Secondary metabolite gene clusters in the entomopathogen fungus Metarhizium anisopliae : genome identification and patterns of expression in a cuticle infection model

Background: The described species from the Metarhizium genus are cosmopolitan fungi that infect arthropod hosts. Interestingly, while some species infect a wide range of hosts (host-generalists), other species infect only a few arthropods (host-specialists). This singular evolutionary trait permits unique comparisons to determine how pathogens and virulence determinants emerge. Among the several virulence determinants that have been described, secondary metabolites (SMs) are suggested to play essential roles during fungal infection. Despite progress in the study of pathogen-host relationships, the majority of genes related to SM production in Metarhizium spp. are uncharacterized, and little is known about their genomic organization, expression and regulation. To better understand how infection conditions may affect SM production in Metarhizium anisopliae, we have performed a deep survey and description of SM biosynthetic gene clusters (BGCs) in M. anisopliae, analyzed RNA-seq data from fungi grown on cattle-tick cuticles, evaluated the differential expression of BGCs, and assessed conservation among the Metarhizium genus. Furthermore, our analysis extended to the construction of a phylogeny for the following three BGCs: a tropolone/citrinin-related compound (MaPKS1), a pseurotin-related compound (MaNRPS-PKS2), and a putative helvolic acid (MaTERP1). Results: Among 73 BGCs identified in M. anisopliae, 20 % were up-regulated during initial tick cuticle infection and presumably possess virulence-related roles. These up-regulated BGCs include known clusters, such as destruxin, NG39x and ferricrocin, together with putative helvolic acid and, pseurotin and tropolone/citrinin-related compound clusters as well as uncharacterized clusters. Furthermore, several previously characterized and putative BGCs were silent or down-regulated in initial infection conditions, indicating minor participation over the course of infection. Interestingly, several up-regulated BGCs were not conserved in host-specialist species from the Metarhizium genus, indicating differences in the metabolic strategies employed by generalist and specialist species to overcome and kill their host. These differences in metabolic potential may have been partially shaped by horizontal gene transfer (HGT) events, as our phylogenetic analysis provided evidence that the putative helvolic acid cluster in Metarhizium spp. originated from an HGT event. Conclusions: Several unknown BGCs are described, and aspects of their organization, regulation and origin are discussed, providing further support for the impact of SM on the Metarhizium genus lifestyle and infection process

Occurrence of pests and diseases in cactus pear genotypes / Ocorrência de pragas e doenças em genótipos de palma forrageira

The objective was to evaluate the occurrence of pests, diseases and mortality rate in nine genotypes of cactus pear (Nopalea cochenillifera) destined for forage production. The genotypes were implanted in a complete randomized block design, with nine treatments and three replications. After 330 days of cultivation, the occurrence of pests and diseases and verification of plant mortality were carried out. Among all the pests and diseases observed in this experimental trial, the most prevalent disease regardless of the evaluated genotype was the anthracnose stain “Colletotrichum gloeosporioides” (49.20%) and the less frequent diseases were the resine “Dothiorella ribis” (6.87%) and soft rot “Erwinia carotovora” (2.58%). The only occurrence pest was the cochineal in scales “Diaspis echinocacti” (22.69%). The Texas (V13) and Negro Michoacan (F07) genotypes showed the highest occurrence of pests and diseases, from 50% of the total plants, followed by anthracnose stain and cochineal in scales. On the contrary, the genotypes Tamazunchale (V12) and California (V14) were not affected by any pest or disease. It was observed that the genotypes Nopalea Uruapan (V20) and Blanco San Pedro (V19), had a lower occurrence of pests and diseases, less than 20% of the total plants. The genotypes that presented the highest mortality rate were Texas (V13), Blanco San Pedro (V19) and Polotitlan (V09), with 80, 70 and 65% mortality rate, respectively. The genotypes Nopalea Uruapan (V20) and California (V14) had the lowest mortality rate (20 and 35%), respectively. The genotypes that were least affected by pests and diseases and had the lowest mortality rate are Tamazunchale (V12), California (V14) and Nopalea Uruapan (V20)

