Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp

Detalhes bibliográficos
Autor(a) principal: Real, Fernando [UNIFESP]
Data de Publicação: 2011
Tipo de documento: Tese
Idioma: por
Título da fonte: Repositório Institucional da UNIFESP
Texto Completo: http://repositorio.unifesp.br/handle/11600/9669
Resumo: Most non-viral intracellular pathogens gain entrance into human and animal host cells by “classic” or modifIed phagocytosis and are thus lodged in phagosomes which they may or not continue to occupy in the course of infection. Their survival and multiplication within host cells depend on modulation of the compositional and functional phenotypes of the phagosomes they occupy, including intravacuolar pH, substrate acquisition through membrane transporters and channels, and fusion with lysosomes, and other cell phagosomes and vesicles. Each phagosome thus exhibits particular features, whose biogenesis is conditioned to different signals triggered by both the host cell and the internalized particle/microorganism. Once internalized by host cells, some pathogens escape the phagosome and assume the host cell cytosol as their intracellular niche. Other pathogens interfere with phagosomal maturation, leading to the development of phagosomes excluded from host cell endocytic and secretory pathways or vacuoles with selective fusogenic properties. During their intracelular lifecycle, protozoan parasites of the genus Leishmania remain enclosed in phagolysosome-like structures called Parasitophorous Vacuoles (PVs). The morphological and biochemical diversity of Leishmania PVs were not extensively studied. Most species – such as L. major, L. donovani and L. braziliensis – are lodged in membrane-bound PVs, containing one or two amastigotes, that undergo fission as parasites divide. The mechanisms involved in PV fission remain to be elucidated. In contrast, species from the L. mexicana complex, such as L. amazonensis, L. mexicana and L. pifanoi, occupy large PVs which may contain many parasites. The present experimental aimed to answer the question: “what is the importance of a speciesspecific PV to Leishmania intracellular parasitism?”. In the experiments herewith described, mouse bone marrow-derived macrophages were co-infected with two Leishmania species to investigate the possibility of fusion between PVs that shelter different parasites, and the consequences of a possible intravacuolar cohabitation on their survival and multiplication. Macrophages were initially infected with L. amazonensis and later on superinfected with L. major, which represent species with different PV size, parasite content and biogenesis. In order to distinguish the two species, macrophages were infected with non-fluorescent L. amazonensis amastigotes and superinfected with either amastigotes or promastigotes of L. major transfected with the fluorescent proteins GFP or DsRed2. Although PVs contacted each other, fusion between L. amazonensis and L. major amastigote PVs was not detected. Leishmania major multiplication and PV fission were not affected by coinfection. In contrast, PVs containing L. major promastigotes fused with pre-established L. amazonensis PVs. In these “chimeric” vacuoles (containing both Leishmania), L. major promastigotes multiplied, however they did not differentiate into amastigotes. The differentiation of L. major promastigotes into amastigotes occurred exclusively within their own, unfused PVs.
