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1.
Microbiol Res ; 227: 126303, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421717

RESUMO

The inhibitory action that a Brevibacillus laterosporus strain isolated from the honeybee body causes against the American Foulbrood (AFB) etiological agent Paenibacillus larvae was studied by in-vitro experiments. A protein fraction isolated from B. laterosporus culture supernatant was involved in the observed inhibition of P. larvae vegetative growth and spore germination. As a result of LC-MS/MS proteomic analyses, the bacteriocin laterosporulin was found to be the major component of this fraction, followed by other antimicrobial proteins and substances including lectins, chaperonins, various enzymes and a number of putative uncharacterized proteins. The results obtained in this study highlight the potential of B. laterosporus as a biological control agent for preserving and improving honeybee health.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Abelhas/microbiologia , Brevibacillus/metabolismo , Paenibacillus larvae/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Brevibacillus/isolamento & purificação , Cromatografia Líquida , Testes de Sensibilidade Microbiana , Proteômica , Espectrometria de Massas em Tandem
2.
Pestic Biochem Physiol ; 158: 54-60, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378361

RESUMO

Extensive planting of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has spurred increasingly rapid evolution of resistance in pests. In the pink bollworm, Pectinophora gossypiella, a devastating global pest, resistance to Bt toxin Cry1Ac produced by transgenic cotton is linked with mutations in a gene (PgCad1) encoding a cadherin protein that binds Cry1Ac in the larval midgut. We previously reported a long non-coding RNA (lncRNA) in intron 20 of cadherin alleles associated with both resistance and susceptibility to Cry1Ac. Here we tested the hypothesis that reducing expression of this lncRNA decreases transcription of PgCad1 and susceptibility to Cry1Ac. Quantitative RT-PCR showed that feeding susceptible neonates small interfering RNAs (siRNAs) targeting this lncRNA but not PgCad1 decreased the abundance of transcripts of both the lncRNA and PgCad1. Moreover, neonates fed the siRNAs had lower susceptibility to Cry1Ac. The results imply that the lncRNA increases transcription of PgCad1 and susceptibility of pink bollworm to Cry1Ac. The results suggest that disruption of lncRNA expression could be a novel mechanism of pest resistance to Bt toxins.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/farmacologia , Caderinas/genética , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Mariposas/efeitos dos fármacos , RNA Longo não Codificante/genética , Transcrição Genética/genética , Animais , Bacillus thuringiensis/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Mariposas/genética , Mariposas/metabolismo , Controle Biológico de Vetores
3.
Exp Parasitol ; 204: 107724, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31279930

RESUMO

Only two drugs are currently available for the treatment of Chagas disease and their effectiveness are unsatisfactory. Photorhabdus luminescens and Xenorhabdus nematophila, two enteric bacteria highly pathogenic to a broad range of insects, have been studied as potential source for bioactive metabolites against protozoa causing neglected tropical diseases. Therefore, we tested the in vitro anti-Trypanosoma cruzi activity of secreted metabolites from these bacteria. The conditioned medium of X. nematophila and P. luminescens showed significant parasiticidal activity in a concentration-dependent manner (IC50XN = 0.34 mg/mL, IC50PL = 1.0 mg/mL). The parasiticidal compound was identified as a small molecule stable to heating and pH changes ranging from 2 to 12. Moreover, anti-Trypanosoma molecules secreted by both bacteria stimulate the trypanocidal activity of macrophages by a mechanism independent of nitric oxide. Summarizing, our studies reveal that P. luminescens and X. nematophila are potential sources of putative novel drugs against Chagas disease.


