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1.
Cell Host Microbe ; 26(3): 412-425.e5, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31492656

RESUMO

Surviving infection requires immune and repair mechanisms. Developing organisms face the additional challenge of integrating these mechanisms with tightly controlled developmental processes. The larval Drosophila midgut lacks dedicated intestinal stem cells. We show that, upon infection, larvae perform limited repair using adult midgut precursors (AMPs). AMPs differentiate in response to damage to generate new enterocytes, transiently depleting their pool. Developmental delay allows for AMP reconstitution, ensuring the completion of metamorphosis. Notch signaling is required for the differentiation of AMPs into the encasing, niche-like peripheral cells (PCs), but not to differentiate PCs into enterocytes. Dpp (TGF-ß) signaling is sufficient, but not necessary, to induce PC differentiation into enterocytes. Infection-induced JAK-STAT pathway is both required and sufficient for differentiation of AMPs and PCs into new enterocytes. Altogether, this work highlights the constraints imposed by development on an organism's response to infection and demonstrates the transient use of adult precursors for tissue repair.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Trato Gastrointestinal/metabolismo , Larva/metabolismo , Animais , Diferenciação Celular , Modelos Animais de Doenças , Drosophila/microbiologia , Drosophila/fisiologia , Proteínas de Drosophila/genética , Enterócitos/metabolismo , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/patologia , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/patologia , Janus Quinases/metabolismo , Larva/imunologia , Larva/microbiologia , Metamorfose Biológica , Pectobacterium carotovorum/patogenicidade , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma
2.
Mar Drugs ; 17(5)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117226

RESUMO

MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomLI144V and MomLV149A. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc). Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc-infected Chinese cabbage. The results indicated that MomL and its mutants (MomLL254R, MomLI144V, MomLV149A) significantly decreased the pathogenicity of Pcc. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.


Assuntos
Aminoácidos/análise , Hidrolases de Éster Carboxílico , Pectobacterium carotovorum/enzimologia , Pectobacterium carotovorum/genética , Engenharia de Proteínas , Substituição de Aminoácidos , Brassica rapa/microbiologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Ativação Enzimática/genética , Mutação , Pectobacterium carotovorum/patogenicidade , Virulência/genética
3.
J Microbiol Biotechnol ; 29(5): 785-793, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31030456

RESUMO

Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is the most damaging disease in Brassica crops around the world. In this study, we developed a molecular marker specific to Xcc race 5. To do this, the available whole genome sequences of Xcc races/strains and Xc subspecies were aligned and identified a highly variable genomic region (XccR5-89.2). Subsequently, a primer set covering the 'XccR5-89.2' region was designed and tested against the genomic DNA of Xcc races/strains, Xc subspecies and other plant-infecting bacterial strains (Pseudomonas syringae pv. maculicola and Erwinia carotovora subsp. carotovora). The results showed that the 'XccR5-89.2' primer pair amplified a 2,172-bp fragment specific to Xcc race 5. Moreover, they also amplified a 1,515-bp fragment for Xcc race 1 and an over 3,000-bp fragment for Xcc race 3. However, they did not amplify any fragments from the remaining Xcc races/strains, subspecies or other bacterial strains. The 'XccR5-89.2' primer pair was further PCR amplified from race-unknown Xcc strains and ICMP8 was identified as race 5 among nine race-unknown Xcc strains. Further cloning and sequencing of the bands amplified from race 5 and ICMP8 with 'XccR5-89.2' primers revealed both carrying identical sequences. The results showed that the 'XccR5-89.2' marker can effectively and proficiently detect, and identify Xcc race 5 from Xcc races/strains, subspecies and other plant-infecting bacteria. To our knowledge, this is the first report for an Xcc race 5-specific molecular marker.


Assuntos
Genes Bacterianos/genética , Doenças das Plantas/microbiologia , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidade , Proteínas de Bactérias , Clonagem Molecular , DNA Bacteriano/genética , Endopeptidases , Marcadores Genéticos , Genoma Bacteriano , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/patogenicidade , Reação em Cadeia da Polimerase/métodos , Pseudomonas syringae/genética , Pseudomonas syringae/patogenicidade , Alinhamento de Sequência , Sequenciamento Completo do Genoma
4.
Mar Biotechnol (NY) ; 21(2): 276-290, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30762152

