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1.
World J Microbiol Biotechnol ; 36(2): 30, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32025825

RESUMO

The objective of this study was to determine whether seeds of Brassica oleracea var. italica (i.e. broccoli, an edible plant) produce defensins that inhibit phytopathogenic fungi and pathogenic bacteria of clinical significance. Crude extracts obtained from broccoli seeds were fractioned by molecular exclusion techniques and analyzed by liquid chromatography-high-resolution mass spectrometry. Two peptides were identified, BraDef1 (10.68 kDa) and BraDef2 (9.9 kDa), which were categorized as Class I defensins based on (a) their primary structure, (b) the presence of four putative cysteine disulfide bridges, and (c) molecular modeling predictions. BraDef1 and BraDef2 show identities of, respectively, 98 and 71%, and 67 and 85%, with defensins from Brassica napus and Arabidopsis thaliana. BraDef (BraDef1 + BraDef2) disrupted membranes of Colletotrichum gloeosporioides and Alternaria alternata and also reduced hyphal growth of C. gloeosporioides by ~ 56% after 120 h of incubation. Pathogenic bacteria (Bacillus cereus 183, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, and Vibrio parahaemolitycus) were susceptible to BraDef, but probiotic bacteria such as Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus casei were not inhibited. To our knowledge, this is the first report of defensins present in seeds of B. oleracea var. italica (i.e. edible broccoli). Our findings suggest an applied value for BraDef1/BraDef2 in controlling phytopathogenic fungi and pathogenic bacteria of clinical significance.


Assuntos
Anti-Infecciosos/farmacologia , Brassica/química , Defensinas/farmacologia , Sequência de Aminoácidos , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Bactérias/efeitos dos fármacos , Cromatografia Líquida , Defensinas/química , Defensinas/isolamento & purificação , Fungos/efeitos dos fármacos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Modelos Moleculares , Extratos Vegetais/química , Sementes/química
2.
PLoS One ; 15(2): e0228876, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32059041

RESUMO

Citrus Huanglongbing (HLB), which is also known as citrus greening, is a destructive disease continuing to devastate citrus production worldwide. Although all citrus varieties can be infected with 'Candidatus Liberibacter asiaticus' (CaLas), a certain level of HLB tolerance of scion varieties can be conferred by some rootstocks. To understand the effects of rootstock varieties on orange fruit under CaLas stress, comparative iTRAQ proteomic profilings were conducted, using fruit from 'Valencia' sweet orange grafted on the sensitive ('Swingle') and tolerant rootstocks (a new selection called '46x20-04-48') infected by CaLas as experimental groups, and the same plant materials without CaLas infection as controls. The symptomatic fruit on 'Swingle' had 573 differentially-expressed (DE) proteins in comparison with their healthy fruit on the same rootstock, whereas the symptomatic fruit on '46x20-04-48' had 263 DE proteins. Many defense-associated proteins were down-regulated in the symptomatic fruit on 'Swingle' rootstock that were seldom detected in the symptomatic fruit on the '46x20-04-48' rootstock, especially the proteins involved in the jasmonate biosynthesis (AOC4), jasmonate signaling (ASK2, RUB1, SKP1, HSP70T-2, and HSP90.1), protein hydrolysis (RPN8A and RPT2a), and vesicle trafficking (SNAREs and Clathrin) pathways. Therefore, we predict that the down-regulated proteins involved in the jasmonate signaling pathway and vesicle trafficking are likely to be related to citrus sensitivity to the CaLas pathogen.


Assuntos
Citrus sinensis/genética , Citrus sinensis/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Rhizobiaceae/patogenicidade , Citrus sinensis/metabolismo , Defensinas/genética , Defensinas/metabolismo , Resistência à Doença/genética , Frutas/genética , Frutas/metabolismo , Frutas/microbiologia , Reguladores de Crescimento de Planta/genética , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Reação em Cadeia da Polimerase , Proteoma/genética , Proteoma/metabolismo , Proteômica , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
PLoS One ; 15(1): e0227606, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935243