Assuntos
Proteínas de Bactérias/farmacologia , Photorhabdus/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Xenorhabdus/química , Análise de Variância , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/uso terapêutico , Bioensaio , Doença de Chagas/tratamento farmacológico , Meios de Cultivo Condicionados , Endopeptidase K/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Temperatura Ambiente , Tripanossomicidas/efeitos adversos , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/crescimento & desenvolvimento
4.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 847-856, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223003

RESUMO

Pectobacterium carotovorum subsp. carotovorum is one of the world's top ten plant pathogens, mainly infecting cruciferous economic crops and ornamental flowers. In this study, an antibacterial gene cpxP (Gene ID: 29704421) was cloned from the genome of Pectobacterium carotovorum subsp. carotovorum, and constructed on the prokaryotic expression plasmid pET-15b, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3), then stability and bacteriostatic experiments of the purified CpxP protein were performed. The final concentration of IPTG was 1 mmol/L, obtaining high-efficiency exogenous expression of the CpxP protein. There was no other protein after purification, and the destined protein exhibited good thermal stability and pH stability. The antibacterial test results showed that the inhibition rate of the CpxP protein on carrot slice was 44.89% while the inhibition rate on potato slice was 59.41%. To further explain its antibacterial mechanism, studying the spatial structure of this protein can provide new ideas for the control of soft rot and new protein pesticide targets.


Assuntos
Bactérias , Proteínas de Bactérias , Proteínas de Membrana , Pectobacterium carotovorum , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Escherichia coli/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/farmacologia , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/metabolismo , Plasmídeos/genética
5.
Eur J Histochem ; 63(2)2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243942

RESUMO

The limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the protocol of a commercial kit developed for quantifying the microtubule-associated protein 1A/1B light chain 3B (LC3), the most reliable marker for autophagosomes currently available. The protocol modifications were set up measuring the autophagic flux in neoplastic (THP-1 cells) and primary cells (peripheral blood mononuclear cells; PBMC) of healthy donors. Moreover, PBMC of active tuberculosis (TB) patients were stimulated with the Mycobacterium tuberculosis purified protein derivatives or infected with live Mycobacterium bovis bacillus Calmette-Guerin (BCG). We found that the baseline median fluorescent intensity (MFI) of THP-1 cells changed depending on the time of sample acquisition to the flow cytometer. To solve this problem, a fixation step was introduced in different stages of the assay's protocol, obtaining more reproducible and sensitive results when a post-LC3 staining fixation was performed, in either THP1 or PBMC. Furthermore, since we found that results are influenced by the type and the dose of the lysosome inhibitor used, the best dose of Chloroquine for LC3 accumulation were set up in either THP-1 cells or PBMC. Finally, applying these experimental settings, we measured the autophagic flux in CD14+ cells from active TB patients' PBMC upon BCG infection. In conclusion, our data indicate that the protocol modifications here described in this work improve the stability and accuracy of a flow cytometry-based assay for the evaluation of autophagy, thus assuring more standardised cell analyses.


Assuntos
Autofagia , Citometria de Fluxo/métodos , Proteínas Associadas aos Microtúbulos/análise , Autofagia/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Cloroquina/farmacologia , Fluorescência , Humanos , Leucócitos Mononucleares/microbiologia , Mycobacterium bovis/química , Coloração e Rotulagem , Células THP-1
6.
Pestic Biochem Physiol ; 157: 219-229, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153472

RESUMO

Photorhabdus akhurstii can produce a variety of proteins that aid this bacterium and its mutualistic nematode vector, Heterorhabditis indica to kill the insect host. Herein, we characterized (by heterologously expressing in E. coli) an open reading frame (1713 bp) of the toxin complex protein, TcaB from P. akhurstii strains IARI-SGHR2 and IARI-SGMS1 and assessed its toxic effect on G. mellonella larvae. The intra-hemocoel injection of purified TcaB (molecular weight-63 kDa) caused fourth instar larval bodies to blacken and die with LD50 values of 67.25 (IARI-SGHR2) and 52.08 (IARI-SGMS1) ng per larva at 12 h. Additionally, oral administration of the toxin caused larval mortality with LD50 values of 709.55 (IARI-SGHR2) and 598.44 (IARI-SGMS1) ng per g diet per larva at 7 days post feeding. Injection of purified TcaB caused loss of viability of fourth instar G. mellonella hemocytes at 6 h post incubation; cells displayed morphological changes typical of apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and disintegration. Injection of TcaB also elevated the phenoloxidase activity in insect hemolymph which triggers an extensive immune response that potentially leads to larval death. Similar to other bacterial toxins TcaB possesses potent biological activity which may enable it to be used as an efficient agent for pest management.