RESUMO

Many Gram-negative aquacultural and agricultural pathogens control virulence factor expression through a quorum-sensing (QS) mechanism involving the production of N-acylhomoserine (AHL) signalling molecules. Thus, the interruption of QS systems by the enzymatic degradation of signalling molecules, known as quorum quenching (QQ), has been proposed as a novel strategy to combat these infections. Given that the symbiotic bacteria of marine invertebrates are considered to be an important source of new bioactive molecules, this study explores the presence of AHL-degrading bacteria among 827 strains previously isolated from the microbiota of anemones and holothurians. Four of these strains (M3-1, M1-14, M3-13 and M9-54-2), belonging to the species Stenotrophomonas maltophilia, were selected on the basis of their ability to degrade a broad range of AHLs, and the enzymes involved in their activity were identified. Strain M9-54-2, which showed the strongest AHL-degrading activity, was selected for further study. High-performance liquid chromatography-mass-spectrometry confirmed that the QQ enzyme is not a lactonase. Strain M9-54-2 degraded AHL accumulation and reduced the production of enzymatic activity in Pectobacterium carotovorum CECT 225T and Vibrio coralliilyticus VibC-Oc-193 in in vitro co-cultivation experiments. The effect of AHL inactivation was confirmed by a reduction in potato tuber maceration and brine shrimp (Artemia salina) mortality caused by P. carotovorum and Vibrio coralliilyticus, respectively. This study strengthens the evidence of marine organisms as an underexplored and promising source of QQ enzymes, useful to prevent infections in aquaculture and agriculture. To our knowledge, this is the first time that anemones and holothurians have been studied for this purpose.


Assuntos
Acil-Butirolactonas/metabolismo , Pectobacterium carotovorum/patogenicidade , Vibrio/patogenicidade , Virulência , Animais , Artemia/microbiologia , Técnicas de Cocultura , Holothuria/microbiologia , Microbiota , Doenças das Plantas/microbiologia , Percepção de Quorum , Anêmonas-do-Mar/microbiologia , Solanum tuberosum/microbiologia , Stenotrophomonas maltophilia , Vibrioses/metabolismo
5.
Phytochemistry ; 159: 75-89, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30597374

RESUMO

Solanum tuberosum, commonly known as the potato, is a worldwide food staple. During harvest, storage, and distribution the crop is at risk of mechanical damage. Wounding of the tuber skin can also become a point of entry for bacterial and fungal pathogens, resulting in substantial agricultural losses. Building on the proposal that potato tubers produce metabolites to defend against microbial infection during early stages of wound healing before protective suberized periderm tissues have developed, we assessed extracts of wound tissues from four potato cultivars with differing skin morphologies (Norkotah Russet, Atlantic, Chipeta, and Yukon Gold). These assays were conducted at 0, 1, 2, 3 and 7 days post wounding against the plant pathogen Erwinia carotovora and a non-pathogenic Escherichia coli strain that served as a control. For each of the potato cultivars, only polar wound tissue extracts demonstrated antibacterial activity. The polar extracts from earlier wound-healing time points (days 0, 1 and 2) displayed notably higher antibacterial activity against both strains than the later wound-healing stages (days 3 and 7). These results support a burst of antibacterial activity at early time points. Parallel metabolite profiling of the extracts revealed differences in chemical composition at different wound-healing time points and allowed for identification of potential marker compounds according to healing stage for each of the cultivars. It was possible to monitor the transformations in the metabolite profiles that could account for the phenomenon of temporal resistance by looking at the relative quantities of various metabolite classes as a function of time.


Assuntos
Antibacterianos/farmacologia , Pectobacterium carotovorum/efeitos dos fármacos , Extratos Vegetais/farmacologia , Tubérculos/metabolismo , Solanum tuberosum/metabolismo , Cicatrização/efeitos dos fármacos , Alcaloides/metabolismo , Aminas/metabolismo , Biomarcadores/metabolismo , Escherichia coli/fisiologia , Testes de Sensibilidade Microbiana , Pectobacterium carotovorum/patogenicidade , Fenóis/metabolismo , Tubérculos/microbiologia , Solanum tuberosum/classificação , Solanum tuberosum/microbiologia , Especificidade da Espécie
6.
Phytopathology ; 109(1): 27-35, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30028233

RESUMO

Thionins are a family of antimicrobial peptides. We performed in silico expression analyses of the 44 rice (Oryza sativa) thionins (OsTHIONs). Modulated expression levels of OsTHIONs under different treatments suggest their involvement in many processes, including biotic, abiotic, and nutritional stress responses, and in hormone signaling. OsTHION15 (LOC_Os06g32600) was selected for further characterization based on several in silico analyses. OsTHION15 in O. sativa subsp. indica 'KDML 105' was expressed in all of the tissues and organs examined, including germinating seed, leaves, and roots of seedlings and mature plants, and inflorescences. To investigate the antimicrobial activity of OsTHION15, we produced a recombinant peptide in Escherichia coli Rosetta-gami (DE3). The recombinant OsTHION15 exhibited inhibitory activities toward rice-pathogenic bacteria such as Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum pv. atroseptica, with minimum inhibitory concentrations of 112.6 and 14.1 µg ml-1, respectively. A significant hyphal growth inhibition was also observed toward Fusarium oxysporum f. sp. cubense and Helminthosporium oryzae. In addition, we demonstrated the in planta antibacterial activity of this peptide in Nicotiana benthamiana against X. campestris pv. glycines. These activities suggest the possible application of OsTHION15 in plant disease control.


