Browsing by Author "Caneschi, Washington Luiz"

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    Açaí (Euterpe oleracea Mart.) modulates oxidative stress resistance in Caenorhabditis elegans by direct and indirect mechanisms.
    (2014) Bonomo, Larissa de Freitas; Silva, David Nunes; Boasquivis, Patrícia Ferreira; Paiva, Franciny Aparecida; Guerra, Joyce Ferreira da Costa; Martins, Talita Alves Faria; Torres, Álvaro Gustavo de Jesus; Paula, Igor Thadeu Borges Raposo de; Caneschi, Washington Luiz; Jacolot, Philippe; Grossin, Nicolas; Tessier, Frederic J.; Boulanger, Eric; Silva, Marcelo Eustáquio; Pedrosa, Maria Lúcia; Oliveira, Riva de Paula
    Açaí (Euterpe oleracea Mart.) has recently emerged as a promising source of natural antioxidants. Despite its claimed pharmacological and nutraceutical value, studies regarding the effects of açaí in vivo are limited. In this study, we use the Caenorhabditis elegans model to evaluate the in vivo antioxidant properties of açaí on an organismal level and to examine its mechanism of action. Supplementation with ac¸aı´ aqueous extract (AAE) increased both oxidative and osmotic stress resistance independently of any effect on reproduction and development. AAE suppressed bacterial growth, but this antimicrobial property did not influence stress resistance. AAE-increased stress resistance was correlated with reduced ROS production, the prevention of sulfhydryl (SH) level reduction and gcs-1 activation under oxidative stress conditions. Our mechanistic studies indicated that AAE promotes oxidative stress resistance by acting through DAF-16 and the osmotic stress response pathway OSR-1/UNC-43/SEK-1. Finally, AAE increased polyglutamine protein aggregation and decreased proteasome activity. Our findings suggest that natural compounds available in AAE can improve the antioxidant status of a whole organism under certain conditions by direct and indirect mechanisms.
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    Analyses of seven new genomes of Xanthomonas citri pv. aurantifolii strains, causative agents of Citrus Canker B and C, show a reduced repertoire of pathogenicity-related genes.
    (2019) Fonseca, Natasha Peixoto; Patané, José Salvatore Leister; Varani, Alessandro de Mello; Felestrino, Érica Barbosa; Caneschi, Washington Luiz; Sanchez, Angelica Bianchini; Assis, Renata de Almeida Barbosa; Garcia, Camila Carrião Machado; Belasque Junior, José; Martins Junior, Joaquim; Facincani, Agda Paula
    Xanthomonas citri pv. aurantifolii pathotype B (XauB) and pathotype C (XauC) are the causative agents respectively of citrus canker B and C, diseases of citrus plants related to the better-known citrus canker A, caused by Xanthomonas citri pv. citri. The study of the genomes of strains of these related bacterial species has the potential to bring new understanding to the molecular basis of citrus canker as well as their evolutionary history. Up to now only one genome sequence of XauB and only one genome sequence of XauC have been available, both in draft status. Here we present two new genome sequences of XauB (both complete) and five new genome sequences of XauC (two complete). A phylogenomic analysis of these seven genome sequences along with 24 other related Xanthomonas genomes showed that there are two distinct and wellsupported major clades, the XauB and XauC clade and the Xanthomonas citri pv. citri clade. An analysis of 62 Type III Secretion System effector genes showed that there are 42 effectors with variable presence/absence or pseudogene status among the 31 genomes analyzed. A comparative analysis of secretion-system and surfacestructure genes showed that the XauB and XauC genomes lack several key genes in pathogenicity-related subsystems. These subsystems, the Types I and IV Secretion Systems, and the Type IV pilus, therefore emerge as important ones in helping explain the aggressiveness of the A type of citrus canker and the apparent dominance in the field of the corresponding strain over the B and C strains.
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    Arsenic resistance in cultured cutaneous microbiota is associated with anuran lifestyles in the Iron Quadrangle, Minas Gerais State, Brazil.