Assuntos
Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Inseticidas/metabolismo , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Photorhabdus/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Hemócitos/efeitos dos fármacos , Photorhabdus/genética
7.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 564-577, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205019

RESUMO

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.


Assuntos
Proteínas de Bactérias/química , N-Acilneuraminato Citidililtransferase/química , Vibrio cholerae/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Domínio Catalítico , Cristalização , Cristalografia por Raios X/métodos , Cistina Difosfato/química , Cistina Difosfato/metabolismo , Ácido N-Acetilneuramínico Citidina Monofosfato/química , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , N-Acilneuraminato Citidililtransferase/farmacologia , N-Acilneuraminato Citidililtransferase/fisiologia , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Ácidos Siálicos/metabolismo
8.
Gene ; 710: 387-398, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31136783

RESUMO

Busseola fusca (Fuller) (Lepidoptera: Noctuidae), a major insect pest of maize in sub-Saharan Africa, has developed high levels of non-recessive resistance to Cry1Ab toxin expressed in genetically modified Bt maize. Multiple resistance mechanisms to various Cry toxins have been identified in Lepidoptera, but no study has yet been done to determine the mechanism of Cry1Ab resistance in B. fusca. Therefore, the larval transcriptome of B. fusca was sequenced, de novo assembled and characterized. Differential expression analysis was performed to compare gene expression profiles of Cry toxin challenged and unchallenged neonate larvae to assess the molecular basis of the defence mechanism employed by this insect. Several genes associated with Cry toxin resistance in other lepidopteran pests were detected in B. fusca. Results suggest that differential expression of metabolic and immune-related genes might explain Cry1Ab toxin defence in this pest (supplemental file). Transcript expression profiles of neonates demonstrated that 33.59% and 60.31% of the 131 differentially expressed genes were upregulated and downregulated in the toxin-challenged neonate larvae, respectively. Transcripts were grouped into two subclusters according to the similarity of their expression patterns. Transcripts in subcluster 1 were moderately upregulated in the toxin-challenged neonate larvae, and, conversely, downregulated in the unchallenged neonate larvae. The solute carrier organic anion transporter, which is involved in insecticide detoxification, was upregulated in the toxin-challenged neonate larvae. Conversely, most of the transcripts in subcluster 2 were moderately downregulated in the toxin-challenged neonate larvae, and upregulated for neonates feeding on non-challenged maize. Four unidentified transcripts were extremely down-regulated in the toxin-challenged neonate larvae, and upregulated in the unchallenged neonate larvae. Further studies are recommended to establish if there is a direct correlation between these differentially expressed genes and the observed resistance. Elucidation of such defence mechanisms is crucial for developing insect resistance management strategies to ensure sustainable use of genetically modified maize in Africa. Nevertheless, this is the first study on gene expression profiles of B. fusca strains challenged with Cry toxin. The transcriptome characterized in this study provides a significant resource base for future studies on B. fusca and contributes to understanding some of the gene regulation and signalling networks involved in the defence of B. fusca against Cry1Ab toxin.


Assuntos
Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Perfilação da Expressão Gênica/métodos , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/genética , Resistência a Inseticidas , Mariposas/genética , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Endotoxinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Hemolisinas/genética , Larva/efeitos dos fármacos , Larva/genética , Redes e Vias Metabólicas , Mariposas/efeitos dos fármacos , Plantas Geneticamente Modificadas/parasitologia , Análise de Sequência de RNA , Zea mays/genética , Zea mays/metabolismo , Zea mays/parasitologia
9.
Food Chem Toxicol ; 129: 376-381, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054996

RESUMO

The ipd072Aa gene from Pseudomonas chlororaphis encodes the IPD072Aa protein which confers protection against certain coleopteran pests when expressed in genetically modified (GM) plants. A weight of evidence approach was used to assess the safety of the IPD072Aa protein. This approach considered the history of safe use of the source organism and bioinformatic comparison of the protein sequence with known allergenic and toxic proteins. The IPD072Aa protein was assessed for resistance to degradation in the presence of simulated gastric fluid containing pepsin as well as heat stability. There was no hazard identified with the IPD072Aa protein. Furthermore, an acute oral toxicity study found no evidence of adverse effects. Collectively, these studies support the human health safety assessment of the IPD072Aa protein.