Assuntos
Oryza/genética , Doenças das Plantas/genética , Tioninas/genética , Oryza/microbiologia , Pectobacterium carotovorum/patogenicidade , Doenças das Plantas/microbiologia , Xanthomonas/patogenicidade
7.
Microbiol Res ; 217: 23-33, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30384906

RESUMO

Four hundred and fifty bacteria were evaluated for antagonistic activity against bacterial soft rot of potato caused by Pectobacterium carotovorum sp strain II16. A strain Ar10 exhibiting potent antagonist activity has been identified as Bacillus amyloliquefaciens on the basis of biochemical and molecular characterization. Cell free supernatant showed a broad spectrum of antibacterial activity against human and phytopathogenic bacteria in the range of 10-60 AU/mL. Incubation of P. carotovorum cells with increasing concentrations of the antibacterial compound showed a killing rate of 94.8 and 96% at MIC and 2xMIC respectively. In addition, the antibacterial agent did not exert haemolytic activity at the active concentration and has been preliminary characterized by TLC and GC-MS as a glycolipid compound. Treatment of potato tubers with strain Ar10 for 72 h significantly reduced the severity of disease symptoms (100 and 85.05% reduction of necrosis deep / area and weight loss respectively). The same levels in disease symptoms severity was also recorded following treatment of potato tubers with cell free supernatant for 1 h. Data suggest that protection against potato soft rot disease may be related to glycolipid production by strain Ar10. The present study affords new alternatives for anti-Pectobacterium carotovorum bioactive compounds against the soft rot disease of potato.


Assuntos
Antibacterianos/farmacologia , Bacillus amyloliquefaciens/metabolismo , Agentes de Controle Biológico/antagonistas & inibidores , Glicolipídeos/antagonistas & inibidores , Pectobacterium carotovorum/efeitos dos fármacos , Doenças das Plantas/prevenção & controle , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Bacillus amyloliquefaciens/classificação , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/isolamento & purificação , Agentes de Controle Biológico/química , Agentes de Controle Biológico/isolamento & purificação , Agentes de Controle Biológico/metabolismo , Endófitos , Glicolipídeos/química , Glicolipídeos/isolamento & purificação , Glicolipídeos/metabolismo , Cinética , Testes de Sensibilidade Microbiana , Pectobacterium carotovorum/isolamento & purificação , Pectobacterium carotovorum/patogenicidade , Doenças das Plantas/microbiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/microbiologia , Solanum tuberosum/microbiologia
8.
Mol Plant Microbe Interact ; 31(11): 1166-1178, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30198820

RESUMO

Hfq is a RNA chaperone and participates in a wide range of cellular processes and pathways. In this study, mutation of hfq gene from Pectobacterium carotovorum subsp. carotovorum PccS1 led to significantly reduced virulence and plant cell wall-degrading enzyme (PCWDE) activities. In addition, the mutant exhibited decreased biofilm formation and motility and greatly attenuated carbapenem production as well as secretion of hemolysin coregulated protein (Hcp) as compared with wild-type strain PccS1. Moreover, a higher level of callose deposition was induced in Nicotiana benthamiana leaves when infiltrated with the mutant. A total of 26 small (s)RNA deletion mutants were obtained among a predicted 27 sRNAs, and three mutants exhibited reduced virulence in the host plant. These results suggest that hfq plays a key role in Pectobacterium virulence by positively impacting PCWDE production, secretion of the type VI secretion system, bacterial competition, and suppression of host plant responses.