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spelling Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania sppHomotypic and Heterotypic Fusion between Leishmania spp. Parasitophorous VacuolesInfecção intracelularLeishmania (L.) amazonensisLeishmania (L.) majorMacrófagosVacúolo parasitóforoFusão de vesículasLeishmaniaMacrophagesLeishmania majorLeishmaniaMost non-viral intracellular pathogens gain entrance into human and animal host cells by “classic” or modifIed phagocytosis and are thus lodged in phagosomes which they may or not continue to occupy in the course of infection. Their survival and multiplication within host cells depend on modulation of the compositional and functional phenotypes of the phagosomes they occupy, including intravacuolar pH, substrate acquisition through membrane transporters and channels, and fusion with lysosomes, and other cell phagosomes and vesicles. Each phagosome thus exhibits particular features, whose biogenesis is conditioned to different signals triggered by both the host cell and the internalized particle/microorganism. Once internalized by host cells, some pathogens escape the phagosome and assume the host cell cytosol as their intracellular niche. Other pathogens interfere with phagosomal maturation, leading to the development of phagosomes excluded from host cell endocytic and secretory pathways or vacuoles with selective fusogenic properties. During their intracelular lifecycle, protozoan parasites of the genus Leishmania remain enclosed in phagolysosome-like structures called Parasitophorous Vacuoles (PVs). The morphological and biochemical diversity of Leishmania PVs were not extensively studied. Most species – such as L. major, L. donovani and L. braziliensis – are lodged in membrane-bound PVs, containing one or two amastigotes, that undergo fission as parasites divide. The mechanisms involved in PV fission remain to be elucidated. In contrast, species from the L. mexicana complex, such as L. amazonensis, L. mexicana and L. pifanoi, occupy large PVs which may contain many parasites. The present experimental aimed to answer the question: “what is the importance of a speciesspecific PV to Leishmania intracellular parasitism?”. In the experiments herewith described, mouse bone marrow-derived macrophages were co-infected with two Leishmania species to investigate the possibility of fusion between PVs that shelter different parasites, and the consequences of a possible intravacuolar cohabitation on their survival and multiplication. Macrophages were initially infected with L. amazonensis and later on superinfected with L. major, which represent species with different PV size, parasite content and biogenesis. In order to distinguish the two species, macrophages were infected with non-fluorescent L. amazonensis amastigotes and superinfected with either amastigotes or promastigotes of L. major transfected with the fluorescent proteins GFP or DsRed2. Although PVs contacted each other, fusion between L. amazonensis and L. major amastigote PVs was not detected. Leishmania major multiplication and PV fission were not affected by coinfection. In contrast, PVs containing L. major promastigotes fused with pre-established L. amazonensis PVs. In these “chimeric” vacuoles (containing both Leishmania), L. major promastigotes multiplied, however they did not differentiate into amastigotes. The differentiation of L. major promastigotes into amastigotes occurred exclusively within their own, unfused PVs.Quase todos os patógenos intracelulares não virais de importância humana e animal penetram nas células hospedeiras por fagocitose “clássica” ou modificada. Por este motivo os fagossomos constituem o primeiro e, às vezes, o último habitat dos patógenos intracelulares. Sua sobrevida e multiplicação na célula hospedeira dependem da modulação do fenótipo composicional e funcional dos fagossomos em que habitam, como o pH intravacuolar, a aquisição de substratos e nutrientes pela presença de canais e transportadores e a fusão com lisossomos, outros fagossomos e vesículas. Cada fagossomo ou fagolisossomo é uma entidade particular, cuja biogênese depende de sinais expressos ou disparados pela célula e pela partícula ou organismo internalizado. Uma vez internalizados pelas células hospedeiras, alguns patógenos escapam do fagossomo e se instalam no citosol. Outros interferem com a maturação dos fagossomos, de forma a excluí-los das vias endocítica e secretória ou gerar vacúolos de capacidade fusogênica seletiva. Os parasitas do gênero Leishmania permanecem, durante todo o ciclo de vida intracelular no hospedeiro mamífero, em estruturas semelhantes