Assuntos
Proteínas de Bactérias/farmacologia , Besouros/efeitos dos fármacos , Pseudomonas chlororaphis/metabolismo , Alérgenos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/toxicidade , Eletroforese em Gel de Poliacrilamida , Controle Biológico de Vetores/métodos , Plantas Geneticamente Modificadas/genética , Testes de Toxicidade , Zea mays/genética
10.
J Microbiol ; 57(7): 626-636, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054134

RESUMO

Beta haemolytic Group A streptococcus (GAS) or Streptococcus pyogenes are strict human pathogens responsible for mild to severe fatal invasive infections. Even with enormous number of reports exploring the role of S. pyogenes exotoxins in its pathogenesis, inadequate knowledge on the biofilm process and the potential role of exotoxins in bacterial dissemination from matured biofilms has been a hindrance in development of effective and targeted treatments. Therefore, the present study was aimed in investigating the uncharted role of these exotoxins in biofilm process. Through our study the putative role of ciaRH in the SpeA dependent ablation of biofilm formation could be speculated and thus helping in bacterial dissemination. The seed-dispersal effect of SpeA was time and concentration dependent and seen to be consistent within various streptococcal species. Transcriptome analysis of SpeA treated S. pyogenes biofilms revealed the involvement of many transcriptional regulators (ciaRH) and response genes (luxS, shr, shp, SPy_0572), hinting towards specific mechanisms underlying the dispersal effect by SpeA. This finding opens up a discussion towards understanding a new mechanism involved in the pathogenesis of Streptococcus pyogenes and might help in understanding the bacterial infections in a better way.


Assuntos
Proteínas de Bactérias , Biofilmes/efeitos dos fármacos , Exotoxinas , Proteínas de Membrana , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/fisiologia , Exotoxinas/farmacologia , Exotoxinas/fisiologia , Perfilação da Expressão Gênica/métodos , Humanos , Proteínas de Membrana/farmacologia , Proteínas de Membrana/fisiologia , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Superantígenos/fisiologia
11.
Nat Chem ; 11(5): 463-469, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011175

RESUMO

Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from non-Staphylococcus aureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing and virulence gene expression in the human pathogen Staphylococcus aureus. However, only a limited number of AIP structures from non-S. aureus staphylococci have been identified to date, as the minute amounts secreted in complex media render it difficult. Here, we report a method for the identification of AIPs by exploiting their thiolactone functionality for chemoselective trapping and enrichment of the compounds from the bacterial supernatant. Standard liquid chromatography mass spectrometry analysis, guided by genome sequencing data, then readily provides the AIP identities. Using this approach, we confirm the identity of five known AIPs and identify the AIPs of eleven non-S. aureus species, and we expect that the method should be extendable to AIP-expressing Gram-positive bacteria beyond the Staphylococcus genus.


Assuntos
Proteínas de Bactérias/análise , Depsipeptídeos/análise , Staphylococcus/química , Sequência de Aminoácidos , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Cisteína/química , Depsipeptídeos/síntese química , Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , Limite de Detecção , Listeria monocytogenes/química , Estrutura Molecular , Percepção de Quorum/efeitos dos fármacos , Staphylococcus/metabolismo
12.
Vet Microbiol ; 232: 96-104, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31030852