Assuntos
Biofilmes/crescimento & desenvolvimento , Calla (Planta)/microbiologia , Fator Proteico 1 do Hospedeiro/metabolismo , Pectobacterium carotovorum/enzimologia , Doenças das Plantas/microbiologia , Sistemas de Secreção Tipo VI/metabolismo , Sequência de Aminoácidos , Calla (Planta)/imunologia , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucanos/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/patogenicidade , Pectobacterium carotovorum/fisiologia , Doenças das Plantas/imunologia , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Alinhamento de Sequência , Sistemas de Secreção Tipo VI/genética , Virulência
9.
PLoS Pathog ; 14(9): e1007279, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30180210

RESUMO

The digestive tract is the first organ affected by the ingestion of foodborne bacteria. While commensal bacteria become resident, opportunistic or virulent bacteria are eliminated from the gut by the local innate immune system. Here we characterize a new mechanism of defense, independent of the immune system, in Drosophila melanogaster. We observed strong contractions of longitudinal visceral muscle fibers for the first 2 hours following bacterial ingestion. We showed that these visceral muscle contractions are induced by immune reactive oxygen species (ROS) that accumulate in the lumen and depend on the ROS-sensing TRPA1 receptor. We then demonstrate that both ROS and TRPA1 are required in a subset of anterior enteroendocrine cells for the release of the DH31 neuropeptide which activates its receptor in the neighboring visceral muscles. The resulting contractions of the visceral muscles favors quick expulsion of the bacteria, limiting their presence in the gut. Our results unveil a precocious mechanism of defense against ingested opportunistic bacteria, whether they are Gram-positive like Bacillus thuringiensis or Gram-negative like Erwinia carotovora carotovora. Finally, we found that the human homolog of DH31, CGRP, has a conserved function in Drosophila.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Proteínas de Drosophila/fisiologia , Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/fisiologia , Hormônios de Inseto/fisiologia , Animais , Animais Geneticamente Modificados , Bacillus thuringiensis/patogenicidade , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Drosophila melanogaster/fisiologia , Feminino , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/fisiopatologia , Humanos , Imunidade Inata , Canais Iônicos , Lactobacillus plantarum/patogenicidade , Contração Muscular/fisiologia , Infecções Oportunistas/microbiologia , Infecções Oportunistas/fisiopatologia , Infecções Oportunistas/prevenção & controle , Pectobacterium carotovorum/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Canal de Cátion TRPA1/fisiologia
10.
Int J Biol Macromol ; 116: 31-36, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29738862

RESUMO

Vitamin B6 (VitB6) is an essential cofactor for >140 biochemical reactions. Also, VitB6 is a potent antioxidant and helps plants cope with both biotic and abiotic stress conditions. However, the role of VitB6 in plant disease resistance has yet to be confirmed using molecular biology approaches. Here, we analyzed the expression patterns of VitB6 biosynthetic genes, including the de novo (PDX1 [PDX1.2 and 1.3] and PDX2) and the salvage (SOS4) pathways during the response to Erwinia carotovora subsp. carotovora. By quantitative PCR, we found that the most significant upregulation in the transcript profile of PDX2, which showed a 9.2-fold increase in expression at 12 h post inoculation (hpi) compared to 24-48 hpi. We also detected significant upregulation of PDX1.2 and PDX1.3, which were 6.6- and 4.3-fold upregulated at 24 hpi compared to 12 hpi, while SOS4 showed only low-level expression. Also, at 24 hpi, a significant increase in superoxide dismutase, catalase, peroxidase, and polyphenol oxidase activities was observed in plants. Our findings confirm that the expression of de novo and salvage pathway genes is induced by E. carotovora and that this plays an important role in the regulation of defense response by modulating cellular antioxidant capacity.


Assuntos
Antioxidantes/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Vitamina B 6/biossíntese , Vitamina B 6/genética , Catalase/metabolismo , Catecol Oxidase/metabolismo , Lycopersicon esculentum/metabolismo , Pectobacterium carotovorum/patogenicidade , Peroxidase/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Superóxido Dismutase/metabolismo , Regulação para Cima/genética
11.
PLoS Pathog ; 14(3): e1006936, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29499056