    (2019) Cordeiro, Isabella Ferreira; Fonseca, Natasha Peixoto; Felestrino, Érica Barbosa; Caneschi, Washington Luiz; Pires, Maria Rita Silvério; Moreira, Leandro Marcio
    The microbiota of the anuran skin contributes significantly to the maintenance and survival of these animals. In the present study, we investigated the composition of culturable microbiota on the skin of frogs from Tripui Ecological Station (TES), a locality in the Brazilian Iron Quadrangle that is naturally contaminated with arsenic. A total of 328 culturable bacterial isolates were obtained from 21 individuals belonging to seven anuran species, captured in different microenvironments within TES. The results indicate that higher numbers of culturable bacterial isolates, as well as higher resistance to arsenic and a higher capacity to produce biofilms, were associated with three frog species that had come into contact with arsenic-contaminated water during their reproductive period. These results raise the possibility that the adaptation of these anurans species to arsenicladen environments may have a direct correlation with a specialized microbiota.
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    Bioprospecção de bactérias de regiões de canga do Quadrilátero Ferrífero : estratégia de busca de alvos com potencial biotecnológico.
    (2018) Caneschi, Washington Luiz; Moreira, Leandro Marcio; Moreira, Leandro Marcio; Silva, Cynthia Canêdo da; Teixeira, Mônica Cristina; Almeida Junior, Nalvo Franco de; Silva, Silvana de Queiroz
    O extrativismo mineral na região do Quadrilátero Ferrífero brasileiro destruiu grandes áreas de terra, dizimando espécies vegetais e sua microbiota associada. Pouco se sabe sobre a microbiota da região. Assim bactérias cultiváveis associadas a plantas e de solos foram investigadas por seu potencial biotecnológico. Amostras foram coletadas de nove espécies de plantas e seis amostras de solos, sendo 65 isolados bacterianos cultiváveis obtidos. Estes representam predominantemente bacilos gram-positivos (70%) capazes de produzir amilases (55%), proteases (63%), celulases (47%), ácido indolacético (AIA) (46%), sideróforos (26%) e solubilizar fosfato (9%). Além disso, 65% destes foram resistentes à ampicilina, 100% eram sensíveis à tetraciclina e 97% tolerantes a altas concentrações de arsênio. Três isolados foram estudados ainda: o isolado FOB3 (Rosenbergiella sp.) produziu altas concentrações de AIA in vitro na ausência de triptofano, demonstrado pela melhora na germinação de planta e taxa de crescimento onde o isolado estava presente. Os isolados C25 (Acinetobacter sp.) e FG3 (Serratia sp.), tiveram plasmídeos extraídos e inseridos em células de E. coli, onde modificaram o perfil fisiológico das cepas transformadas. A cepa E. coli :: pFG3B apresentou a maior capacidade de produção de biofilme, além de aumento na taxa de replicação, tolerância ao arsênio e atividade de catalase, além de aumentar a integridade do DNA na presença de arsênio. A cepa Serratia sp. teve seu genoma completo sequenciado pela plataforma PacBio sendo identificada como Serratia liquefaciens cepa FG3 (SlFG3). Foi identificado um cromossomo de 5,7 Mpb (5398 genes) e dois plasmídeos com 159 Kbp (179 genes) e 125 Kpb (146 genes). A análise comparativa do genoma de SlFG3 foi realizada com 33 outras espécies de Serratia com genomas completos e revelou a presença de 18 supostas inserções de fagos, permitindo identificar 311 genes únicos para o SlFG3 e um core genome de 417 famílias de proteínas. A análise filogenômica baseada no core genome permitiu agrupar SlFG3 em um clado com outras três linhagens de S. liquefaciens (FDAARGOS125, HUMV21 e ATCC27592) e Serratia proteamaculans 568 (Sp568), que compartilham um core de 3998 famílias de proteínas ortólogas. Entre esses cinco genomas, 75 genes são compartilhados apenas em SlFG3 e S. protemaculans 568, ambos isolados de plantas, e 643 famílias de proteínas são exclusivas de SlFG3. A análise desses genes revelou a presença de uma via completa relacionada à degradação de protocatecuato e compostos cloroaromáticos. Além disso, SlFG3 possui um repertório diversificado de genes associados a NRPs / PKS, um conjunto completo de síntese de celulose e metabolismo oxidativo completo e reparo de DNA. Finalmente, SlFG3 ainda possui genes relacionados à tiolação de RNA e DNA, inserida em uma região de fago que pode proteger os ácidos nucléicos contra diferentes condições de estresse. Esses achados resumem que o SlFG3 parece estar bem adaptada à diferentes situações de estresse impostas por condições extremas além de ser extremamente interessante no estudo de mecanismos molecularese composição gênica que pode ser melhor explorado com alto potencial biotecnológico.
