Genotyping and descriptive proteomics of a potential zoonotic canine strain of Giardia duodenalis, infective to mice

The zoonotic potential of giardiasis, as proposed by WHO since the late 70's, has been largely confirmed in this century. The genetic assemblages A and B of Giardia duodenalis are frequently isolated from human and canine hosts. Most of the assemblage A strains are not infective to adult mice, which can limit the range of studies regarding to biology of G. duodenalis, including virulence factors and the interaction with host immune system. This study aimed to determine the infectivity in mice of an assemblage A Giardia duodenalis strain (BHFC1) isolated from a dog and to classify the strain in sub-assemblages (Al, All, AIII) through the phylogenetic analysis of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. In addition, the proteomic profile of soluble and insoluble protein fractions of trophozoites was analyzed by 2D-electrophoresis. Accordingly, trophozoites of BHFC1 were highly infective to Swiss mice. The phylogenetic analysis of tpi and gdh revealed that BHFC1 clustered to sub-assemblage Al. The proteomic map of soluble and insoluble protein fractions led to the identification of 187 proteins of G. duodenalis, 27 of them corresponding to hypothetical proteins. Considering both soluble and soluble fractions, the vast majority of the identified proteins (n = 82) were classified as metabolic proteins, mainly associated with carbon and lipid metabolism, including 53 proteins with catalytic activity. Some of the identified proteins correspond to antigens while others can be correlated with virulence. Besides a significant complementation to the proteomic data of G. duodenalis, these data provide an important source of information for future studies on various aspects of the biology of this parasite, such as virulence factors and host and pathogen interactions1110CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE MINAS GERAIS - FAPEMIGsem informaçã

Genotyping and Descriptive Proteomics of a Potential Zoonotic Canine Strain of Giardia duodenalis, Infective to Mice

Submitted by Sandra Infurna ([email protected]) on 2017-03-02T18:57:50Z No. of bitstreams: 1 alex_chapeaurouge_etal_IOC_2016.pdf: 1813108 bytes, checksum: d2d4db74321df407e1868919c06835d6 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-03-02T19:11:30Z (GMT) No. of bitstreams: 1 alex_chapeaurouge_etal_IOC_2016.pdf: 1813108 bytes, checksum: d2d4db74321df407e1868919c06835d6 (MD5)Made available in DSpace on 2017-03-02T19:11:30Z (GMT). No. of bitstreams: 1 alex_chapeaurouge_etal_IOC_2016.pdf: 1813108 bytes, checksum: d2d4db74321df407e1868919c06835d6 (MD5) Previous issue date: 2016Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Universidade de Campinas. Centro de Biologia Molecular e Engenharia Genética. Campinas, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.Universidade Federal de Minas Gerais. Departamento de Biologia Geral. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Georgetown University. Biology Department. Washington, USA.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.The zoonotic potential of giardiasis, as proposed by WHO since the late 70's, has been largely confirmed in this century. The genetic assemblages A and B of Giardia duodenalis are frequently isolated from human and canine hosts. Most of the assemblage A strains are not infective to adult mice, which can limit the range of studies regarding to biology of G. duodenalis, including virulence factors and the interaction with host immune system. This study aimed to determine the infectivity in mice of an assemblage A Giardia duodenalis strain (BHFC1) isolated from a dog and to classify the strain in sub-assemblages (AI, AII, AIII) through the phylogenetic analysis of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. In addition, the proteomic profile of soluble and insoluble protein fractions of trophozoites was analyzed by 2D-electrophoresis. Accordingly, trophozoites of BHFC1 were highly infective to Swiss mice. The phylogenetic analysis of tpi and gdh revealed that BHFC1 clustered to sub-assemblage AI. The proteomic map of soluble and insoluble protein fractions led to the identification of 187 proteins of G. duodenalis, 27 of them corresponding to hypothetical proteins. Considering both soluble and soluble fractions, the vast majority of the identified proteins (n = 82) were classified as metabolic proteins, mainly associated with carbon and lipid metabolism, including 53 proteins with catalytic activity. Some of the identified proteins correspond to antigens while others can be correlated with virulence. Besides a significant complementation to the proteomic data of G. duodenalis, these data provide an important source of information for future studies on various aspects of the biology of this parasite, such as virulence factors and host and pathogen interactions

Mapping Alterations Induced by Long-Term Axenic Cultivation of Leishmania amazonensis Promastigotes With a Multiplatform Metabolomic Fingerprint Approach