RESUMO

Gut immunity is regulated by intricate and dynamic mechanisms to ensure homeostasis despite a constantly changing microbial environment. Several regulatory factors have been described to participate in feedback responses to prevent aberrant immune activity. Little is, however, known about how transcriptional programs are directly tuned to efficiently adapt host gut tissues to the current microbiome. Here we show that the POU/Oct gene nubbin (nub) encodes two transcription factor isoforms, Nub-PB and Nub-PD, which antagonistically regulate immune gene expression in Drosophila. Global transcriptional profiling of adult flies overexpressing Nub-PB in immunocompetent tissues revealed that this form is a strong transcriptional activator of a large set of immune genes. Further genetic analyses showed that Nub-PB is sufficient to drive expression both independently and in conjunction with nuclear factor kappa B (NF-κB), JNK and JAK/STAT pathways. Similar overexpression of Nub-PD did, conversely, repress expression of the same targets. Strikingly, isoform co-overexpression normalized immune gene transcription, suggesting antagonistic activities. RNAi-mediated knockdown of individual nub transcripts in enterocytes confirmed antagonistic regulation by the two isoforms and that both are necessary for normal immune gene transcription in the midgut. Furthermore, enterocyte-specific Nub-PB expression levels had a strong impact on gut bacterial load as well as host lifespan. Overexpression of Nub-PB enhanced bacterial clearance of ingested Erwinia carotovora carotovora 15. Nevertheless, flies quickly succumbed to the infection, suggesting a deleterious immune response. In line with this, prolonged overexpression promoted a proinflammatory signature in the gut with induction of JNK and JAK/STAT pathways, increased apoptosis and stem cell proliferation. These findings highlight a novel regulatory mechanism of host-microbe interactions mediated by antagonistic transcription factor isoforms.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/imunologia , Proteínas de Homeodomínio/metabolismo , Homeostase , Imunidade Inata/imunologia , Intestinos/imunologia , Fatores do Domínio POU/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/microbiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Intestinos/microbiologia , Masculino , NF-kappa B/genética , NF-kappa B/metabolismo , Fatores do Domínio POU/genética , Pectobacterium carotovorum/patogenicidade , Isoformas de Proteínas
12.
Cell Host Microbe ; 23(3): 338-352.e5, 2018 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-29503179

RESUMO

DUOX, a member of the NADPH oxidase family, acts as the first line of defense against enteric pathogens by producing microbicidal reactive oxygen species. DUOX is activated upon enteric infection, but the mechanisms regulating DUOX activity remain incompletely understood. Using Drosophila genetic tools, we show that enteric infection results in "pro-catabolic" signaling that initiates metabolic reprogramming of enterocytes toward lipid catabolism, which ultimately governs DUOX homeostasis. Infection induces signaling cascades involving TRAF3 and kinases AMPK and WTS, which regulate TOR kinase to control the balance of lipogenesis versus lipolysis. Enhancing lipogenesis blocks DUOX activity, whereas stimulating lipolysis via ATG1-dependent lipophagy is required for DUOX activation. Drosophila with altered activity in TRAF3-AMPK/WTS-ATG1 pathway components exhibit abolished infection-induced lipolysis, reduced DUOX activation, and enhanced susceptibility to enteric infection. Thus, this work uncovers signaling cascades governing inflammation-induced metabolic reprogramming and provides insight into the pathophysiology of immune-metabolic interactions in the microbe-laden gut epithelia.


Assuntos
Sistema Digestório/imunologia , Drosophila/imunologia , Oxidases Duais/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Inflamação/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Sistemas CRISPR-Cas/genética , Técnicas de Cultura de Células , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Enterócitos/metabolismo , Feminino , Microbioma Gastrointestinal , Edição de Genes , Regulação da Expressão Gênica , Homeostase , Imunidade Inata , Metabolismo dos Lipídeos , Lipólise , MAP Quinase Quinase Quinase 1/metabolismo , Masculino , Pectobacterium carotovorum/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/metabolismo
13.
J Appl Microbiol ; 124(6): 1580-1588, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29437273

RESUMO

AIMS: Isolation and characterization of pectolytic bacteria associated with soft rot disease of potatoes in Nakuru, Kenya, to provide the basis for the development of disease control measures. METHODS AND RESULTS: Potato tubers showing symptoms of soft rot were collected from different farms in Molo and Mau Narok regions within Nakuru county. Isolation was done using crystal violet pectate medium (CVPM). Out of the 71 isolates that showed growth on CVPM, pathogenicity tests revealed that 36 of them had the ability to macerate tissues of potato tubers. All the isolates yielded a fragment of approximately 1500 bp after 16S rDNA amplification. Using the BIOLOG microbial identification system, 20 bacterial isolates were identified as Pectobacterium carotovorum subsp. carotovorum, 7 were Pseudomonas fluorescens B while 9 were Ps. fluorescens A. Y1/Y2 primers successfully amplified pectate lyase-encoding (pel) gene, approximately 434 bp, in all the 20 P. carotovorum species. The virulence of the isolated strains to cause disease, according to pectinolytic tests, varied with change in incubation temperature of the test samples. Pectobacterium carotovorum strains were the most virulent at 30°C while disease severity due to infection by Ps. fluorescens A strains was high at 20°C compared to the other isolates. CONCLUSION: This study reveals the identity of pectolytic bacterial species from two genera, Pectobacterium and Pseudomonas, as causative agents of potato soft rot in Nakuru, Kenya. SIGNIFICANCE AND IMPACT OF THE STUDY: Research findings from this study will aid in developing suitable risk mitigation methods for adoption by farmers to prevent losses due to soft rot.