RESUMO

Cardiovascular disease (CVD) risks persist in patients despite treatment. CVD susceptibility also varies with sex and ethnicity and is not entirely explained by conventional CVD risk factors. The aim of the present study was to identify novel CVD candidate markers in circulating Peripheral blood mononuclear cells (PBMCs) and plasma from Arab obese subjects with and without CVD using proteomic approaches. Human adults with confirmed CVD (n = 208) and matched non-CVD controls (n = 152) living in Kuwait were examined in the present cross-sectional study. Anthropometric and classical biochemical parameters were determined. We employed a shotgun proteomic profiling approach on PBMCs isolated from a subset of the groups (n = 4, each), and differentially expressed proteins selected between the two groups were validated at the mRNA level using RT-PCR (n = 6, each). Plasma levels of selected proteins from the proteomics profiling: Proteinase-3 (PR3), Annexin-A3 (ANX3), Defensin (DEFA1), and Matrix Metalloproteinase-9 (MMP9), were measured in the entire cohort using human enzyme-linked immunosorbent assay kits and were subsequently correlated with various clinical parameters. Out of the 1407 we identified and quantified from the proteomics profiling, 47 proteins were dysregulated with at least twofold change between the two subject groups. Among the differentially expressed proteins, 11 were confirmed at the mRNA levels. CVD influenced the levels of the shortlisted proteins (MMP9, PR3, ANX3, and DEFA1) in the PBMCs and plasma differentially. Despite the decreased levels of both protein and mRNA in PBMCs, PR3 circulating levels increased significantly in patients with CVD and were influenced by neither diabetes nor statin treatment. No significant changes were; however, observed in the DEFA1, MMP9, and ANX3 levels in plasma. Multivariate logistic regression analysis revealed that only PR3 was independently associated with CVD. Our results suggest that the dysregulation of PR3 levels in plasma and PBMCs reflects underlying residual CVD risks even in the treated population. More prospective and larger studies are required to establish the role of PR3 in CVD progression.


Assuntos
Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/metabolismo , Mieloblastina/metabolismo , Adulto , Anexina A3/análise , Anexina A3/sangue , Anexina A3/metabolismo , Árabes , Estudos Transversais , Defensinas/análise , Defensinas/sangue , Defensinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Kuweit/epidemiologia , Leucócitos/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/sangue , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Mieloblastina/análise , Mieloblastina/sangue , Plasma/metabolismo , Estudos Prospectivos , Proteômica , RNA Mensageiro/genética
4.
Nanoscale ; 11(48): 23366-23381, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31793603

RESUMO

One of the most important causes of failure in tumour treatment is the development of resistance to therapy. Cancer cells can develop the ability to lose sensitivity to anti-neoplastic drugs during reciprocal crosstalk between cells and their interaction with the tumour microenvironment (TME). Cell-to-cell communication regulates a cascade of interdependent events essential for disease development and progression and can be mediated by several signalling pathways. Exosome-mediated communication is one of the pathways regulating these events. Tumour-derived exosomes (TDE) are believed to have the ability to modulate TMEs and participate in multidrug resistance mechanisms. In this work, we studied the effect of the natural defensin from common bean, PvD1, on the formation of exosomes by breast cancer MCF-7 cells, mainly the modulatory effect it has on the level of CD63 and CD9 tetraspanins. Moreover, we followed the interaction of PvD1 with biological and model membranes of selected composition, by biophysical and imaging techniques. Overall, the results show that PvD1 induces a dual effect on MCF-7 derived exosomes: the peptide attenuates the recruitment of CD63 and CD9 to exosomes intracellularly and binds to the mature exosomes in the extracellular environment. This work uncovers the exosome-mediated anticancer action of PvD1, a potential nutraceutical agent.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Defensinas/farmacologia , Exossomos/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Neoplasias da Mama/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Exossomos/metabolismo , Feminino , Humanos , Células MCF-7 , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismo
5.
BMC Vet Res ; 15(1): 465, 2019 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-31864349

RESUMO

BACKGROUND: Despite being one of the major causes of infertility in mares, the mechanisms responsible for equine endometrosis are still unclear and controversial. In the last few years, many investigations focused on local immune response modulation. Since it is generally accepted that endometrial fibrosis increases with age, we hypothesize that older mares could show altered local immune modulation, initiating a pro-inflammatory and tissue remodeling cascade of events that could lead to endometrosis. The aim of this study, indeed, is to evaluate and describe the local gene expression of genes involved in acute inflammatory response and fibrosis (COL1A1, COL3A1, TNFA, MMP9, IL6, TGFB1 and TGFBR1), together with others associated to immune modulation (DEFB4B, IDO1 and FOXP3), in uterine specimens from mares of different age. RESULTS: Twenty-five Standardbred mares were involved in the study with age ranging from 7 to 19 years (mean 10.40 ± 4.42). They were divided by age into two groups: G1 (n = 15, less than 10 years old) and G2 (N = 10, greater than 11 years old). Specimens from the uterus' right horn-body junction were collected and processed for histology evaluation and RT-qPCR assay.Gene expression of DEFB4B, MMP9 and TNFA was higher in younger mares, suggesting a balance in immune modulation and tissue remodeling. Interleukin-6 and COL3A1 gene expressions were greater in older animals, probably indicating inflammatory pathways activation and fibrosis increase. Although no differences in fibrosis and inflammation distribution could be found with histological examination among G1 and G2, our results suggest a possible involvement of DEF4BB in regulating the local immune response in younger mare's uterus (G1); age may contribute to the dis-regulation of DEFB4B transcription and, indirectly, influence the extracellular matrix homeostasis. Transcription of IDO1 and FOXP3 genes, instead, does not seem to be age related, or to be involved in local immune-response and tissue remodeling functions. CONCLUSIONS: Further investigations are needed in order to clarify the interactions between the expression of DEFB4B, IL6, TNFA, COL3A1 and MMP9 and other local signals of immune-modulation and tissue remodeling, in mares in a prospective study design.


