Estudo da Variabilidade Genética de Leishmania (Viannia) braziliensis em Minas Gerais, Brasil

Submitted by Nuzia Santos ([email protected]) on 2016-01-07T16:38:39Z No. of bitstreams: 1 Dissertacao_BCM_JeronimoMarteletoNunesRugani (1).pdf: 1205673 bytes, checksum: 9782a5cd43a0368938ad2c3c5d706db3 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-01-07T16:38:51Z (GMT) No. of bitstreams: 1 Dissertacao_BCM_JeronimoMarteletoNunesRugani (1).pdf: 1205673 bytes, checksum: 9782a5cd43a0368938ad2c3c5d706db3 (MD5)Made available in DSpace on 2016-01-07T16:38:51Z (GMT). No. of bitstreams: 1 Dissertacao_BCM_JeronimoMarteletoNunesRugani (1).pdf: 1205673 bytes, checksum: 9782a5cd43a0368938ad2c3c5d706db3 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.No Brasil, de todas as espécies do gênero Leishmania, a mais frequentemente encontrada parasitando o homem é a Leishmania (Viannia) braziliensis. Esta espécie pode causar um amplo espectro de manifestações, desde lesões únicas ao envolvimento mucoso, sendo esta última a complicação mais séria. Análises que visam acessar a variabilidade genética de Leishmania (Viannia) braziliensis são essenciais para o estudo de possíveis correlações entre manifestações clínicas de leishmaniose tegumentar americana (LTA) com parasitos geneticamente variantes e sua origem geográfica. O objetivo do estudo é analisar a variabilidade genética de isolados de L. braziliensis provenientes de diversas regiões de Minas Gerais. Foi realizado o diagnóstico clínico e molecular de indivíduos portadores de manifestações típicas e atípicas de várias regiões endêmicas do estado e os isolados separados em dois grupos amostrais: o grupo 1 contendo amostras de variadas macrorregiões do estado; e grupo 2 composto por amostras isoladas na terra indígena Xakriabá, em São João das Missões. A identificação específica de todas as amostras como L. braziliensis foi confirmada utilizando a técnica de PCR-g6pd. A análise da variabilidade genética foi realizada para os marcadores genéticos hsp70, Cpb, ITS1, g6pd e 6pgd. Na PCR-RFLP do hsp70 foram observados dois perfis de restrição: todas as amostras do grupo 1 e as cepas MG15 e MG16 do grupo 2 tiveram perfil de restrição indistinguível ao da cepa L. braziliensis referência, enquanto a maioria as amostras do grupo 2 exibiram perfil de restrição variante. O fragmento obtido pela PCR do hsp70 foi sequenciado e foram observados polimorfismos inclusive no sítio de restrição da enzima HaeIII. Na PCR-RFLP do Cpb, as amostras do grupo 1 e as cepas MG15 e MG16 do grupo 2 tiveram perfil de restrição indistinguível ao da cepa referência, enquanto a maioria das amostras do grupo 2 apresentaram perfil de restrição correspondente às demais espécies do subgênero Viannia. O sequenciamento do fragmento revelou polimorfismos inclusive no sítio de restrição da enzima TaqI. Na PCR-RFLP do ITS1 foi observado que as amostras do grupo 1 e as cepas MG15 e MG16 exibiram perfil de restrição semelhante a L. guyanensis, enquanto as amostras do grupo 2 perfil de L. braziliensis. As cepas MG19 e MG27 (grupo 2) exibiram perfis de restrição diferentes das cepas referência utilizadas. O sequenciamento do fragmento da PCR-6pgd exibiu polimorfismos que diferenciam entre as espécies L. braziliensis e L. guyanensis nas amostras estudadas. Os perfis de restrição e as sequências foram utilizadas em análises estatísticas de classificação por similaridade por partição e hierárquico. A análise de partição corroborou a divisão das amostras em dois grupos, sugerindo uma maior variabilidade genética entre as amostras do grupo 2. As análises aglomerativas suportaram a de partição onde foi observada associação do grupo 2 com a origem geográfica e presença de manifestações atípicas de LTA não sendo observada associação com número de lesões. As sequências foram utilizadas em análises