RESUMO

The bovine endometrium is constantly challenged with pathogenic bacteria, especially with Escherichia coli. In previous studies, we showed that prostaglandin E2 (PGE2) synthesis was increased in E. coli-infected bovine endometrial tissue, which promoted the development of inflammatory damage. However, the molecular mechanism underlying this accumulation of PGE2 remained undefined. Lipoprotein (LP) is one of critical outer membrane protein in E. coli, which regulates inflammatory response. In this study, we determined the role of LP in PGE2 accumulation in bovine endometrial tissue by infecting the tissue with wild endometrial pathogenic E. coli and E. coli LP deletion mutant (JE5505) strains. We demonstrate that JE5505 was less effective than pathogenic E. coli in inducing the production of PGE2,IL-6, TNF-α, HMGB-1, and HABP1 and that the induction of cytokines was dependent on the activation of MAPKs, as revealed by rapid phosphorylation of ERK1/2/NF-κB in the endometrial tissues, furthermore, LP also induced PGE2 synthessis and cytokine secretion. Additionally, ERK and NF-κB inhibitors significantly inhibited PGE2 production and cytokine secretion and reduced or attenuated tissue damage in JE5505-infected and LP induced endometrial tissues. What is more important, we reported PGE2 introduction increased the expression of pro-inflammatory factors and DAMPs in E. coli-infected bovine endometrial tissue. Taken together, these results indicate that LP is involved in the accumulation of PGE2 through the activation of the ERK/NF-κB pathway that induces the production of pro-inflammatory factors and damage-associated molecular patterns (DAMPs) in E. coli-infected bovine endometrial tissue. These results should help in better understanding and management of postpartum inflammatory diseases in dairy cows.


Assuntos
Dinoprostona/biossíntese , Escherichia coli/patogenicidade , Inflamação/patologia , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Animais , Proteínas de Bactérias/farmacologia , Bovinos , Citocinas , Endométrio/imunologia , Infecções por Escherichia coli/patologia , Feminino , Lipoproteínas/farmacologia , Transdução de Sinais
13.
Mar Drugs ; 17(2)2019 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-30813318

RESUMO

Wound healing is a dynamic and complex process. The proliferation and migration of dermal fibroblasts are crucial for wound healing. Recent studies have indicated that the extracts from Spirulina platensis have a positive potential for wound healing. However, its underlying mechanism is not fully understood. Our previous study showed that spirulina crude protein (SPCP) promoted the viability of human dermal fibroblast cell line (CCD-986sk cells). In this study, we further investigated the wound healing effect and corresponding mechanisms of SPCP on CCD-986sk cells. Bromodeoxyuridine (BrdU) assay showed that SPCP promoted the proliferation of CCD-986sk cells. The wound healing assay showed that SPCP promoted the migration of CCD-986sk cells. Furthermore, cell cycle analysis demonstrated that SPCP promoted CCD-986sk cells to enter S and G2/M phases from G0/G1 phase. Western blot results showed that SPCP significantly upregulated the expression of cyclin D1, cyclin E, cyclin-dependent kinase 2 (Cdk2), cyclin-dependent kinase 4 (Cdk4), and cyclin-dependent kinase 6 (Cdk6), as well as inhibited the expression of CDK inhibitors p21 and p27 in CCD-986sk cells. In the meanwhile, SPCP promoted the phosphorylation and activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt). However, the phosphorylation of Akt was significantly blocked by PI3K inhibitor (LY294002), which in turn reduced the SPCP-induced proliferation and migration of CCD-986sk cells. Therefore, the results presenting in this study suggested that SPCP can promote the proliferation and migration of CCD-986sk cells; the PI3K/Akt signaling pathway play a positive and important role in these processes.


Assuntos
Proteínas de Bactérias/farmacologia , Fibroblastos/efeitos dos fármacos , Spirulina/química , Cicatrização/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Transdução de Sinais
14.
Mol Cell Biochem ; 457(1-2): 179-189, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30911956

RESUMO

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that triggers several survival mechanisms against the host immune system. Many studies show that the diverse components of Mtb can modulate apoptosis in various types of cells differently. So far, apoptosis induced by ESAT-6, an early secreted antigenic target of 6-kDa of Mtb, has been studied but the details of molecular mechanism and signaling pathway remain incompletely defined. This study investigated the role of recombinant ESAT-6 in inducing apoptosis in primary bone marrow-derived macrophages (BMDMs) of mice using Annexin V/PI assay with FACS analysis and Western blotting technique. It has been found that ESAT-6-induced apoptosis in BMDMs in a dose- and time-dependent pattern. Apoptosis induced by ESAT-6 was mainly via the intrinsic pathway with elevated protein levels of cleaved caspase-9 and -3. Furthermore, ESAT-6 also induced Bim activation during this process. Interestingly, this event was TLR2-dependent since the effect of ESAT-6 on apoptosis vanished in BMDM from mice with TLR2 deficiency. Furthermore, ROS generation and MAPKs phosphorylation induced by ESAT-6 were also involved in caspase-9 and caspase-3 activation. Taken together, these data suggest that ESAT-6-mediated apoptosis is involved in ROS-MAPKs signaling and further activating the intrinsic pathway, which provides new insights into the basic physiology of macrophage death in tuberculosis.