Assuntos
Envelhecimento , Defensinas/metabolismo , Endométrio/metabolismo , Cavalos/fisiologia , Animais , Cruzamento , Defensinas/genética , Feminino , Fibrose , Expressão Gênica , Cavalos/genética , Cavalos/metabolismo , Inflamação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária
6.
Int J Mol Sci ; 21(1)2019 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-31877866

RESUMO

There is a growing interest in the complex role of host defense peptides (HDPs) in the pathophysiology of several immune-mediated inflammatory diseases. The physicochemical properties and selective interaction of HDPs with various receptors define their immunomodulatory effects. However, it is quite challenging to understand their function because some HDPs play opposing pro-inflammatory and anti-inflammatory roles, depending on their expression level within the site of inflammation. While it is known that HDPs maintain constitutive host protection against invading microorganisms, the inducible nature of HDPs in various cells and tissues is an important aspect of the molecular events of inflammation. This review outlines the biological functions and emerging roles of HDPs in different inflammatory conditions. We further discuss the current data on the clinical relevance of impaired HDPs expression in inflammation and selected diseases.


Assuntos
Imunidade Adaptativa/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Bactérias/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/classificação , Peptídeos Catiônicos Antimicrobianos/genética , Bactérias/classificação , Catelicidinas/genética , Catelicidinas/imunologia , Catelicidinas/metabolismo , Defensinas/genética , Defensinas/imunologia , Defensinas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Inflamação/genética , Inflamação/microbiologia
7.
J Pept Sci ; 25(12): e3223, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31713951

RESUMO

Previously Os, a 22 amino acid sequence of a defensin from the soft tick Ornithodoros savignyi, was found to kill Gram-positive and Gram-negative bacteria at low micromolar concentrations. In this study, we evaluated synthetic peptide analogues of Os for antibacterial activity with an aim to identify minimalized active peptide sequences and in so doing obtain a better understanding of the structural requirements for activity. Out of eight partially overlapping sequences of 10 to 12 residues, only Os(3-12) and Os(11-22) exhibit activity when screened against Gram-positive and Gram-negative bacteria. Carboxyamidation of both peptides increased membrane-mediated activity, although carboxyamidation of Os(11-22) negatively impacted on activity against Staphylococcus aureus. The amidated peptides, Os(3-12)NH2 and Os(11-22)NH2 , have minimum bactericidal concentrations of 3.3 µM against Escherichia coli. Killing was reached within 10 minutes for Os(3-12)NH2 and only during the second hour for Os(11-22)NH2 . In an E. coli membrane liposome system, both Os and Os(3-12)NH2 were identified as membrane disrupting while Os(11-22)NH2 was less active, indicating that in addition to membrane permeabilization, other targets may be involved in bacterial killing. In contrast to Os, the membrane disruptive effect of Os(3-12)NH2 did not diminish in the presence of salt. Neither Os nor its amidated derivatives caused human erythrocyte haemolysis. The contrasting killing kinetics and effects of amidation together with structural and liposome leakage data suggest that the 3-12 fragment relies on a membrane disruptive mechanism while the 11-22 fragment involves additional target mechanisms. The salt-resistant potency of Os(3-12)NH2 identifies it as a promising candidate for further development.


Assuntos
Amidas/farmacologia , Antibacterianos/farmacologia , Defensinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Amidas/química , Animais , Antibacterianos/química , Bacillus subtilis/efeitos dos fármacos , Defensinas/química , Relação Dose-Resposta a Droga , Escherichia coli/efeitos dos fármacos , Cinética , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/química , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade
8.
Nat Commun ; 10(1): 4853, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649262

RESUMO

Few secreted proteins involved in plant infection common to necrotrophic bacteria, fungi and oomycetes have been identified except for plant cell wall-degrading enzymes. Here we study a family of iron-binding proteins that is present in Gram-negative and Gram-positive bacteria, fungi, oomycetes and some animals. Homolog proteins in the phytopathogenic bacterium Dickeya dadantii (IbpS) and the fungal necrotroph Botrytis cinerea (BcIbp) are involved in plant infection. IbpS is secreted, can bind iron and copper, and protects the bacteria against H2O2-induced death. Its 1.7 Å crystal structure reveals a classical Venus Fly trap fold that forms dimers in solution and in the crystal. We propose that secreted Ibp proteins binds exogenous metals and thus limit intracellular metal accumulation and ROS formation in the microorganisms.