filogenéticas onde foi observado que tanto utilizando somente L. guyanensis quanto outras espécies filogeneticamente mais distantes de L. braziliensis como outgroup, a divisão em grupos proposta foi suportada. A partir do painel de amostras de L. braziliensis estudado conclui-se que em Minas Gerais observamos a presença de um grupo de amostras geneticamente variantes, associadas ao perfil atípico de lesões e provenientes da região norte do estado.Leishmania (Viannia) braziliensis is the most common species of Leishmania genus which parasites humans in Brazil. This species may present a huge spectrum of clinical symptoms, from single wounds to serious mucosal involvement, which is the most severe complication. Analyzes that assess the genetic variability of L. braziliensis are essential to clarify a possible correlation between atypical clinical manifestations of LTA with parasites genetically variants and their geographical distribution, and contribute to population structure studies of this species in Minas Gerais state. The aim of this study was to analyze the genetic variability of Leishmania (V.) braziliensis isolates from different regions of Minas Gerais State. Clinical and molecular diagnosis of patients with typical and atypical lesions from various endemic areas of Minas Gerais were carried out and the isolates were separeted into two sample groups: group 1 containing samples from various geographical regions of the state; and group 2 composed b isolates from Xakriabá Indigenous Reserve localized in São João das Missões district. The specific identification of samples was performed with PCR-G6PD method and L. braziliensis was confirmed in all of them. The analysis of genetic variability was carried out using genetic markers hsp70, Cpb, ITS1, G6PD and 6PGD. In PCR-RFLP of hsp70 two restriction patterns were observed: all samples from group 1 and MG15 and MG16 isolates of group 2 showed indistinguishable restriction profile to L. braziliensis reference strain, while most of the Group 2 samples exhibited a variant restriction profile. The hsp70 amplicon was sequenced and polymorphisms were observed even at the HaeIII restriction enzyme’s site. In PCR-RFLP of Cpb, samples of group 1 and MG15 and MG16 strains of group 2 restriction profile were indistinguishable to the reference strain L. braziliensis, while most of the group 2 samples showed restriction profiles identical to other species of the subgenus L . (Viannia). The sequenced fragment showed polymorphisms including one at restriction site of TaqI enzyme. Results of ITS1 PCR-RFLP from group 1 and MG15 and MG16 strains were a restriction profile similar to L. guyanensis specie, while Group 2 samples presented L. braziliensis profile. The MG19 and MG27 strains (group 2) exhibited different patterns of those reference strains. The sequencing of the PCR-6pgd showed inter-species polymorphisms. Restriction profiles and sequences were used in hierarchical, partition and similarity statistical analyzes. The partition analysis confirmed the division of samples into two groups, suggesting greater genetic variability between samples of group 2. The clustering analysis supported the partition where was observed an association of group to the geographical origin and presence of atypical manifestations of LTA, but no association was observed with number of wounds. The sequences were used in phylogenetic analyzes and was observed that both using only L. guyanensis as other more phylogenetically distant species L. braziliensis as outgroup, the division into two groups proposal was supported. Considering the L. braziliensis samples panel studied in this project it is possible to conclud that there is a group of samples genetically variants associated with atypical profile of wounds from the northern region of Minas Gerais state