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    Biotechnological potential of plant growth-promoting bacteria from the roots and rhizospheres of endemic plants in ironstone vegetation in southeastern Brazil.
    (2018) Felestrino, Érica Barbosa; Vieira, Izadora Tabuso; Caneschi, Washington Luiz; Cordeiro, Isabella Ferreira; Assis, Renata de Almeida Barbosa; Lemes, Camila Gracyelle de Carvalho; Fonseca, Natasha Peixoto; Sanchez, Angelica Bianchini; Caicedo Cepeda, Juan Carlos; Ferro, Jesus Aparecido; Garcia, Camila Carrião Machado; Carmo, Flávio Fonseca do; Kamino, Luciana Hiromi Yoshino; Moreira, Leandro Marcio
    Microorganisms associated with plants have a great biotechnological potential, but investigations of these microorganisms associated with native plants in peculiar environments has been incipient. The objective of this study was to analyze the plant growth-promoting bacteria potential of cultivable bacteria associated with rare plants from the ferruginous rocky fields of the Brazilian Iron Quadrangle. The roots and rhizospheres of nine endemic plants species and samples of a root found in a lateritiric duricrust (canga) cave were collected, the culturable bacteria isolated and prospected for distinct biotechnological and ecological potentials. Out of the 148 isolates obtained, 8 (5.4%) showed potential to promote plant growth, whereas 4 (2.7%) isolates acted as biocontrol agents against Xanthomonas citri pathotype A (Xac306), reducing the cancrotic lesions by more than 60% when co-inoculated with this phytopathogen in Citrus sinensis plants. Moreover, other 4 (2.7%) isolates were classified as potential bioremediation agents, being able to withstand high concentrations of arsenite (5 mM As3+) and arsenate (800 mM As5+), by removing up to 35% and 15% of this metalloid in solution, respectively. These same four isolates had a positive influence on the growth of both the roots and the aerial parts when inoculated with tomato seeds in the soil contaminated with arsenic. This is the first time that an investigation highlights the potentialities of bacteria associated with rare plants of ferruginous rocky fields as a reservoir of microbiota of biotechnological and ecological interest, highlighting the importance of conservation of this area that is undergoing intense anthropic activity
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    Brazilian ironstone plant communities as reservoirs of culturable bacteria with diverse biotechnological potential.