Assuntos
Arabidopsis/metabolismo , Cobre/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Ferro/metabolismo , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Anti-Infecciosos Locais/farmacologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Botrytis/genética , Botrytis/metabolismo , Proteínas de Transporte/metabolismo , Defensinas/genética , Dimerização , Gammaproteobacteria/efeitos dos fármacos , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas de Ligação ao Ferro/genética , Doenças das Plantas/genética , Sideróforos/genética , Sideróforos/metabolismo
9.
mBio ; 10(5)2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641083

RESUMO

Big defensins, ancestors of ß-defensins, are composed of a ß-defensin-like C-terminal domain and a globular hydrophobic ancestral N-terminal domain. This unique structure is found in a limited number of phylogenetically distant species, including mollusks, ancestral chelicerates, and early-branching cephalochordates, mostly living in marine environments. One puzzling evolutionary issue concerns the advantage for these species of having maintained a hydrophobic domain lost during evolution toward ß-defensins. Using native ligation chemistry, we produced the oyster Crassostrea gigas BigDef1 (Cg-BigDef1) and its separate domains. Cg-BigDef1 showed salt-stable and broad-range bactericidal activity, including against multidrug-resistant human clinical isolates of Staphylococcus aureus We found that the ancestral N-terminal domain confers salt-stable antimicrobial activity to the ß-defensin-like domain, which is otherwise inactive. Moreover, upon contact with bacteria, the N-terminal domain drives Cg-BigDef1 assembly into nanonets that entrap and kill bacteria. We speculate that the hydrophobic N-terminal domain of big defensins has been retained in marine phyla to confer salt-stable interactions with bacterial membranes in environments where electrostatic interactions are impaired. Those remarkable properties open the way to future drug developments when physiological salt concentrations inhibit the antimicrobial activity of vertebrate ß-defensins.IMPORTANCE ß-Defensins are host defense peptides controlling infections in species ranging from humans to invertebrates. However, the antimicrobial activity of most human ß-defensins is impaired at physiological salt concentrations. We explored the properties of big defensins, the ß-defensin ancestors, which have been conserved in a number of marine organisms, mainly mollusks. By focusing on a big defensin from oyster (Cg-BigDef1), we showed that the N-terminal domain lost during evolution toward ß-defensins confers bactericidal activity to Cg-BigDef1, even at high salt concentrations. Cg-BigDef1 killed multidrug-resistant human clinical isolates of Staphylococcus aureus Moreover, the ancestral N-terminal domain drove the assembly of the big defensin into nanonets in which bacteria are entrapped and killed. This discovery may explain why the ancestral N-terminal domain has been maintained in diverse marine phyla and creates a new path of discovery to design ß-defensin derivatives active at physiological and high salt concentrations.


Assuntos
Antibacterianos/química , Defensinas/química , Nanoestruturas/química , Animais , Antibacterianos/farmacologia , Crassostrea/efeitos dos fármacos , Humanos , Imunidade Inata , Espectroscopia de Ressonância Magnética , Staphylococcus aureus/efeitos dos fármacos
10.
Andrologia ; 51(11): e13437, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31637753

RESUMO

Pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) and nuclear oligomerization domain (NOD) receptors along with antimicrobial proteins and peptides (AMPs) are crucial for innate immunity. The pathology of insulin-dependent diabetes mellitus is associated with the disrupted expression of TLRs, NODs and AMPs in the kidney, lungs and other organs. However, such a relation in the male reproductive tract is not yet investigated. In this study, we analysed the expression pattern of Tlr1-13, Nod1/2 receptors and AMPs (ß-defensins and defensin-like proteins of the Sperm-Associated Antigen 11 (Spag11) family) in the male reproductive tissues (caput, cauda and testis) obtained from diabetic or insulin-treated diabetic or untreated control rats. Alterations in the expression pattern of Tlr1-13, Nod 1/ 2, Defb1, 2, 21, 24, 27, 30 and Spag11a/ c/ t were observed under diabetic conditions. Administration of insulin to diabetic rats could modulate the expression pattern of only some these genes. Results of our study indicate perturbed gene expression profile of Tlrs, Nod1/2, Defbs and Spag11 isoforms in the epididymis and testis of diabetic rats, and this could be one of the important reasons for the increased risk of infections in the male genital tract.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Defensinas/metabolismo , Diabetes Mellitus Experimental/metabolismo , Epididimo/metabolismo , Testículo/metabolismo , Receptores Toll-Like/metabolismo , Aloxano , Animais , Antígenos de Superfície/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Epididimo/efeitos dos fármacos , Glicopeptídeos/metabolismo , Insulina/farmacologia , Insulina/uso terapêutico , Masculino , Ratos Wistar , Testículo/efeitos dos fármacos
11.
Amino Acids ; 51(10-12): 1633-1648, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31654210