Assuntos
Pectobacterium carotovorum , Doenças das Plantas/microbiologia , Pseudomonas fluorescens , Solanum tuberosum/microbiologia , Quênia , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/patogenicidade , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/patogenicidade
14.
Biochem Biophys Res Commun ; 496(2): 462-467, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29337064

RESUMO

Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) for CGA biosynthesis has not been isolated and functionally characterized in potato. In this work, we cloned StC3'H from potato and showed that it catalyzed the formation of caffeoylshikimate and CGA (caffeoylquinate) from p-coumaroyl shikimate and p-coumaroyl quinate, respectively, but was inactive towards p-coumaric acid in in vitro enzyme assays. When the expression of StC3'H proteins was blocked through antisense (AS) inhibition under the control of a tuber-specific patatin promoter, moderate changes in tuber yield as well as phenolic metabolites in the core tuber tissue were observed for several AS lines. On the other hand, the AS and control potato lines exhibited similar responses to a bacterial pathogen Pectobacterium carotovorum. These results suggest that StC3'H is implicated in phenolic metabolism in potato. They also suggest that CGA accumulation in the core tissue of potato tubers is an intricately controlled process and that additional C3'H activity may also be involved in CGA biosynthesis in potato.


Assuntos
Ácido Clorogênico/metabolismo , Oxigenases de Função Mista/genética , Proteínas de Plantas/genética , Tubérculos/enzimologia , Solanum tuberosum/enzimologia , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Ácido Clorogênico/análogos & derivados , Clonagem Molecular , Expressão Gênica , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Pectobacterium carotovorum/patogenicidade , Pectobacterium carotovorum/fisiologia , Pichia/genética , Pichia/metabolismo , Proteínas de Plantas/metabolismo , Tubérculos/genética , Tubérculos/microbiologia , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ácido Chiquímico/análogos & derivados , Ácido Chiquímico/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/microbiologia
15.
Mol Plant Pathol ; 19(1): 35-48, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27671364

RESUMO

The identification of phytopathogen proteins that are differentially expressed during the course of the establishment of an infection is important to better understand the infection process. In vitro approaches, using plant extracts added to culture medium, have been used to identify such proteins, but the biological relevance of these findings for in planta infection are often uncertain until confirmed by in vivo studies. Here, we compared the proteins of Pectobacterium carotovorum ssp. carotovorum strain PccS1 differentially expressed in Luria-Bertani medium supplemented with extracts of the ornamental plant Zantedeschia elliotiana cultivar 'Black Magic' (in vitro) and in plant tissues (in vivo) by two-dimensional electrophoresis coupled with mass spectrometry. A total of 53 differentially expressed proteins (>1.5-fold) were identified (up-regulated or down-regulated in vitro, in vivo or both). Proteins that exhibited increased expression in vivo but not in vitro, or in both conditions, were identified, and deletions were made in a number of genes encoding these proteins, four of which (clpP, mreB, flgK and eda) led to a loss of virulence on Z. elliotiana, although clpP and mreB were later also shown to be reduced in growth in rich and minimal media. Although clpP, flgK and mreB have previously been reported as playing a role in virulence in plants, this is the first report of such a role for eda, which encodes 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, a key enzyme in Entner-Doudoroff metabolism. The results highlight the value of undertaking in vivo as well as in vitro approaches for the identification of new bacterial virulence factors.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/patogenicidade , Doenças das Plantas/microbiologia , Zantedeschia/microbiologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Eletroforese em Gel Bidimensional , Genes Bacterianos , Mutação/genética , Óperon/genética , Doenças das Plantas/genética , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espectrometria de Massas em Tandem , Transcrição Genética , Regulação para Cima/genética , Virulência/genética
16.
Microb Pathog ; 113: 348-356, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29126952