Dementia in Latin America : paving the way towards a regional action plan

Regional challenges faced by Latin American and Caribbean countries (LACs) to fight dementia, such as heterogeneity, diversity, political instabilities, and socioeconomic disparities, can be addressed more effectively grounded in a collaborative setting based on the open exchange of knowledge. In this work, the Latin American and Caribbean Consortium on Dementia (LAC-CD) proposes an agenda for integration to deliver a Knowledge to Action Framework (KtAF). First, we summarize evidence-based strategies (epidemiology, genetics, biomarkers, clinical trials, nonpharmacological interventions, networking and translational research) and align them to current global strategies to translate regional knowledge into actions with transformative power. Then, by characterizing genetic isolates, admixture in populations, environmental factors, and barriers to effective interventions and mapping these to the above challenges, we provide the basic mosaics of knowledge that will pave the way towards a KtAF. We describe strategies supporting the knowledge creation stage that underpins the translational impact of KtAF

Recombinant Mycobacterium smegmatis expressing CMX pretein induces immune response against Mycobacterium tuberculosis in BALB/c mice

Submitted by Jaqueline Silva ([email protected]) on 2014-10-23T20:48:04Z No. of bitstreams: 2 Dissertação - Fábio Muniz de Oliveira - 2014.pdf: 12287155 bytes, checksum: 58eb4d1e227a17283f27cf610577402a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5)Approved for entry into archive by Jaqueline Silva ([email protected]) on 2014-10-23T20:49:02Z (GMT) No. of bitstreams: 2 Dissertação - Fábio Muniz de Oliveira - 2014.pdf: 12287155 bytes, checksum: 58eb4d1e227a17283f27cf610577402a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5)Made available in DSpace on 2014-10-23T20:49:02Z (GMT). No. of bitstreams: 2 Dissertação - Fábio Muniz de Oliveira - 2014.pdf: 12287155 bytes, checksum: 58eb4d1e227a17283f27cf610577402a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-28Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPqFor hundreds years tuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis (Mtb), has been a global public health problem. Even after the development of the vaccine BCG, in 1921, tuberculosis control continues on slow pace. This comes to be as a result of the variable efficacy (from 0 to 80%) presented by the vaccine in the protection against TB in adults. Therefore, the development of a new vaccine against TB is necessary. In this study, it was evaluated a recombinant vaccine composed of Mycobacterium smegmatis expressing the CMX fusion protein (mc2- CMX), formed from three antigen epitopes of Mtb: Ag85C, MPT51 and HspX. M. smegmatis mc2 155 was transformed with pLA71-CMX by electroporation, and the presence of the CMX protein was confirmed by imuno blotting. BALB/c mice were distributed in four groups: saline, infection, BCG and mc2-CMX. The groups were immunized with their respective vaccines in two moments with an interval of fifteen days and the animal blood was collected fifteen days after the last immunization. Thirty days after the last immunization, the animals were challenged with Mtb H37Rv (intravenously) and thirty days after the challenge, the blood was collected to perform ELISA test. Seventy days after the challenge, the lungs from all mice were collected to obtain cells for flow-cytometry, histological analysis and also to determine the bacillary burden. The immunization with mc2-CMX induced higher levels of antibodies of IgG1 (1,910±0,70) and IgG2a (0,139±0,020) class anti-CMX when compared with BCG group (0,646±0,19 and 0,413±0,24; respectively, p<0,05). These results demonstrated the relevance of CMX antigen in the immunogenicity of the recombinant vaccine. Seventy days after the challenge, the amount of T CD4 cells in the lung producing Th1- type cytokines was assessed. It was observed a significant increase in the percentage of T CD4 cells positive for IFN-γ and TNF-α in the immunized mice with mc2-CMX vaccine, when compared with the group immunized with BCG. Mice challenged with Mtb presented significant higher percentage of IL-2 producer cells when compared with the non-immunized group. However, only the mice immunized with the vaccine mc2- CMX presented significant higher percentage when compared with the infection group. The immune response induced by the vaccine was effective in the control of Mtb infection, confirmed by the histological