Submitted by Nuzia Santos ([email protected]) on 2020-02-04T17:07:30Z No. of bitstreams: 1 Mapping Alterations Induced .pdf: 15523213 bytes, checksum: a485f8b44283ace87e344a06f1a2edea (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2020-02-04T17:13:46Z (GMT) No. of bitstreams: 1 Mapping Alterations Induced .pdf: 15523213 bytes, checksum: a485f8b44283ace87e344a06f1a2edea (MD5)Made available in DSpace on 2020-02-04T17:13:46Z (GMT). No. of bitstreams: 1 Mapping Alterations Induced .pdf: 15523213 bytes, checksum: a485f8b44283ace87e344a06f1a2edea (MD5) Previous issue date: 2019Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil / Centro de Metabolómica y Bioanálisis. Unidad Metabolómica. Interacciones y Bioanálisis (UMIB), Universidad CEU. San Pablo, Boadilla del Monte, Spain.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil / Centro de Metabolómica y Bioanálisis. Unidad Metabolómica. Interacciones y Bioanálisis (UMIB), Universidad CEU. San Pablo, Boadilla del Monte, Spain.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Biologia Geral. Laboratório de Biotecnologia e Marcadores Moleculares. Belo Horizonte, MG, Brasil.Manchester Institute of Biotechnology. The University of Manchester. Manchester, United Kingdom.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Fundação 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 Morfologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Morfologia. Belo Horizonte, MG, Brasil.Centro de Metabolómica y Bioanálisis. Unidad Metabolómica. Interacciones y Bioanálisis (UMIB), Universidad CEU. San Pablo, Boadilla del Monte, Spain.Centro de Metabolómica y Bioanálisis. Unidad Metabolómica. Interacciones y Bioanálisis (UMIB), Universidad CEU. San Pablo, Boadilla del Monte, Spain.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Belo Horizonte, MG, Brasil.Leishmaniases are widespread neglected diseases with an incidence of 1.6 million new cases and 40 thousand deaths per year. Leishmania parasites may show distinct, species-specific patterns of virulence that lead to different clinical manifestations. It is well known that successive in vitro passages (SIVP) lead to the attenuation of virulence, but neither the metabolism nor the pathways involved in these processes are well understood. Herein, promastigotes of a virulent L. amazonensis strain recently isolated from mice was compared to SIVP derived and attenuated promastigotes, submitted to 10, 40, and 60 axenic passages and named R10, R40, and R60, respectively. In vitro assays and in vivo tests were performed to characterize and confirmed the attenuation profiles. A metabolomic fingerprint comparison of R0, R10, and R60 was performed by means of capillary electrophoresis, liquid and gas chromatography coupled to mass spectrometry. To validate the metabolomic data, qPCR for selected loci, flow cytometry to measure aPS exposure, sensitivity to antimony tartrate and ROS production assays were conducted. The 65 identified metabolites were clustered in biochemical categories and mapped in eight metabolic pathways: ABC transporters; fatty acid biosynthesis; glycine, serine and threonine metabolism; β-alanine metabolism; glutathione metabolism; oxidative phosphorylation; glycerophospholipid metabolism and lysine degradation. The obtained metabolomic data correlated with previous proteomic findings of the SVIP parasites and the gene expression of 13 selected targets. Late SIVP cultures were more sensitive to SbIII produced more ROS and exposed less phosphatidylserine in their surface. The correspondent pathways were connected to build a biochemical map of the most significant alterations involved with the process of attenuation of L. amazonensis. Overall, the reported data pointed out to a very dynamic and continuous metabolic reprogramming process, accompanied by changes in energetic, lipid and redox metabolisms, membrane remodeling and reshaping of parasite-host cells interactions, causing impacts in chemotaxis, host inflammatory responses and infectivity at the early stages of infection

2D protein map showing the spots of the soluble protein fraction (Proteins 1 to 429) and the numerical distribution of localized proteins.

<p>The protein identification correspondent to each number is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164946#pone.0164946.s003" target="_blank">S3 Table</a>.</p

2D protein map showing the spots of the insoluble fraction (Proteins 430 to 903) and the numerical distribution of localized proteins.

<p>The protein identification correspondent to each number is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164946#pone.0164946.s003" target="_blank">S3 Table</a>.</p

Classification of the identified proteins by molecular function (A), biological process (B) and protein class (C).

<p>This classification is suggested by gene ontology and Panther^® and provides a general characterization of a group of proteins. The numbers presented in each graph correspond to the number of proteins.</p

Number of trophozoites or cysts recovered from Swiss mice gut after infection with <i>G</i>. <i>duodenalis</i> strains BHCF1 and Portland-1.

<p>Mice were inoculated with 1 x 10⁶ trophozoites through intra-gastric route. Parasites were recovered from gut and counted in Neubauer chamber. Numbers correspond to average and standard deviation of parasite counting’s obtained for each group, from three independent experiments, with minimal number of 3 mice per group, in each experiment.</p

Presence of transmembrane domains and signal peptide for the annoted proteins identified in insoluble and soluble protein fractions.

<p>Presence of transmembrane domains and signal peptide for the annoted proteins identified in insoluble and soluble protein fractions.</p

Consensus phylogenetic relationships of <i>G</i>. <i>duodenalis</i> with Bayesian posterior probabilities using a Markov chain Monte Carlo sampling technique, for <i>bg</i>, <i>tpi</i>, <i>and gdh</i> gene sequences of <i>G</i>. <i>duodenalis</i>.

<p>Markov chains were run for 6,000,000 iterations and the trees were sampled every 100 iterations. The GTR model was used with gamma correction. Sequences from <i>Giardia muris</i>, <i>Giardia microti and Giardia ardeae</i> were employed as outgroups.</p