Assuntos
Antígenos de Bactérias/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Macrófagos/metabolismo , Mycobacterium tuberculosis/química , Animais , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Macrófagos/patologia , Masculino , Camundongos
15.
Mar Drugs ; 17(3)2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30871149

RESUMO

Fibrinolytic enzymes have received more attention due to their medicinal potential for thrombolytic diseases. The aim of this study is to characterize the in vitro fibrinolytic nature of purified protease producing Streptomyces radiopugnans VITSD8 from marine brown tube sponges Agelas conifera. Three varieties of sponge were collected from the Rameshwaram Sea coast, Tamil Nadu, India. The fibrinolytic activity of Streptomyces sp. was screened and determined by casein plasminogen plate and fibrin plate methods respectively. The crude caseinolytic protease was purified using ammonium sulfate fractionation, affinity and ion-exchange chromatography. Based on the morphological, biochemical, and molecular characterization, the isolate VITSD8 was confirmed as Streptomyces radiopugnans. Maltose and peptone were found to be the best carbon and nitrogen sources for the production of fibrinolytic protease. The carbon and nitrogen source peptone showed (781 U/mL) enzyme activity. The optimum pH and temperature for fibrinolytic protease production was found to be 7.0 and 33 °C respectively. The purified enzyme showed a maximum specific activity of 3891 U. The blood clot lysis activity was compared with the standard, and it was concluded that a minimum of 0.18 U (10 µL) of purified protease was required to dissolve the blood clot. This is the first report which exploits the fibrinolytic protease activity of Streptomyces radiopugnans VITSD8 extracted from a marine sponge. Hence the investigation suggests a potential benefit of purified fibrinolytic protease which will serve as an excellent clot buster alternative.


Assuntos
Proteínas de Bactérias/química , Endopeptidases/biossíntese , Fibrinolíticos/química , Peptídeo Hidrolases/química , Poríferos/microbiologia , Streptomyces/química , Streptomyces/enzimologia , Animais , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/farmacologia , Filogenia , Espectroscopia de Infravermelho com Transformada de Fourier , Trombose/tratamento farmacológico
16.
Protein Pept Lett ; 26(6): 414-422, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-30919769

RESUMO

BACKGROUND: Genome sequence analysis (GenBank access No.: FN667742.1) shows that Xenorhabdus nematophila ATCC19061 contains one gene (Xn-cbp) encoding chitin binding protein (Xn-CBP). OBJECTIVE: The present work aims to clarify the characteristics and function of Xn-CBP from X. nematophila HB310. METHODS: In this study, the Xn-cbp gene was cloned and expressed in Escherichia coli BL21 (DE3). Substrate binding assays were performed to explain the ability of Xn-CBP combined with the polysaccharide. The insecticidal toxicity of Xn-CBP against the second-instar larvae of Helicoverpa armigera was determined by feeding method. Besides, the antifungal activity of Xn-CBP against Coniothyrium diplodiella, Verticillium dahlia, and Fusarium oxysporum was tested by spore germination assay and hyphal extension assay. RESULTS: Xn-CBP encoded 199 amino acids with a calculated mass of 28 kDa, which contained a signal peptide and a chitin binding domain. The Bmax and Kd values of Xn-CBP to colloidal chitin were 2.46 and 4.08, respectively. Xn-CBP had insecticidal activity against the H. armigera with a growth inhibition rate of 84.08%. Xn-CBP had the highest spore germination inhibitory effect on C. diplodiella with the inhibition rate of 83.11%. The hyphal growth inhibition rate of Xn-CBP to F. oxysporum, 41.52%, was higher than the other two fungi. CONCLUSION: The Xn-CBP had the highest binding ability to colloidal chitin and it showed insecticidal activity and antifungal activity. The present study laid a foundation for further exploitation and utilization of X. nematophila.