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake.

Submitted by Nuzia Santos ([email protected]) on 2019-08-29T14:03:01Z No. of bitstreams: 1 Antimony resistance in Leishmania .pdf: 1634456 bytes, checksum: aef9a32186654027ee883cd5b35c81b1 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2019-08-29T14:08:25Z (GMT) No. of bitstreams: 1 Antimony resistance in Leishmania .pdf: 1634456 bytes, checksum: aef9a32186654027ee883cd5b35c81b1 (MD5)Made available in DSpace on 2019-08-29T14:08:25Z (GMT). No. of bitstreams: 1 Antimony resistance in Leishmania .pdf: 1634456 bytes, checksum: aef9a32186654027ee883cd5b35c81b1 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Fisiologia e Biofísica. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brasil.BACKGROUND: In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES: The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS: The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS: Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS: There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure

Molecular Detection of Leishmania in Phlebotomine Sand Flies (Diptera: Psychodidae) from a Cutaneous Leishmaniasis Focus at Xakriabá Indigenous Reserve, Brazil

Submitted by Paloma Shiambukuro ([email protected]) on 2015-12-03T19:59:57Z No. of bitstreams: 1 Rego et al (2015) Molecular detection of Leishmania.pdf: 696299 bytes, checksum: f841a3811c921e45f6fb8006744821f7 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-12-15T13:08:29Z (GMT) No. of bitstreams: 1 Rego et al (2015) Molecular detection of Leishmania.pdf: 696299 bytes, checksum: f841a3811c921e45f6fb8006744821f7 (MD5)Made available in DSpace on 2015-12-15T13:08:29Z (GMT). No. of bitstreams: 1 Rego et al (2015) Molecular detection of Leishmania.pdf: 696299 bytes, checksum: f841a3811c921e45f6fb8006744821f7 (MD5) Previous issue date: 2015Fundação Oswaldo. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrasilFundação Oswaldo. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrasilFundação Oswaldo. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrasilFundação Oswaldo. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrasilFundação Oswaldo. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrasilFundação Oswaldo. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrasilAutochthonous cases of American cutaneous leishmaniasis (ACL) have been reported since 2001 in the Xakriabá Indigenous Reserve located in the municipality of São João das Missões in northern Minas Gerais state, Brazil. In order to study the presence of Leishmania DNA in phlebotomine sand flies, six entomological collections were carried out from July 2008 through July 2009, using 40 light traps placed in peridomicile areas of 20 randomly selected houses. From October 2011 through August 2012, another six collections were carried out with 20 light traps distributed among four trails (five traps per trail) selected for a previous study of wild and synanthropic hosts of Leishmania. A total of 4,760 phlebotomine specimens were collected belonging to ten genera and twenty-three species. Single female specimens or pools with up to ten specimens of the same locality, species and date, for Leishmania detection by molecular methods. Species identification of parasites was performed with ITS1 PCR-RFLP using HaeIII enzyme and genetic sequencing for SSU rRNA target. The presence of Leishmania DNA was detected in eleven samples from peridomicile areas: Lu. longipalpis (two), Nyssomyia intermedia (four), Lu. renei (two), Lu. ischnacantha, Micropygomyia goiana and Evandromyia lenti (one pool of each specie). The presence of Leishmania DNA was detected in twelve samples from among the trails: Martinsmyia minasensis (six), Ny. intermedia (three), Mi. peresi (two) and Ev. lenti (one). The presence of Leishmania infantum DNA in Lu. longipalpis and Leishmania braziliensis DNA in Ny. intermediasupport the epidemiological importance of these species of sand flies in the cycle of visceral and cutaneous leishmaniasis, respectively. The results also found other species associated with Leishmania DNA, such as Mt. minasensis and Ev. lenti, which may participate in a wild and/or synanthropic cycle of Leishmania transmission in the studied area

Location of study area.

<p>The location of the municipality of São João das Missões in northern Minas Gerais, Brazil. The native village of Imbaúbas located in the Xakriabá Indigenous Reserve, where the study was performed, is indicated.</p

Detection of <i>Leishmania</i> in sand flies collected from trails in the Xakriabá Indigenous Reserve, Brazil, during the study period.

<p>Sand flies were pooled with up to ten specimens of the same locality, species and date, and ITS1 PCR was performed with total DNA extracted from these pools. The figure represents an ethidium bromide-stained 2% agarose gel in which the amplicons were submitted to electrophoresis. Lanes: MW, molecular weight marker—100 bp; lanes 1–4, positive controls of <i>Le</i>. <i>amazonensis</i> (IFLA/BR/67/PH8), <i>Le</i>. <i>braziliensis</i> (MHOM/BR/75/M2903), <i>Le</i>. <i>infantum</i> (MHOM/BR/74/PP75), <i>Le</i>. <i>guyanensis</i> (MHOM/BR/75/M4147) respectively; 5–14, phlebotomine positive pools; NC, negative control.</p

Species identification of <i>Leishmania</i> from sand flies collected from trails in the Xakriabá Indigenous Reserve, Brazil, during the study period.

<p>Sand flies were grouped in pools of up to ten specimens of the same species, locality, and date, and ITS1 PCR-RFLP was performed with total DNA extracted from these pools. The figure represents an ethidium bromide-stained 4% agarose gel in which the amplicons were submitted to electrophoresis. Lanes: MW, molecular weight marker—100 bp; lanes 1–4, positive controls of <i>Le</i>. <i>amazonensis</i> (IFLA/BR/67/PH8), <i>Le</i>. <i>braziliensis</i> (MHOM/BR/75/M2903), <i>Le</i>. <i>infantum</i> (MHOM/BR/74/PP75), <i>Le</i>. <i>guyanensis</i> (MHOM/BR/75/M4147) respectively; 5–14, phlebotomine positive pools.</p

Survey of Sand Flies (Diptera: Psychodidae) in an Environmentally Protected Area in Brazil.