    (2018) Caneschi, Washington Luiz; Felestrino, Érica Barbosa; Fonseca, Natasha Peixoto; Villa, Morghana Marina; Lemes, Camila Gracyelle de Carvalho; Cordeiro, Isabella Ferreira; Assis, Renata de Almeida Barbosa; Sanchez, Angelica Bianchini; Vieira, Izadora Tabuso; Kamino, Luciana Hiromi Yoshino; Carmo, Flávio Fonseca do; Garcia, Camila Carrião Machado; Moreira, Leandro Marcio
    Extensive mineral extractivism in the Brazilian Iron Quadrangle (IQ) region has destroyed large areas of land, decimating plant species, and their associated microbiota. Very little is known about the microbiota of the region; hence, cultivable bacteria associated with plants of its soils were investigated for their biotechnological potential. Samples were collected from nine plant species and six soils, and 65 cultivable bacterial isolates were obtained. These represent predominantly gram-positive bacilli (70%) capable of producing amylases (55%), proteases (63%), cellulases (47%), indole acetic acid (IAA) (46%), siderophores (26%), and to solubilize phosphate (9%). In addition, 65% of these were resistant to ampicillin, 100% were sensitive to tetracycline, and 97% were tolerant to high arsenic concentrations. Three isolates were studied further: the isolate FOB3 (Rosenbergiella sp.) produced high concentrations of IAA in vitro in the absence of tryptophan – shown by the significant improvement in plant germination and growth rate where the isolate was present. For isolates C25 (Acinetobacter sp.) and FG3 (Serratia sp.), plasmids were purified and inserted into Escherichia coli cells where they modified the physiological profile of the transformed strains. The E. coli::pFG3B strain showed the highest capacity for biofilm production, as well as an increase in the replication rate, arsenic tolerance and catalase activity. Moreover, this strain increased DNA integrity in the presence of arsenic, compared to the wild-type strain. These results help to explain the importance of bacteria in maintaining plant survival in ferruginous, rocky soils, acting as plant growth promoters, and to highlight the biotechnological potential of these bacteria. IMPORTANCE The Iron Quadrangle region is responsible for ∼60% of all Brazilian iron production and, at the same time, is responsible for housing a wide diversity of landscapes, and consequently, a series of endemic plant species and dozens of rare species – all of which have been poorly studied. Studies exploring the microbiota associated with these plant species are limited and in the face of the continuous pressure of extractive action, some species along with their microbiota are being decimated. To understand the potential of this microbiota, we discovered that cultivable bacterial isolates obtained from plants in the ferruginous rocky soil of the Iron Quadrangle region have diverse biotechnological potential, revealing a genetic ancestry still unknown.
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    Complete genome sequence and analysis of Alcaligenes faecalis strain Mc250, a new potential plant bioinoculant.
    (2020) Felestrino, Érica Barbosa; Sanchez, Angelica Bianchini; Caneschi, Washington Luiz; Lemes, Camila Gracyelle de Carvalho; Assis, Renata de Almeida Barbosa; Cordeiro, Isabella Ferreira; Fonseca, Natasha Peixoto; Villa, Morghana Marina; Vieira, Izadora Tabuso; Kamino, Luciana Hiromi Yoshino; Carmo, Flávio Fonseca do; Silva, Aline Maria da; Thomas, Andrew Maltez; Patané, José Salvatore Leister; Ferreira, Fernanda Carla; Freitas, Leandro Grassi de; Varani, Alessandro de Mello; Ferro, Jesus Aparecido; Silva, Robson Soares; Almeida Junior, Nalvo Franco de; Garcia, Camila Carrião Machado; Setubal, João Carlos; Moreira, Leandro Marcio
    Here we present and analyze the complete genome of Alcaligenes faecalis strain Mc250 (Mc250), a bacterium isolated from the roots of Mimosa calodendron, an endemic plant growing in ferruginous rupestrian grasslands in Minas Gerais State, Brazil. The genome has 4,159,911 bp and 3,719 predicted protein-coding genes, in a single chromosome. Comparison of the Mc250 genome with 36 other Alcaligenes faecalis genomes revealed that there is considerable gene content variation among these strains, with the core genome representing only 39% of the protein-coding gene repertoire of Mc250. Mc250 encodes a complete denitrification pathway, a network of pathways associated with phenolic compounds degradation, and genes associated with HCN and siderophores synthesis; we also found a repertoire of genes associated with metal internalization and metabolism, sulfate/sulfonate and cysteine metabolism, oxidative stress and DNA repair. These findings reveal the genomic basis for the adaptation of this bacterium to the harsh environmental conditions from where it was isolated. Gene clusters associated with ectoine, terpene, resorcinol, and emulsan biosynthesis that can confer some competitive advantage were also found. Experimental results showed that Mc250 was able to reduce (~60%) the virulence phenotype of the plant pathogen Xanthomonas citri subsp. citri when co-inoculated in Citrus sinensis, and was able to eradicate 98% of juveniles and stabilize the hatching rate of eggs to 4% in two species of agricultural nematodes. These results reveal biotechnological potential for the Mc250 strain and warrant its further investigation as a biocontrol and plant growth-promoting bacterium.