Assuntos
Antifúngicos/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Quitina/química , Xenorhabdus/química , Animais , Antifúngicos/farmacologia , Ascomicetos/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Escherichia coli/genética , Inseticidas/química , Larva/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Ligação Proteica
17.
Mol Plant Microbe Interact ; 32(8): 961-971, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30830835

RESUMO

The type VI secretion system (T6SS) is used by gram-negative bacteria to translocate effectors that can antagonize other bacterial cells. Models predict the variation in collections of effector and cognate immunity genes determine competitiveness and can affect the dynamics of populations and communities of bacteria. However, the outcomes of competition cannot be entirely explained by compatibility of effector-immunity (EI) pairs. Here, we characterized the diversity of T6SS loci of plant-pathogenic Agrobacterium tumefaciens and showed that factors other than EI pairs can impact interbacterial competition. All examined strains encode T6SS active in secretion and antagonism against Escherichia coli. The spectra of EI pairs as well as compositions of gene neighborhoods are diverse. Almost 30 in-planta competitions were tested between different genotypes of A. tumefaciens. Fifteen competitions between members of different species-level groups resulted in T6SS-dependent suppression in in-planta growth of prey genotypes. In contrast, ten competitions between members within species-level groups resulted in no significant effect on the growth of prey genotypes. One strain was an exceptional case and, despite encoding a functional T6SS and toxic effector protein, could not compromise the growth of the four tested prey genotypes. The data suggest T6SS-associated EI pairs can influence the competitiveness of strains of A. tumefaciens, but genetic features have a significant role on the efficacy of interbacterial antagonism.


Assuntos
Agrobacterium tumefaciens , Variação Genética , Interações Hospedeiro-Patógeno , Sistemas de Secreção Tipo VI , Agrobacterium tumefaciens/fisiologia , Proteínas de Bactérias/farmacologia , Escherichia coli/efeitos dos fármacos , Sistemas de Secreção Tipo VI/metabolismo
18.
Microb Pathog ; 130: 146-155, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30826430

RESUMO

Application of antibiotics to combat bacterial diseases in fish has been criticized due to likely emergence of drug resistance. Therefore, investigation of new bioactive compounds from natural sources has been taken into account. This study was designed to purify and characterize the bioactive compound in the cell free supernatant (CFSs) of autochthonous gut bacteria (Bacillus methylotrophicus KU556164, B. amyloliquefaciens KU556165, Pseudomonas fluorescens KU556166 and B. licheniformis KU556167) isolated from rohu, Labeo rohita. CFSs were antagonistic to fish pathogenic Aeromonas spp., moderately thermo-tolerant and active in wide range of pH (5-11). Antibacterial activity of the CFSs was reduced by the action of proteases (e.g., Proteinase K and Trypsin), indicating proteinaceous nature of the bioactive compound like the bacteriocins. Three-step purification procedure resulted in recovery of 16.97%, 18.04%, 33.33% and 6.38% activity of the antimicrobial protein produced by B. methylotrophicus, B. amyloliquefaciens, P. fluorescens and B. licheniformis, respectively. Purification at each step revealed decrease in protein content with gradual increase in the specific activity of the antimicrobial protein. The purified antibacterial compound ranged between 18.2 and 25.6 kDa. Identification through MALDI-TOF MS/MS and database search through Mascot search engine predicted that the bactericidal compound belonged to either alkaline proteases, or, transcriptional regulator and some hypothetical proteins. Apart from potential technological application of the antibacterial compound, the present study might show promise for application of gut-associated bacteriocinogenic bacteria to control diseases in fish caused by pathogenic bacteria.