RESUMO

Three bacterial isolates were isolated from infected potato tubers showing soft and brown rots like symptoms as well as one isolate from infected peach tree showing crown gall symptom. The morphological, biochemical and molecular assays proved that bacterial isolates belonging to Pectobacterium carotovorum subsp. carotovorum, Ralstonia solanacearum, Dickeya spp. and Agrobacterium tumefaciens. The acetone (AcE) and n-butanol (ButE) extracts of Callistemon viminalis flowers and essential oil from aerial parts of Conyza dioscoridis as well as ButE of Eucalyptus camaldulensis bark are evaluated at different concentrations against the growth of the isolated bacteria. The diameter of inhibition zone (IZ) and the minimum inhibitory concentrations (MICs) are compared. Results indicated that the highest IZ values were 20.0 mm and 18.3 mm for E. camaldulensis bark ButE and C. viminalis flower ButE, respectively, against P. carotovorum; 16.3 mm and 16.0 mm for E. camaldulensis bark ButE and C. viminalis flower ButE, respectively, against R. solanacearum; 18.5 mm for C. viminalis flower AcE and C. dioscoridis aerial parts EO against Dickeya spp.; and 15.0 mm for C. viminalis flower AcE against A. tumefaciens. MICs ranged from <16 µg/mL for D. solani to >4000 µg/mL for A. tumefaciens. It was proved that C. viminalis flowers AcE contains mainly 5-hydroxymethylfurfural (20.6%), palmitic acid (18.5%), and pyrogallol (16.4%); while C. viminalis flower ButE contains palmitic acid (36.3%), 2-hydroxymyristic acid (9.4%), 5-hydroxymethylfurfural (7.2%), and shikimic acid (6.6%); whereas E. camaldulensis bark ButE contains 8-nonynoic acid methyl ester (45.6), camphor (30.9%), menthol (8.8%), and 1,8-cineole (eucalyptol) (8.2%), whilst the EO of C. dioscoridis aerial parts comprises Z-(13,14-epoxy)tetradec-11-en-1-ol acetate (11.6%), γ-elemene (10.2%), tau.-muurolol (7.1%), and cadina-3,9-diene (4.7%). It can be concluded that phytochemical extracts of C. viminalis, E. camaldulensis and C. dioscoridis demonstrated strong to moderate antibacterial effects against the studied plant bacterial pathogens.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Conyza/química , Eucalyptus/química , Myrtaceae/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Agrobacterium tumefaciens/efeitos dos fármacos , Agrobacterium tumefaciens/patogenicidade , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Egito , Flores/química , Testes de Sensibilidade Microbiana , Óleos Voláteis/farmacologia , Pectobacterium/efeitos dos fármacos , Pectobacterium/patogenicidade , Pectobacterium carotovorum/efeitos dos fármacos , Pectobacterium carotovorum/patogenicidade , Compostos Fitoquímicos/química , Doenças das Plantas/microbiologia , Óleos Vegetais/química , Óleos Vegetais/farmacologia , Raízes de Plantas/microbiologia , Ralstonia solanacearum/efeitos dos fármacos , Ralstonia solanacearum/patogenicidade , Solanum tuberosum/microbiologia
17.
Pak J Biol Sci ; 20(3): 147-153, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29023006

RESUMO

BACKGROUND: Pectobacterium carotovorum subsp. carotovorum (Pcc) is a soft rot bacterium which upon entry into the plant macerates plant tissues by producing plant cell wall degrading enzymes. It has a wide host range which includes carrot, potato, tomato, leafy greens, squash and other cucurbits, onion, green peppers and cassava. During plant-microbe interactions, one of the ways of plant response to pathogen infection is through the small RNA silencing mechanism. Under pathogen attack the plant utilizes microRNAs to regulate gene expression by means of mediating gene silencing at transcriptional and post-transcriptional level. This study aims to assess for the first time, the expression profile of some stress-responsive miRNA and differential expression pattern of their target genes in Arabidopsis thaliana inoculated with Pcc. MATERIALS AND METHODS: Leaves of five weeks old Arabidopsis thaliana plants were infected with Pcc and the quantitative real time-PCR, was used to investigate after 0, 24, 48 and 72 h post infection, the expression profiling of the stress-responsive miRNAs which include: miR156, miR159, miR169, miR393, miR396 miR398, miR399 and miR408 along with their target genes which include: Squamosa promoter-binding-like protein, myb domain protein 101, nuclear factor Y subunit A8, concanavalin A-like lectin protein kinase, growth regulating factor 4, copper superoxide dismutase, ubiquitin-protein ligase and plantacyanin respectively. RESULTS: The findings showed that the overexpression of 6 miRNAs at 24, 48 and 72 h after infection resulted in the repression of their target genes and the expression of 2 miRNAs didn't affect their target genes. CONCLUSION: These results provide the first indication of the miRNAs role in response to the infection of Pcc in A. thaliana and open new vistas for a better understanding of miRNA regulation of plant response to Pcc.