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    Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.
    (2019) Fonseca, Natasha Peixoto; Felestrino, Érica Barbosa; Caneschi, Washington Luiz; Sanchez, Angelica Bianchini; Cordeiro, Isabella Ferreira; Lemes, Camila Gracyelle de Carvalho; Assis, Renata de Almeida Barbosa; Carvalho, Flávia Maria de Souza; Ferro, Jesus Aparecido; Varani, Alessandro de Mello; Belasque Junior, José; Setubal, João Carlos; Telles, Guilherme Pimentel; Aguena, Deiviston da Silva; Almeida Junior, Nalvo Franco de; Moreira, Leandro Marcio
    Background. In Citrus cultures, three species of Xanthomonas are known to cause distinct diseases. X. citri subsp. citri patothype A, X. fuscans subsp. aurantifolii pathotypes B and C, and X. alfalfae subsp. citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently available for proper and early detection of these diseases in the fields. The present study aimed to develop a new molecular diagnostic method based on genomic sequences derived from the four species of Xanthomonas. Results. Using comparative genomics approaches, primers were synthesized for the identification of the four causative agents of citrus diseases. These primers were validated for their specificity to their target DNA by both conventional and multiplex PCR. Upon evaluation, their sensitivity was found to be 0.02 ng/µl in vitro and 1.5 × 104 CFU ml−1 in infected leaves. Additionally, none of the primers were able to generate amplicons in 19 other genomes of Xanthomonas not associated with Citrus and one species of Xylella, the causal agent of citrus variegated chlorosis (CVC). This denotes strong specificity of the primers for the different species of Xanthomonas investigated in this study. Conclusions. We demonstrated that these markers can be used as potential candidates for performing in vivo molecular diagnosis exclusively for citrus-associated Xanthomonas. The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism.
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    Isolamento e purificação de sideróforos bacterianos : uma abordagem educacional multidisciplinar.
    (2019) Caneschi, Washington Luiz; Sanchez, Angelica Bianchini; Pereira, Josielda Gomes; Garcia, Camila Carrião Machado; Moreira, Leandro Marcio
    As bactérias promovem a captação ambiental de ferro pela produção e secreção de sideróforos, que quelam o ferro e são posteriormente internalizadas para fornecer esse essencial metal ao metabolismo celular. Neste estudo, descrevemos uma sequência didática que permitirá aos estudantes de graduação isolar, purificar parcialmente e classificar os sideróforos produzidos por bactérias de diferentes ambientes. Essa proposta foi avaliada com alunos do mestrado profissional em ensino de ciências abordando parâmetros metodológicos e aprendizagem conceitual associado à metodologia. Os resultados mostraram que essa sequência didática pode trabalhar temáticas bioquímicas em conjunto com uma abordagem multidisciplinar e de baixo custo, permitindo a discussão de questões-chave em diferentes áreas do conhecimento biológico que é frequentemente negligenciada em estudos universitários.
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    Serratia liquefaciens FG3 isolated from a metallophyte plant sheds light on the evolution and mechanisms of adaptive traits in extreme environments.
