Navegando por Autor "Nogueira, Guilherme Bicalho"
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Item Bovicin HC5 and nisin reduce Staphylococcus aureus adhesion to polystyrene and change the hydrophobicity profile and Gibbs free energy of adhesion(International Journal of Food Microbiology, 2014-11-03) Pimentel-Filho, Natan de Jesus; Martins, Mayra Carla de Freitas; Nogueira, Guilherme Bicalho; Mantovani, Hilário Cuquetto; Vanetti, Maria Cristina DantasStaphylococcus aureus is an opportunistic pathogen often multidrug-resistant that not only causes a variety of human diseases, but also is able to survive on biotic and abiotic surfaces through biofilm communities. The best way to inhibit biofilm establishment is to prevent cell adhesion. In the present study, subinhibitory concentrations of the bacteriocins bovicin HC5 and nisin were tested for their capability to interfere with the adhesion of S. aureus to polystyrene. Subinhibitory dosages of the bacteriocins reduced cell adhesion and this occurred probably due to changes in the hydrophobicity of the bacterial cell and polystyrene surfaces. After treatment with bovicin HC5 and nisin, the surfaces became more hydrophilic and the free energy of adhesion (∆Gadhesion) between bacteria and the polystyrene surface was unfavorable. The transcriptional level of selected genes was assessed by RT-qPCR approach, revealing that the bacteriocins affected the expression of some important biofilm associated genes (icaD, fnbA, and clfB) and rnaIII, which is involved in the quorum sensing mechanism. The conditioning of food-contact surfaces with bacteriocins can be an innovative and powerful strategy to prevent biofilms in the food industry. The results are relevant for food safety as they indicate that bovicin HC5 and nisin can inhibit bacterial adhesion and consequent biofilm establishment, since cell adhesion precedes biofilm formation.Item Caracterização estrutural e funcional do gene pacCl, que codifica o regulador transcricional de resposta ao pH, de Colletotrichum lindemuthianum, agente causal da antracnose do feijoeiro(Universidade Federal de Viçosa, 2009-02-18) Nogueira, Guilherme Bicalho; Queiroz, Marisa Vieira de; http://lattes.cnpq.br/3681276749465961Desde o contato inicial até o estabelecimento dos sintomas da doença, C. lindemuthianum regula a expressão diferencial de genes essenciais para a patogenicidade que vão possibilitar a colonização do tecido hospedeiro e a finalização do seu ciclo de infecção. Nesse trabalho, o gene pacCl, que codifica o regulador transcricional de resposta ao pH ambiental, foi isolado e caracterizado estrutural e funcionalmente. Uma sequência de 2.546 pb foi isolada do banco genômico de C. lindemuthianum, correspondente ao gene pacCl. Esta sequência apresenta uma ORF de 1.746 pb, codificando, portanto, uma proteína de 581 resíduos de aminoácidos. Foram identificados na sequência, três íntrons putativos, contendo 60, 58 e 74 pb, cada um deles. O alinhamento múltiplo da sequência deduzida da proteína, com as sequências disponíveis nos bancos de dados, demonstrou elevado grau de identidade e similaridade da proteína identificada com o regulador transcricional PacC/Him101 de fungos filamentosos e leveduras, respectivamente. Além disso, a proteína PacCl se mostrou eficiente como marcador filogenético em fungos, pois agrupou de forma coerente os organismos taxonomicamente relacionados. A hibridização de DNA usando o gene pacCl como sonda, mostrou que o gene encontra- se em uma única cópia no genoma de C. lindemuthianum. Do mesmo modo, a análise do mutante Mutpac2 por hibridização de DNA, confirmou que o gene pacCl havia sido inativado por um evento de recombinação homóloga do tipo troca gênica. Por fim, a técnica de PCR quantitativo em tempo real mostrou que o gene pacCl é expresso em todas as fases do ciclo de infecção, porém, os maiores níveis foram encontrados no final da fase necrotrófica. Pode-se sugerir que um dos fatores responsáveis por esse aumento, seja a alcalinização do tecido vegetal, decorrente do envelhecimento do tecido, e/ou até mesmo da própria atividade do fungo, que em muitos casos secreta substâncias alcalinizantes, como por exemplo, amônia.Item Genome organization and assessment of high copy number and increased expression of pectinolytic genes from Penicillium griseoroseum: a potential heterologous system for protein production(Journal of Industrial Microbiology & Biotechnology, 2014-01-08) Teixeira, Janaina Aparecida; Nogueira, Guilherme Bicalho; Queiroz, Marisa Vieira de; Araújo, Elza Fernandes deThe fungus Penicillium griseoroseum has the potential for application on an industrial scale as a host for the production of homologous and heterologous proteins, mainly because it does not produce some mycotoxins or secrete proteases under the growth conditions for pectinase production. However, for the fungus to be used effectively as an expression heterologous system, an understanding of the organization of its genome, as well as the mechanisms of gene expression and protein production, is required. In the present study, the size of the P. griseoroseum genome was estimated to be 29.8–31.5 Mb, distributed among four chromosomes. An analysis of plg1 and pgg2 pectinolytic genes expression and copy number in recombinant multi-copy strains of P. griseoroseum demonstrated that an increase in the number of gene copies could increase enzyme production, but the transcription could be affected by the gene integration position. Placing a copy of the plg1 gene under the control of the gpd promoter of Aspergillus nidulans yielded a 200-fold increase in transcription levels compared to the endogenous gene, and two copies of the pgg2 gene produced an 1100-fold increase compared with the endogenous gene. These results demonstrated that transcription, translation, and protein secretion in the fungus P. griseoroseum respond to an increased number of gene copies in the genome. The processing capacity and efficiency of protein secretion in P. griseoroseum are consistent with our premise that this fungus can be used for the industrial-scale production of several enzymes.Item The histidine kinase slnCl1 of Colletotrichum lindemuthianum as a pathogenicity factor against Phaseolus vulgaris L(Microbiological Research, 2019-02) Nogueira, Guilherme Bicalho; Santos, Leandro Vieira dos; Queiroz, Casley Borges de; Corrêa, Thamy Lívia Ribeiro; Menicucci, Renato Pedrozo; Bazzolli, Denise Mara Soares; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira deColletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes.Item PacCl, a pH-responsive transcriptional regulator, is essential in the pathogenicity of Colletotrichum lindemuthianum, a causal agent of anthracnose in bean plants(European Journal of Plant Pathology, 2014-08-08) Nogueira, Guilherme Bicalho; Soares, Marcos Antônio; Bazzolli, Denise Mara Soares; Araújo, Elza Fernandes de; Langin, Thierry; Queiroz, Marisa Vieira deIn fungi, the expression of genes encoding proteins related to parasitism is regulated by several factors, including pH. This study reports the structural and functional characterization of the pacCl gene, which encodes the transcription factor PacC of C. lindemuthianum. The pacCl gene showed reduced expression in acidic pH, and its transcription was activated by elevated extracellular pH. The importance of this gene was demonstrated by the development of a pacC1 disruption mutant line of C. lindemuthianum. The mutant line was able to penetrate the host tissue through differentiation of primary hyphae. However, it was not able to cause maceration on the infected plant tissue. The results suggest that PacCl is a regulator of gene activation, and its expression is required for fungal growth in alkaline conditions, as well as for the transcription of genes necessary for the passage from the biotrophic to the necrotrophic phase.