Assuntos
Arabidopsis/genética , Arabidopsis/microbiologia , MicroRNAs/genética , Pectobacterium carotovorum/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , RNA de Plantas/genética , Estresse Fisiológico , Transcriptoma , Arabidopsis/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/microbiologia , Fatores de Tempo
18.
Exp Cell Res ; 361(2): 225-235, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29074370

RESUMO

Pluripotent stem cell activity is essential to maintain regeneration and homeostasis in the Drosophila midgut following environmental challenges. Although multiple pathways have been implicated in epithelial renewal, the underlying regulatory mechanisms and correlations between relevant genes and pathways remain elusive. In this study, we show that the zinc finger protein CG12744 plays an important role in the differentiation and regeneration of epithelial cells in response to oral infection with Erwinia carotovora carotovora 15. Knocking down CG12744 in enteroblasts decreased the post-infection proportion of enteroblasts and enterocytes and increased the post-infection number of enteroendocrine cells. In addition, in precursors, CG12744 affected the Osa, jun-N-terminal kinase and bone morphogenetic protein signaling pathways to control enterocyte differentiation. Finally, CG12744 maintained epithelial architecture and cell fate in enterocytes following an acute infectious challenge.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Pectobacterium carotovorum/fisiologia , Dedos de Zinco/genética , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/imunologia , Diferenciação Celular , Proteínas de Ligação a DNA/imunologia , Proteínas de Drosophila/imunologia , Drosophila melanogaster/imunologia , Drosophila melanogaster/microbiologia , Enterócitos/imunologia , Enterócitos/microbiologia , Células Enteroendócrinas/imunologia , Células Enteroendócrinas/metabolismo , Células Enteroendócrinas/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Intestinos/microbiologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Masculino , Pectobacterium carotovorum/patogenicidade , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Regeneração/genética , Regeneração/imunologia , Transdução de Sinais , Dedos de Zinco/imunologia
19.
Phytopathology ; 107(11): 1322-1330, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28853642

RESUMO

Pectobacterium carotovorum subsp. carotovorum strain PccS1, a bacterial pathogen causing soft rot disease of Zantedeschia elliotiana (colored calla), was investigated for virulence genes induced by the host plant. Using a promoter-trap transposon (mariner), we obtained 500 transposon mutants showing kanamycin resistance dependent on extract of Z. elliotiana. One of these mutants, PM86, exhibited attenuated virulence on both Z. elliotiana and Brassica rapa subsp. pekinensis. The growth of PM86 was also reduced in minimal medium (MM), and the reduction was restored by adding plant extract to the MM. The gene containing the insertion site was identified as rplY. The deletion mutant ΔrplY, exhibited reduced virulence, motility and plant cell wall-degrading enzyme production but not biofilm formation. Analysis of gene expression and reporter fusions revealed that the rplY gene in PccS1 is up-regulated at both the transcriptional and the translational levels in the presence of plant extract. Our results suggest that rplY is induced by Z. elliotiana extract and is crucial for virulence in P. carotovorum subsp. carotovorum.


Assuntos
Proteínas de Bactérias/metabolismo , Pectobacterium carotovorum/patogenicidade , Extratos Vegetais/farmacologia , Zantedeschia/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Extratos Vegetais/química , Virulência
20.
ACS Chem Biol ; 12(8): 2070-2077, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28612602

RESUMO

Bacterial pathogens utilize numerous signals to identify the presence of their host and coordinate changes in gene expression that allow for infection. Within plant pathogens, these signals typically include small molecules and/or proteins from their plant hosts and bacterial quorum sensing molecules to ensure sufficient bacterial cell density for successful infection. In addition, bacteria use environmental signals to identify conditions when the host defenses are weakened and potentially to signal entry into an appropriate host/niche for infection. A globin coupled sensor protein (GCS), termed PccGCS, within the soft rot bacterium Pectobacterium carotovorum ssp. carotovorum WPP14 has been identified as an O2 sensor and demonstrated to alter virulence factor excretion and control motility, with deletion of PccGCS resulting in decreased rotting of a potato host. Using small molecules that modulate bacterial growth and quorum sensing, PccGCS signaling also has been shown to modulate quorum sensing pathways, resulting in the PccGCS deletion strain being more sensitive to plant-derived phenolic acids, which can function as quorum sensing inhibitors, and exhibiting increased N-acylhomoserine lactone (AHL) production. These findings highlight a role for GCS proteins in controlling key O2-dependent phenotypes of pathogenic bacteria and suggest that modulating GCS signaling to limit P. carotovorum motility may provide a means to decrease rotting of plant hosts.


Assuntos
Globinas/química , Oxigênio , Pectobacterium carotovorum/fisiologia , Percepção de Quorum , Globinas/metabolismo , Modelos Biológicos , Pectobacterium carotovorum/patogenicidade , Transdução de Sinais , Virulência
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