and chains. The penetration depth into the membrane is determined by the angular size of the peptide's hydrophilic region. We demonstrate that only peptides with a small angular size of their hydrophilic region have an intrinsic tendency to render the Gaussian modulus more positive, and we identify conditions at which the Gaussian modulus adopts a positive sign upon increasing the peptide concentration. Our model allows us to also incorporate electrostatic interactions between cationic peptides and anionic lipids on the level of the linear Debye-Hückel model. We show that electrostatic interactions tend to shift the Gaussian modulus toward more positive values. Steric and electrostatic lipid-peptide interactions jointly decrease the effective interaction strength in the headgroup region of the host membrane thus suggesting a generic mechanisms of how certain amphipathic peptides are able to induce the formation of saddle curvatures.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Peptídeos Catiônicos Antimicrobianos/metabolismo , Ligação Proteica , Domínios Proteicos
19.
Int J Mol Sci ; 21(7)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32244587

RESUMO

Peptidoglycan recognition proteins (PGRPs) play an important role in the defense against invading microbes via the recognition of the immunogenic substance peptidoglycan (PGN). Bees possess fewer PGRPs than Drosophila melanogaster and Anopheles gambiae but retain two important immune pathways, the Toll pathway and the Imd pathway, which can be triggered by the recognition of Dap-type PGN by PGRP-LCx with the assistance of PGRP-LCa in Drosophila. There are three isoforms of PGRP-LC including PGRP-LCx, PGRP-LCa and PGRP-LCy in Drosophila. Our previous study showed that a single PGRP-LC exists in bumblebees. In this present study, we prove that the bumblebee Bombus lantschouensis PGRP-LC (Bl-PGRP-LC) can respond to an infection with Gram-negative bacterium Escherichia coli through binding to the Dap-type PGNs directly, and that E. coli infection induces the quick and strong upregulation of PGRP-LC, abaecin and defensin. Moreover, the Bl-PGRP-LC exhibits a very strong affinity for the Dap-type PGN, much stronger than the affinity exhibited by the PGRP-LC from the more eusocial honeybee Apis mellifera (Am-PGRP-LC). In addition, mutagenesis experiments showed that the residue His390 is the anchor residue for the binding to the Dap-type PGN and forms a hydrogen bond with MurNAc rather than meso-Dap, which interacts with the anchor residue Arg413 of PGRP-LCx in Drosophila. Therefore, bumblebee PGRP-LC possesses exclusive characteristics for the immune response among insect PGRPs.


Assuntos
Abelhas/imunologia , Proteínas de Transporte/imunologia , Escherichia coli/imunologia , Peptidoglicano/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Abelhas/química , Abelhas/microbiologia , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Defensinas/genética , Defensinas/imunologia , Defensinas/metabolismo , Proteínas de Drosophila/imunologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Drosophila melanogaster/metabolismo , Escherichia coli/fisiologia , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/imunologia , Bactérias Gram-Positivas/fisiologia , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Proteínas de Insetos/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Homologia de Sequência de Aminoácidos
20.
Anim Sci J ; 91(1): e13365, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32285581

RESUMO

The present study was conducted to examine whether colostrum supplementation in peripartum goats increases the antimicrobial peptides in their milk. Goats were orally administered 2 ml of colostrum whey products (colostrum group) or water (control group) daily, from 2 weeks before until 2 weeks after kidding. Body weights of mothers and kids were measured. Blood, milk, and fecal samples were collected from the mothers, and blood samples were collected from the kids. Concentrations of milk antimicrobial peptides (beta-defensin, cathelicidin, lactoferrin, S100A7, lactoperoxidase, and immunoglobulin A [IgA]) were determined. IgA and nutritional parameters (glucose, total cholesterol, triglyceride, ketone bodies, and non-esterified fatty acids) were also determined in the blood of mothers and kids. Milk IgA and lactoferrin concentrations were higher in the colostrum group than in the control group. Conversely, lower milk concentrations of S100A7 were observed in the colostrum group than that in the control group. Plasma IgA concentrations were higher for kids from the colostrum group than for those from the control group. These results suggest that oral administration of colostrum in pregnant goats increases IgA concentration in postpartum milk, which can subsequently improve the health of their kids.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Peptídeos Catiônicos Antimicrobianos/metabolismo , Colostro , Dieta/veterinária , Suplementos Nutricionais , Lactoferrina/metabolismo , Leite/metabolismo , Proteínas do Soro do Leite/administração & dosagem , beta-Defensinas/metabolismo , Administração Oral , Animais , Feminino , Cabras , Imunoglobulina A/metabolismo , Lactoperoxidase/metabolismo , Período Periparto , Gravidez
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