    (2019) Caneschi, Washington Luiz; Sanchez, Angelica Bianchini; Felestrino, Érica Barbosa; Lemes, Camila Gracyelle de Carvalho; Cordeiro, Isabella Ferreira; Fonseca, Natasha Peixoto; Villa, Morghana Marina; Vieira, Izadora Tabuso; Moraes, Lauro Ângelo Gonçalves de; Assis, Renata de Almeida Barbosa; Carmo, Flávio Fonseca do; Kamino, Luciana Hiromi Yoshino; Silva, Robson Soares; Ferro, Jesus Aparecido; Ferro, Maria Inês Tiraboschi; Ferreira, Rafael Marini; Santos, Vera Lúcia; Silva, Ubiana de Cássia Mourão; Almeida Junior, Nalvo Franco de; Varani, Alessandro de Mello; Garcia, Camila Carrião Machado; Setubal, João Carlos; Moreira, Leandro Marcio
    Serratia liquefaciens strain FG3 (SlFG3), isolated from the flower of Stachytarpheta glabra in the Brazilian ferruginous fields, has distinctive genomic, adaptive, and biotechnological potential. Herein, using a combination of genomics and molecular approaches, we unlocked the evolution of the adaptive traits acquired by S1FG3, which exhibits the second largest chromosome containing the largest conjugative plasmids described for Serratia. Comparative analysis revealed the presence of 18 genomic islands and 311 unique protein families involved in distinct adaptive features. S1FG3 has a diversified repertoire of genes associated with Nonribosomal peptides (NRPs/PKS), a complete and functional cluster related to cellulose synthesis, and an extensive and functional repertoire of oxidative metabolism genes. In addition, S1FG3 possesses a complete pathway related to protocatecuate and chloroaromatic degradation, and a complete repertoire of genes related to DNA repair and protection that includes mechanisms related to UV light tolerance, redox process resistance, and a laterally acquired capacity to protect DNA using phosphorothioation. These findings summarize that SlFG3 is well-adapted to different biotic and abiotic stress situations imposed by extreme conditions associated with ferruginous fields, unlocking the impact of the lateral gene transfer to adjust the genome for extreme environments, and providing insight into the evolution of prokaryotes.
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    O tratamento com extrato aquoso de açaí (Euterpe oleracea Mart.) protege o Caenorhabditis elegans contra exposição aguda à Acrilamida.
    (2014) Caneschi, Washington Luiz; Oliveira, Riva de Paula
    Acrilamida (AAM) é um composto tóxico presente em quantidades relevantes em alimentos ricos em carboidratos que passaram por tratamento térmico a altas temperaturas. Estudos in vivo e in vitro mostraram que AAM pode induzir efeitos neurotóxicos, genotóxicos, carcinogênicos, distúrbios reprodução e no desenvolvimento. O uso de compostos naturais provindos da dieta com efeitos antioxidantes pode fornecer uma estratégia de combate à toxicidade causada pela AAM. O Açaí, Euterpe oleracea Mart., é um fruto da América do Sul comumente usado em bebidas além de ser usado na medicina popular, sendo rico em compostos fitoquímicos, particularmente antocianinas, proantocianidinas e outros flavonóides. Neste projeto, nós avaliamos o possível efeito protetor do extrato aquoso de Açaí (EAA) 100 mg/ml sobre os efeitos tóxicos provocados pela exposição aguda no C. elegans à AAM. Para avaliar a toxicidade da exposição aguda à AAM no C. elegans, nós avaliamos parâmetros biológicos, bioquímicos e genéticos. A exposição aguda à AAM reduziu os tamanho corporal dos animais, o número total de progênie, a longevidade, aumentou a produção de EROs e ativou a expressão de genes de detoxificação gst-4::GFP e gcs-1::GFP, ativou a localização nuclear de DAF-16::GFP, mas não ativou a expressão de sod-3. Para avaliar se o tratamento com EAA possui algum efeito protetor sob a toxicidade induzida pela AAM, os animais foram previamente tratados com EAA e em seguida expostos à AAM. Nossas análises revelaram que o tratamento com EAA impediu a redução corporal dos animais, aumentou o tempo de vida, reduziu a produção de EROs, e preveniu a ativação de gcs-1 por AAM. Já foi demostrado que a ativação de gst-4::GFP por AAM é dependente do fator de transcrição SKN- 1/Nrf2 Nós observamos uma redução parcial da expressão de gcs-1::GFP em animais skn-1(RNAi) tratados com AAM, o que não foi observado para animais daf-16(RNAi), sugerindo que a indução de gcs-1::GFP pela AAM depende parcialmente de SKN-1. O pré-tratamento com EAA reduziu a expressão de gcs- 1::GFP nos animais daf-16(RNAi) tratados com AAM mas não nos animais skn- 1(RNAi) sugerindo que SKN-1 não interfere na expressão de gcs-1 promovido pelo EAA. Por fim, os resultados deste projeto indicam que o pré-tratamento do C. elegans com EAA é capaz de reduzir os efeitos tóxicos induzidos pela exposição aguda à AAM.