RESUMO

For medical use of proteins and peptide-based drugs, it is desirable to have small biologically active sequences because they improve stability, reduce side effects, and production costs. Several plant defensins have their biological activities imparted by a sequence named γ-core. Vu-Def, a Vigna unguiculata defensin, has activity against Leishmania amazonensis, which is one etiological agent of leishmaniasis and for which new drugs are needed. Our intention was to understand if the region comprising the Vu-Def γ-core is responsible for the biological activity against L. amazonensis and to unveil its mechanism of action. Different microbiological assays with L. amazonensis in the presence of the synthetic peptide A36,42,44γ32-46Vu-Def were done, as well as ultrastructural and fluorescent analyses. A36,42,44γ32-46Vu-Def showed biological activity similar to Vu-Def. A36,42,44γ32-46Vu-Def (74 µM) caused 97% inhibition of L. amazonensis culture and parasites were unable to regrow in fresh medium. The cells of the treated parasites showed morphological alterations by ultrastructural analysis and fluorescent labelings that corroborate with the data of the organelles alterations. The general significance of our work is based on the description of a small synthetic peptide, A36,42,44γ32-46Vu-Def, which has activity on L. amazonensis and that the interaction between A36,42,44γ32-46Vu-Def-L. amazonensis results in parasite inhibition by the activation of an apoptotic-like cell death pathway.


Assuntos
Apoptose/efeitos dos fármacos , Defensinas/química , Leishmania/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Vigna/química , Sequência de Aminoácidos , Defensinas/farmacologia , Leishmania/crescimento & desenvolvimento , Modelos Moleculares , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Sementes/química
12.
Planta ; 250(5): 1757-1772, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31428874

RESUMO

MAIN CONCLUSION: The recombinant EcgDf1 defensin has an antimicrobial effect against both plant and human pathogens. In silico analyses predict that EcgDf1 is prone to form dimers capable of interacting with the membranes of microorganisms. Plant defensins comprise a large family of antimicrobial peptides (AMP) with a wide range of biological functions. They are cysteine-rich molecules, highly sequence diverse but with a conserved and stable structure. In this work, a defensin gene (EcgDf1) was isolated from Erythrina crista-galli, a legume tree native from South America. The predicted peptide presents eight cysteines, with a γ-core motif GXCX3-9C and six cysteines distributed like the typical defensin αß motif. The mature EcgDf1 coding sequence was heterologously expressed in Escherichia coli strains and purified by affinity chromatography. Possible dimer and oligomers of EcgDf1 were visible in SDS electrophoresis. Moreover, its 3D structure, determined by homology modeling, docking, and molecular dynamics simulations, was found to be compatible with the formation of homodimers between the ß3 and ß1-loop-α1, leaving the ß2-loop-ß3 free to interact with lipid membranes. The purified recombinant peptide inhibited the growth of several critical plant and human pathogens, like the opportunistic fungi Candida albicans and Aspergillus niger and the plant pathogens Clavibacter michiganensis ssp. michiganensis, Penicillium expansum, Botrytis cinerea, and Alternaria alternata. EcgDf1 is a promising candidate for the development of antimicrobial products for use in agriculture and medicine.


Assuntos
Anti-Infecciosos/farmacologia , Aspergillus niger/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Defensinas/farmacologia , Fabaceae/genética , Anti-Infecciosos/metabolismo , Simulação por Computador , Cisteína , Defensinas/genética , Defensinas/metabolismo , Dimerização , Fabaceae/química , Simulação de Dinâmica Molecular , Proteínas de Plantas/genética , Proteínas Recombinantes , Árvores
13.
Fish Shellfish Immunol ; 93: 1084-1092, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31449980