Assuntos
Antibacterianos/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Produtos Biológicos/metabolismo , Cyprinidae/microbiologia , Microbioma Gastrointestinal , Aeromonas/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antibiose , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Concentração de Íons de Hidrogênio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
19.
J Agric Food Chem ; 67(11): 3168-3178, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30799619

RESUMO

In this study we report a secretory protein that was purified from Serratia marcescens strain S3 isolated from soil from the tobacco rhizosphere. Subsequent mass spectrometry and annotation characterized the protein as secretory alkaline metalloprotease (SAMP). SAMP plays a crucial role in inhibiting Tobacco mosaic virus (TMV). Transmission electron microscopy (TEM), dynamic light scattering (DLS), confocal microscopy, and microscale thermophoresis (MST) were employed to investigate the anti-TMV mechanism of SAMP. Our results demonstrated that SAMP, as a hydrolytic metal protease, combined and hydrolyzed TMV coat proteins to destroy the virus particles. This study is the first to investigate the antiviral effects of a S. marcescens metalloprotease, and our finding suggests that S. marcescens-S3 may be agronomically useful as a disease-controlling factor active against Tobacco mosaic virus.


Assuntos
Antivirais/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Metaloproteases/farmacologia , Serratia marcescens/enzimologia , Antivirais/isolamento & purificação , Antivirais/metabolismo , Proteínas de Bactérias/isolamento & purificação , Metaloproteases/isolamento & purificação , Metaloproteases/metabolismo , Serratia marcescens/química , Serratia marcescens/genética , Vírus do Mosaico do Tabaco/efeitos dos fármacos , Vírus do Mosaico do Tabaco/crescimento & desenvolvimento
20.
J Anim Sci ; 97(4): 1712-1721, 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30753508

RESUMO

The objective was to test the hypothesis that the standardized total tract digestibility (STTD) of Ca and the response to microbial phytase on STTD of Ca and apparent total tract digestibility (ATTD) of P in diets fed to gestating sows are constant throughout gestation. The second objective was to test the hypothesis that retention of Ca and P does not change during gestation. Thirty-six gestating sows (parity = 3.3 ± 1.5; d of gestation = 7 d) were allotted to 4 diets. Two diets containing 0 or 500 units of microbial phytase per kilogram were based on corn, potato protein concentrate, and calcium carbonate. Two Ca-free diets were also formulated without or with microbial phytase to estimate basal endogenous loss of Ca. Daily feed allowance was 1.5 times the maintenance energy requirement. Sows were housed individually in gestation stalls and fed a common gestation diet, but they were moved to metabolism crates from days 7 to 20 (early gestation), days 49 to 62 (midgestation), and again from days 91 to 104 (late gestation). When sows were in metabolism crates, they were fed experimental diets and feces and urine were quantitatively collected for 4 d after 4 d of adaptation. Results indicated that outcomes were not influenced by the interaction between period of gestation and dietary phytase. The basal endogenous loss of Ca was greater (P < 0.05) by sows in early gestation than by sows in mid- or late-gestation, but supplementation of microbial phytase to the Ca-free diet decreased (P < 0.01) the basal endogenous loss of Ca and tended (P = 0.099) to increase ATTD of P. Supplementation of microbial phytase did not affect ATTD of DM, STTD of Ca, Ca retention, ATTD of P, or P retention in sows fed the calcium carbonate-containing diet. The ATTD of DM was not affected by period of gestation, but the ATTD of Ca, the ATTD of P, and the retention of Ca were least (P < 0.05) in midgestation, followed by early and late gestation, respectively, and the STTD of Ca in midgestation was also reduced (P < 0.05) compared with sows in early or late gestation. Phosphorus retention was greater (P < 0.05) in late gestation than in the earlier periods. In conclusion, Ca retention was less negative and ATTD of P tended to increase with supplementation of microbial phytase to the Ca-free diet regardless of gestation period. The basal endogenous loss, STTD of Ca, ATTD of P, and retention of Ca and P in gestating sows change during gestation with the greatest digestibility values observed in late gestation.


Assuntos
6-Fitase/farmacologia , Carbonato de Cálcio/metabolismo , Cálcio na Dieta/metabolismo , Fósforo na Dieta/metabolismo , Suínos/fisiologia , Ração Animal/análise , Animais , Proteínas de Bactérias/farmacologia , Dieta/veterinária , Digestão , Fezes/química , Feminino , Trato Gastrointestinal/metabolismo , Gravidez , Zea mays
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