Submitted by Nuzia Santos ([email protected]) on 2016-02-29T13:50:29Z No. of bitstreams: 1 Survey of Sand Flies.pdf: 485619 bytes, checksum: f8d45630b2b8a572a9f83ac70b93519c (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-02-29T13:54:19Z (GMT) No. of bitstreams: 1 Survey of Sand Flies.pdf: 485619 bytes, checksum: f8d45630b2b8a572a9f83ac70b93519c (MD5)Made available in DSpace on 2016-02-29T13:54:19Z (GMT). No. of bitstreams: 1 Survey of Sand Flies.pdf: 485619 bytes, checksum: f8d45630b2b8a572a9f83ac70b93519c (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Grupo de Estudos em Leishmanioses. Belo Horizonte, MG, BrazilBrazil is one of the most important endemic areas for leishmaniasis worldwide. Protected areas that are tourist attractions likely present an important risk of transmission of cutaneous leishmaniasis (CL). Furthermore, with the geographical expansion of visceral leishmaniasis (VL), several studies have recorded the occurrence of its vector, Lutzomyia longipalpis, and cases of human and canine VL in such tourist areas. The Parque Estadual do Sumidouro is an environmentally protected area located in the Brazilian Cerrado biome and in an important area endemic for leishmaniasis in the state of Minas Gerais. The purpose of this study was to monitor the sand fly fauna in areas of tourist activity in the park. Sampling was performed every month, from September 2011 to August 2013, using CDC light traps at six sites of differing environmental characteristics. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A total of 4,675 sand fly specimens of 25 species belonging to nine genera were collected. The most abundant species were Micropygomyia quinquefer, Lutzomyia renei and Pintomyia pessoai, although only Pi. pessoai is implicated in the transmission of Leishmania braziliensis. The species accumulation curve reached saturation on the 16th sampling event. Species richness, diversity and evenness differed among the sampled areas. The seasonal curve was not determined by a single unique species, and no single species was the most abundant in all environments sampled. The main vector of Leishmania (Leishmania) infantum, Lutzomyia longipalpis, accounted for only 5.35% of the specimens collected. Proven or suspected vectors of Leishmania (Viannia) braziliensis were recorded, and one female of the cortellezzii complex tested positive for Le. braziliensis DNA. Even with a low infection rate (0.62%), these data indicate the circulation of the parasite and reinforce the need for entomological and epidemiological surveillance in the park and its surroundings

Environmental characteristics of the sand fly collection sites in PES from September 2011 to August 2013.

<p>Environmental characteristics of the sand fly collection sites in PES from September 2011 to August 2013.</p

Leishmania Lipophosphoglycan Triggers Caspase-11 and the Non-canonical Activation of the NLRP3 Inflammasome

Submitted by Nuzia Santos ([email protected]) on 2019-10-18T17:46:51Z No. of bitstreams: 1 Leishmania Lipophosphoglycan Triggers.pdf: 6570102 bytes, checksum: 1cc17d11c4468270e12be56a2360905e (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2019-10-18T17:51:32Z (GMT) No. of bitstreams: 1 Leishmania Lipophosphoglycan Triggers.pdf: 6570102 bytes, checksum: 1cc17d11c4468270e12be56a2360905e (MD5)Made available in DSpace on 2019-10-18T17:51:32Z (GMT). No. of bitstreams: 1 Leishmania Lipophosphoglycan Triggers.pdf: 6570102 bytes, checksum: 1cc17d11c4468270e12be56a2360905e (MD5) Previous issue date: 2019Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos. Ribeirão Preto, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos. Ribeirão Preto, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos. Ribeirão Preto, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos. Ribeirão Preto, SP, Brasil.Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos. Ribeirão Preto, SP, Brasil.National Institute of Biological Sciences. Beijing, China.Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brasil.Department of Molecular Microbiology. Washington University School of Medicine. Saint Louis, MO, USA.National Institute of Biological Sciences. Beijing, China.Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos. Ribeirão Preto, SP, Brasil.Activation of the NLRP3 inflammasome by Leishmania parasites is critical for the outcome of leishmaniasis, a disease that affects millions of people worldwide. We investigate the mechanisms involved in NLRP3 activation and demonstrate that caspase-11 (CASP11) is activated in response to infection by Leishmania species and triggers the non-canonical activation of NLRP3. This process accounts for host resistance to infection in macrophages and in vivo. We identify the parasite membrane glycoconjugate lipophosphoglycan (LPG) as the molecule involved in CASP11 activation. Cytosolic delivery of LPG in macrophages triggers CASP11 activation, and infections performed with Lpg1(-/-) parasites reduce CASP11/NLRP3 activation. Unlike bacterial LPS, purified LPG does not activate mouse CASP11 (or human Casp4) in vitro, suggesting the participation of additional molecules for LPG-mediated CASP11 activation. Our data identify a parasite molecule involved in CASP11 activation, thereby establishing the mechanisms underlying inflammasome activation in response to Leishmania species

Species accumulation curve, abundance, and species richness of the sand fly fauna of PES from September 2011 to August 2013.

<p>Species accumulation curve, abundance, and species richness of the sand fly fauna of PES from September 2011 to August 2013.</p