RESUMO

As a family of negatively feedback regulating factors, the suppressor of cytokine signaling (SOCS) can depress cytokine signal transduction, and eventually modulate growth, development, differentiation, and immune response. In the present study, a SOCS homologue (designated as CgSOCS6) was identified from oyster Crassostrea gigas. The open reading frame of CgSOCS6 cDNA was of 1167 bp encoding a peptide of 388 amino acid residues with a central Src homology 2 (SH2) domain, a conserved C-terminal SOCS box, and a nucleus localization sequence (NLS) in its N-terminus. The deduced amino acid sequence of CgSOCS6 shared 37.9-45.5% similarity with other SOCS6/7 family members. In the unrooted phylogenetic tree, CgSOCS6 was clustered with EsSOCS6 from Chinese mitten crab Eriocheir sinensis and assigned into the SOCS6/7 group. The mRNA transcripts of CgSOCS6 were constitutively distributed in all the tested tissues, with the highest level in hemocytes. After lipopolysaccharide (LPS) stimulation, the mRNA expression of CgSOCS6 in hemocytes was significantly up-regulated to the highest level at 6 h (8.48-fold compared to the control group, p < 0.01), and then kept at a relatively higher level from 12 h to 72 h. CgSOCS6 protein could be translocated into the hemocyte nucleus after LPS stimulation. The mRNA expressions of interleukin 17-4 (CgIL17-4), CgIL17-5, and defensin (CgDefh1) in the hemocytes of CgSOCS6-knockdown oysters increased significantly (2.55-fold, 2.68-fold, 4.68-fold of that in EGFP-RNAi oysters, p < 0.05, p < 0.05, p < 0.001, respectively) after LPS stimulation. These findings suggested that CgSOCS6 was involved in the oyster immune response by regulating the expressions of CgIL17-4, CgIL17-5, and CgDefh1.


Assuntos
Crassostrea/genética , Crassostrea/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Sequência de Aminoácidos , Animais , Defensinas/genética , Defensinas/imunologia , Perfilação da Expressão Gênica , Interleucina-17/genética , Interleucina-17/imunologia , Lipopolissacarídeos/farmacologia , Filogenia , Alinhamento de Sequência , Proteínas Supressoras da Sinalização de Citocina/química
14.
Mol Plant Microbe Interact ; 32(12): 1649-1664, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31425003

RESUMO

Antimicrobial peptides play a pivotal role in the innate immunity of plants. Defensins are cysteine-rich antifungal peptides with multiple modes of action. A novel Oleaceae-specific defensin gene family has been discovered in the genome sequences of wild and cultivated species of a perennial olive tree, Olea europaea. OefDef1.1, a member of this defensin family, potently inhibits the in-vitro growth of ascomycete fungal pathogens Botrytis cinerea and three Fusarium spp. OefDef1.1 rapidly permeabilizes the plasma membrane of the conidial and germling cells of B. cinerea. Interestingly, it induces reactive oxygen species and translocates to the cytoplasm only in the germlings but not in the conidia. In medium containing a high concentration of Na1+, antifungal activity of OefDef1.1 is significantly reduced. Surprisingly, a chimeric OefDef1.1 peptide containing the γ-core motif of a Medicago truncatula defensin, MtDef4, displays Na1+-tolerant antifungal activity. In a phospholipid-protein overlay assay, the chimeric peptide exhibits stronger binding to its phosphoinositide partners than OefDef1.1 and is also more potent in inhibiting gray mold disease on the surface of Nicotiana benthamiana and lettuce leaves than OefDef1.1. Significant differences are observed among the four ascomycete pathogens in their responses to OefDef1.1 in growth medium with or without the elevated concentration of Na1+. The varied responses of closely related ascomycete pathogens to this defensin have implications for engineering disease resistance in plants.


Assuntos
Defensinas , Fusarium , Olea , Defensinas/metabolismo , Defensinas/farmacologia , Fusarium/efeitos dos fármacos , Alface/microbiologia , Olea/imunologia , Olea/microbiologia , Tabaco/microbiologia
15.
Ticks Tick Borne Dis ; 10(6): 101269, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31445875

RESUMO

Tick innate immunity involves humoral and cellular responses. Among the humoral effector molecules in ticks are the defensins which are a family of small peptides with a conserved γ-core motif that is crucial for their antimicrobial activity. Defensin families have been identified in several hard and soft tick species. However, little is known about the presence and antimicrobial activity of defensins from the Australian paralysis tick Ixodes holocyclus. In this study the I. holocyclus transcriptome was searched for the presence of defensins. Unique and non-redundant defensin sequences were identified and designated as holosins 1 - 5. The antimicrobial activity of holosins 2 and 3 and of the predicted γ-cores of holosins 1-4 (HoloTickCores 1-4), was assessed using Gram-negative and Gram-positive bacteria as well as the fungus Fusarium graminearum and the yeast Candida albicans. All holosins had molecular features that are conserved in other tick defensins. Furthermore holosins 2 and 3 were very active against the Gram-positive bacteria Staphylococcus aureus and Listeria grayi. Holosins 2 and 3 were also active against F. graminearum and C. albicans and 5 µM of peptide abrogate the growth of these microorganisms. The activity of the synthetic γ-cores was lower than that of the mature defensins apart from HoloTickCore 2 which had activity comparable to mature holosin 2 against the Gram-negative bacterium Escherichia coli. This study reveals the presence of a multigene defensin family in I. holocyclus with wide antimicrobial activity.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Defensinas/genética , Defensinas/imunologia , Ixodes/genética , Ixodes/imunologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antifúngicos/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Austrália , Candida albicans/efeitos dos fármacos , Defensinas/química , Fusarium/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Filogenia , Alinhamento de Sequência , Transcriptoma
16.
J Immunol Res ; 2019: 9708769, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31355298