analysis and the bacillar burden determined. The groups vaccinated with mc2-CMX and BCG presented a significant reduction of the lung lesion induced by the Mtb infection, and also lung bacterial load, when compared with the infection group. Thus, the recombinant vaccine mc2-CMX presents potential characteristics to be used in the prevention of TB.Há séculos a tuberculose (TB), doença infectocontagiosa causada por Mycobacterium tuberculosis (Mtb), vem sendo um problema de saúde pública mundial. Mesmo após o surgimento da vacina BCG em 1921, o controle da tuberculose continua a passos lentos. Isso se deve à eficácia variável de 0 a 80% apresentada pela vacina na proteção contra TB em indivíduos adultos. Deste modo, o desenvolvimento de uma nova vacina contra a TB é necessário. Neste estudo, avaliou-se uma vacina recombinante composta por Mycobacterium smegmatis expressando a proteína de fusão CMX (mc2-CMX), formada por três antígenos do Mtb: Ag85C, MPT51 e HspX. M. smegmatis mc2 155 foi transformado com pLA71-CMX por eletroporação, sendo a expressão da proteína CMX confirmada por imunoblot. Camundongos BALB/c foram distribuídos em quatro grupos: salina, infecção, BCG e mc2-CMX. Os grupos foram imunizados com suas respectivas vacinas em dois momentos com intervalos de 15 dias, e o sangue de todos os animais coletado quinze dias após a última imunização. Trinta dias após a imunização, os animais foram desafiados com Mtb H37Rv (via endovenosa) e trinta dias após o desafio, o sangue foi coletado para realização de ELISA. Setenta dias após desafio, o pulmão e o baço de todos os camundongos foi coletado para obtenção de células para realização de citometria, histopatológico e determinação da carga bacilar. A imunização com o mc2-CMX induziu níveis maiores de anticorpos da classe IgG1 (1,910±0,70) e IgG2a (0,139±0,020) anti-CMX quando comparado com o grupo BCG (0,646±0,19 e 0,413±0,24, respectivamente, p<0,05). Estes resultados demonstram a relevância do antígeno CMX na imunogenicidade da vacina recombinante. Após setenta dias do desafio, a quantidade de células T CD4 produtoras de citocinas do tipo Th1 foi analisada no pulmão. Foi observado um aumento significativo na porcentagem de células T CD4 positivas para IFN-γ e TNF-α nos camundongos imunizados com vacina mc2-CMX, quando comparado com o grupo BCG. Camundongos desafiados com Mtb apresentaram porcentagens maiores de células produtoras de IL-2, quando comparado com o grupo não desafiado. Todavia, somente os camundongos imunizados com a vacina mc2-CMX apresentaram porcentagens significativamente maiores em comparação ao grupo infecção. A resposta imune observada foi efetiva no controle da infecção por Mtb, sendo isto confirmado quando os pulmões dos camundongos foram analisados histologicamente e a carga bacilar determinada. Os grupos vacinados com as vacinas mc2-CMX e BCG apresentaram uma redução significativa da lesão pulmonar induzida pela infecção por Mtb, e também da carga bacilar no pulmão, quando comparados com o grupo infecção. Conclui-se que mc2-CMX tem um bom potencial para ser explorado como vacina contra a TB

Mycobacterium bovis PknG R242P Mutation Results in Structural Changes with Enhanced Virulence in the Mouse Model of Infection

Mycobacterium bovis is the causative agent of tuberculosis in domestic and wild animal species and sometimes in humans, presenting variable degrees of pathogenicity. It is known that PknG is involved in the first steps of Mycobacterium tuberculosis macrophage infection and immune evasion. We questioned whether M. bovispknG genes were conserved among mycobacteria and if natural genetic modifications would affect its virulence. We discovered a single mutation at a catalytic domain (R242P) of one M. bovis isolate and established the relation between the presence of R242P mutation and enhanced M. bovis virulence. Here, we demonstrated that R242P mutation alters the PknG protein conformation to a more open ATP binding site cleft. It was observed that M. bovis with PknG mutation resulted in increased growth under stress conditions. In addition, infected macrophages by M. bovis (R242P) presented a higher bacterial load compared with M. bovis without the pknG mutation. Furthermore, using the mouse model of infection, animals infected with M. bovis (R242P) had a massive innate immune response migration to the lung that culminated with pneumonia, necrosis, and higher mortality. The PknG protein single point mutation in its catalytic domain did not reduce the bacterial fitness but rather increased its virulence