a fagolisossomos denominadas vacúolos parasitóforos (VPs). A diversidade morfológica e bioquímica desses vacúolos foi pouco estudada. Os VPs formados pelas espécies mais estudadas – (L.) L. major, L. (L.) donovani e L. (V.) braziliensis - abrigam uma ou duas formas amastigotas e apresentam pouco espaço vacuolar livre. À medida que os amastigotas se dividem, os VPs que os hospedam fissionam, porém os mecanismos envolvidos neste processo são desconhecidos. Já parasitas do complexo (L.) (L.) mexicana, incluindo L. (L.) amazonensis, L. (L.) mexicana e L. (L.) pifanoi, ocupam VPs espaçosos contendo mais de um amastigota. O presente estudo experimental teve como objetivo responder à questão: “qual a importância de um VP espécie-específico para o parasitismo intracelular de Leishmania?”. Nos experimentos descritos, macrófagos derivados de precursores de medula óssea de camundongo foram coinfectados por duas espécies de Leishmania para investigar a possibilidade de fusão entre VPs que hospedam diferentes parasitas e as consequências de uma possível coabitação intravacuolar sobre a viabilidade e multiplicação dos dois parasitas. Os macrófagos foram inicialmente infectados com L. (L.) amazonensis e em seguida superinfectados por L. (L.) major, espécies que desenvolvem VPs de tamanho, número de parasitas e biogênese distintos. Para permitir o reconhecimento inequívoco da espécie dos parasitas, os macrófagos foram infectados por amastigotas não fluorescentes de L. (L.) amazonensis e superinfectados com amastigotas ou promastigotas de L. (L.) major que expressam proteínas fluorescentes GFP ou DsRed2. Constatamos que os VPs contendo amastigotas de L. (L.) major aderiram aos espaçosos VPs de L. (L.) amazonensis mas a fusão entre os vacúolos não foi detectada. A multiplicação da L. (L.) major e a fissão de seus VPs nos macrófagos superinfectados não foram afetadas pela infecção pelos dois parasitas. Já os VPs contendo promastigotas da L. (L.) major se fundiram com os VPs da L. (L.) amazonensis. Nestes VPs “quiméricos” (contendo ambas as espécies de parasitas) os promastigotas de L. (L.) major dividiram-se mas não se diferenciaram em amastigotas. Essa diferenciação só ocorreu nos pequenos VPs que abrigavam exclusivamente a L. (L.) major.TEDEBV UNIFESP: Teses e dissertaçõesUniversidade Federal de São Paulo (UNIFESP)Rabinovitch, Michel [UNIFESP]Universidade Federal de São Paulo (UNIFESP)Real, Fernando [UNIFESP]2015-07-22T20:50:16Z2015-07-22T20:50:16Z2011-02-22info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersion122 p.application/pdfapplication/pdfREAL, Fernando Roberto Oliveira. Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp. 2011. Tese (Doutorado) - Universidade Federal de São Paulo (UNIFESP), São Paulo, 2011.Publico-0033a.pdfPublico-0033b.pdfhttp://repositorio.unifesp.br/handle/11600/9669porinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP2024-07-30T04:28:36Zoai:repositorio.unifesp.br/:11600/9669Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestbiblioteca.csp@unifesp.bropendoar:34652024-07-30T04:28:36Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false
dc.title.none.fl_str_mv Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp
Homotypic and Heterotypic Fusion between Leishmania spp. Parasitophorous Vacuoles
title Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp
spellingShingle Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp
Real, Fernando [UNIFESP]
Infecção intracelular
Leishmania (L.) amazonensis
Leishmania (L.) major
Macrófagos
Vacúolo parasitóforo
Fusão de vesículas
Leishmania
Macrophages
Leishmania major
Leishmania
title_short Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp
title_full Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp
title_fullStr Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp
title_full_unstemmed Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp
title_sort Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp
author Real, Fernando [UNIFESP]
author_facet Real, Fernando [UNIFESP]
author_role author
dc.contributor.none.fl_str_mv Rabinovitch, Michel [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
dc.contributor.author.fl_str_mv Real, Fernando [UNIFESP]
dc.subject.por.fl_str_mv Infecção intracelular
Leishmania (L.) amazonensis
Leishmania (L.) major
Macrófagos
Vacúolo parasitóforo
Fusão de vesículas
Leishmania
Macrophages
Leishmania major
Leishmania
topic Infecção intracelular
Leishmania (L.) amazonensis
Leishmania (L.) major
Macrófagos
Vacúolo parasitóforo
Fusão de vesículas
Leishmania
Macrophages
Leishmania major
Leishmania