RESUMO

Myeloid-derived suppressor cells (MDSCs) are present in the human lung microenvironment, and they may be involved in the local inflammatory process in chronic obstructive pulmonary disease (COPD). Chronic inflammation in COPD may induce immunogenic cell death of structural airway cells, causing the release of damage-associated molecular patterns (DAMPs). DAMPs may activate the innate and adaptive immune system. The relationship between MDSCs and DAMPs in COPD is poorly described in the available literature. Objectives. (1) Assessment of MDSC percentage and DAMP concentration in bronchoalveolar lavage fluid (BALF) and peripheral blood. (2) Analysis of the relationship between MDSC percentage and chosen DAMPs. Patients and Methods. 30 COPD patients were included. Using monoclonal antibodies directly conjugated with fluorochromes in flow cytometry, MDSCs were assessed in BALF and peripheral blood. The concentration of DAMPs was estimated using sandwich ELISA. Using the Bradford method, the total protein concentrations were evaluated. Results. The percentage of MDSCs among MC in BALF correlated well with the concentration of defensin and heat shock protein 27. Assessing the percentage of MDSCs among all leukocytes in BALF, we revealed a significant correlation with the concentration of defensin, hyaluronic acid, and surfactant protein A. No dependencies occurred between DAMPs and MDSCs in peripheral blood. Conclusion. MDSCs and DAMPs occur in the COPD patient lung microenvironment. Significant correlations between them found in BALF may indicate their influence on the local inflammatory process in COPD. These relationships allow better understanding of the inflammatory process in COPD.


Assuntos
Líquido da Lavagem Broncoalveolar/imunologia , Pulmão/metabolismo , Células Supressoras Mieloides/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Idoso , Líquido da Lavagem Broncoalveolar/química , Defensinas/metabolismo , Feminino , Proteínas de Choque Térmico/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Inflamação/fisiopatologia , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/metabolismo , Células Supressoras Mieloides/química , Células Supressoras Mieloides/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo
17.
Elife ; 82019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31358113

RESUMO

Antimicrobial peptides (AMPs) are small cationic molecules best known as mediators of the innate defence against microbial infection. While in vitro and ex vivo evidence suggest AMPs' capacity to kill cancer cells, in vivo demonstration of an anti-tumour role of endogenous AMPs is lacking. Using a Drosophila model of tumourigenesis, we demonstrate a role for the AMP Defensin in the control of tumour progression. Our results reveal that Tumour Necrosis Factor mediates exposure of phosphatidylserine (PS), which makes tumour cells selectively sensitive to the action of Defensin remotely secreted from tracheal and fat tissues. Defensin binds tumour cells in PS-enriched areas, provoking cell death and tumour regression. Altogether, our results provide the first in vivo demonstration for a role of an endogenous AMP as an anti-cancer agent, as well as a mechanism that explains tumour cell sensitivity to the action of AMPs.


Assuntos
Morte Celular , Defensinas/metabolismo , Fatores Imunológicos/metabolismo , Neoplasias/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Modelos Animais de Doenças , Drosophila , Análise de Sobrevida
18.
Microb Pathog ; 135: 103648, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31356928

RESUMO

Norovirus is a highly infectious human pathogen that causes acute foodborne diseases worldwide. As global diet patterns have begun to incorporate a higher consumption of fresh agricultural products, the internalization of norovirus into plants has emerged as a potential threat to human health. Here, we demonstrated that murine norovirus (MNV1) was internalized into Arabidopsis in multiple phases, and this internalization was correlated with Arabidopsis innate immunity responses. Under hydroponic conditions, continuous treatment of MNV1 retarded root growth and facilitated flower development of Arabidopsis without causing necrotic lesions. Examination of viral titers and RNA levels revealed that MNV1 was internalized into Arabidopsis in at least three different phases. In response to MNV1 treatment, the Arabidopsis defensive marker PR1 (a salicylic acid signaling marker) was transiently up-regulated at the early stage. PDF1.2, a jasmonic acid signaling marker, exhibited a gradual induction over time. Noticeably, Arabidopsis RNS1 (T2 ribonuclease) was rapidly induced by MNV1 and exhibited anti-correlation with the internalization of MNV1. Exposure to recombinant Arabidopsis RNS1 protein reduced the viral titers and degraded MNV1 RNA in vitro. In conclusion, the internalization of MNV1 into Arabidopsis was fluctuated by mutual interactions that were potentially regulated by Arabidopsis immune systems containing RNS1.