description Most non-viral intracellular pathogens gain entrance into human and animal host cells by “classic” or modifIed phagocytosis and are thus lodged in phagosomes which they may or not continue to occupy in the course of infection. Their survival and multiplication within host cells depend on modulation of the compositional and functional phenotypes of the phagosomes they occupy, including intravacuolar pH, substrate acquisition through membrane transporters and channels, and fusion with lysosomes, and other cell phagosomes and vesicles. Each phagosome thus exhibits particular features, whose biogenesis is conditioned to different signals triggered by both the host cell and the internalized particle/microorganism. Once internalized by host cells, some pathogens escape the phagosome and assume the host cell cytosol as their intracellular niche. Other pathogens interfere with phagosomal maturation, leading to the development of phagosomes excluded from host cell endocytic and secretory pathways or vacuoles with selective fusogenic properties. During their intracelular lifecycle, protozoan parasites of the genus Leishmania remain enclosed in phagolysosome-like structures called Parasitophorous Vacuoles (PVs). The morphological and biochemical diversity of Leishmania PVs were not extensively studied. Most species – such as L. major, L. donovani and L. braziliensis – are lodged in membrane-bound PVs, containing one or two amastigotes, that undergo fission as parasites divide. The mechanisms involved in PV fission remain to be elucidated. In contrast, species from the L. mexicana complex, such as L. amazonensis, L. mexicana and L. pifanoi, occupy large PVs which may contain many parasites. The present experimental aimed to answer the question: “what is the importance of a speciesspecific PV to Leishmania intracellular parasitism?”. In the experiments herewith described, mouse bone marrow-derived macrophages were co-infected with two Leishmania species to investigate the possibility of fusion between PVs that shelter different parasites, and the consequences of a possible intravacuolar cohabitation on their survival and multiplication. Macrophages were initially infected with L. amazonensis and later on superinfected with L. major, which represent species with different PV size, parasite content and biogenesis. In order to distinguish the two species, macrophages were infected with non-fluorescent L. amazonensis amastigotes and superinfected with either amastigotes or promastigotes of L. major transfected with the fluorescent proteins GFP or DsRed2. Although PVs contacted each other, fusion between L. amazonensis and L. major amastigote PVs was not detected. Leishmania major multiplication and PV fission were not affected by coinfection. In contrast, PVs containing L. major promastigotes fused with pre-established L. amazonensis PVs. In these “chimeric” vacuoles (containing both Leishmania), L. major promastigotes multiplied, however they did not differentiate into amastigotes. The differentiation of L. major promastigotes into amastigotes occurred exclusively within their own, unfused PVs.
publishDate 2011
dc.date.none.fl_str_mv 2011-02-22
2015-07-22T20:50:16Z
2015-07-22T20:50:16Z
dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format doctoralThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv REAL, Fernando Roberto Oliveira. Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp. 2011. Tese (Doutorado) - Universidade Federal de São Paulo (UNIFESP), São Paulo, 2011.
Publico-0033a.pdf
Publico-0033b.pdf
http://repositorio.unifesp.br/handle/11600/9669
identifier_str_mv REAL, Fernando Roberto Oliveira. Fusão Homotípica e Heterotípica entre Vacúolos Parasitóforos de Leishmania spp. 2011. Tese (Doutorado) - Universidade Federal de São Paulo (UNIFESP), São Paulo, 2011.
Publico-0033a.pdf
Publico-0033b.pdf
url http://repositorio.unifesp.br/handle/11600/9669
dc.language.iso.fl_str_mv por
language por
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 122 p.
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Universidade Federal de São Paulo (UNIFESP)
publisher.none.fl_str_mv Universidade Federal de São Paulo (UNIFESP)
dc.source.none.fl_str_mv reponame:Repositório Institucional da UNIFESP
instname:Universidade Federal de São Paulo (UNIFESP)
instacron:UNIFESP
instname_str Universidade Federal de São Paulo (UNIFESP)
instacron_str UNIFESP
institution UNIFESP
reponame_str Repositório Institucional da UNIFESP
collection Repositório Institucional da UNIFESP
repository.name.fl_str_mv Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)
repository.mail.fl_str_mv biblioteca.csp@unifesp.br
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