Assuntos
Arabidopsis/imunologia , Arabidopsis/virologia , Norovirus/fisiologia , Plântula/imunologia , Plântula/virologia , Internalização do Vírus , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos , Defensinas/metabolismo , Doenças Transmitidas por Alimentos/virologia , Imunidade Inata , Camundongos , Oxilipinas , Desenvolvimento Vegetal , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/virologia , Proteínas Recombinantes , Ribonucleases/genética , Ribonucleases/metabolismo , Plântula/genética , Plântula/metabolismo , Regulação para Cima , Carga Viral
19.
Islets ; 11(4): 89-101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31242128

RESUMO

The gut microbiota can play a role in pancreatitis and, likely, in the development of type 1 diabetes (T1D). Anti-microbial peptides and secretory proteins are important mediators of the innate immune response against bacteria but their expression in the human pancreas is not fully known. In this study, immunohistochemistry was used to analyze the expression of seven anti-microbial peptides (Defensin α1, α4, ß1-4 and Cathelicidin) and two secretory proteins with known antimicrobial properties (REG3A and GP2) in pancreatic and duodenal biopsies from 10 non-diabetic organ donors and one organ donor that died at onset of T1D. Immunohistochemical data was compared with previously published whole-transcriptome data sets. Seven (Defensin α1, ß2, ß3, α4, GP2, Cathelicidin, and REG3A) host defense molecules showed positive staining patterns in most non-diabetic organ donors, whereas two (Defensin ß1 and ß4) were negative in all non-diabetic donors. Two molecules (Defensin α1 and GP2) were restricted to the exocrine pancreas whereas two (Defensin ß3, α4) were only expressed in islet tissue. Cathelicidin, ß2, and REG3A were expressed in both islets and exocrine tissue. The donor that died at onset of T1D had generally less positivity for the host defense molecules, but, notably, this pancreas was the only one where defensin ß1 was found. Neither donor age, immune-cell infiltration, nor duodenal expression correlated to the pancreatic expression of host defense molecules. In conclusion, these findings could have important implications for the inflammatory processes in diabetes and pancreatitis as we find several host defense molecules expressed by the pancreatic tissue.


Assuntos
Defensinas/metabolismo , Proteínas Ligadas por GPI/metabolismo , Pâncreas/metabolismo , Proteínas Associadas a Pancreatite/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Defensinas/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Feminino , Proteínas Ligadas por GPI/genética , Expressão Gênica , Humanos , Imuno-Histoquímica , Lactente , Masculino , Proteínas Associadas a Pancreatite/genética , Doadores de Tecidos , Adulto Jovem
20.
Int J Mol Sci ; 20(13)2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31252518

RESUMO

Aedes aegypti (L.) is the primary vector of emergent mosquito-borne viruses, including chikungunya, dengue, yellow fever, and Zika viruses. To understand how these viruses interact with their mosquito vectors, an analysis of the innate immune system response was conducted. The innate immune system is a conserved evolutionary defense strategy and is the dominant immune system response found in invertebrates and vertebrates, as well as plants. RNA-sequencing analysis was performed to compare target transcriptomes of two Florida Ae. aegypti strains in response to chikungunya virus infection. We analyzed a strain collected from a field population in Key West, Florida, and a laboratory strain originating from Orlando. A total of 1835 transcripts were significantly expressed at different levels between the two Florida strains of Ae. aegypti. Gene Ontology analysis placed these genes into 12 categories of biological processes, including 856 transcripts (up/down regulated) with more than 1.8-fold (p-adj (p-adjust value) ≤ 0.01). Transcriptomic analysis and q-PCR data indicated that the members of the AaeCECH genes are important for chikungunya infection response in Ae. aegypti. These immune-related enzymes that the chikungunya virus infection induces may inform molecular-based strategies for interruption of arbovirus transmission by mosquitoes.


Assuntos
Aedes/imunologia , Imunidade Inata , Transcriptoma , Aedes/genética , Aedes/virologia , Animais , Vírus Chikungunya/patogenicidade , Defensinas/genética , Defensinas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo
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