Distinct mitochondrial HSP70 homologues conserved in various Leishmania species suggest novel biological functions

Campos, Rodrigo M.; Nascimento, Mirna; Ferraz, J. Cândido; Pereira, Mariana M.C.; Rocha, Pollyanna O.; Thompson, Glória M.; Cysne-Finkelstein, Léa; Figueiredo, Regina Célia Bressan Queiroz de; Neto, Osvaldo P. de Melo

doi:10.1016/j.molbiopara.2008.04.013

Cited by 15 publications

(19 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Images were produced in Fluoview software (FV300 version 5). For immunolocalization studies of the mitochondrial HSP70 protein, the parasites were fixed with 2% paraformaldehyde, permeabilized with Triton X100 (Sigma) and reacted with an anti-rabbit HSP70 antibody as a first antibody (kindly provided by Dr Osvaldo de Melo Neto, Centro de Pesquisas Aggeu Magalhães, Fiocruz, Recife, Brazil; [72]) and an anti-rabbit Alexa 594 as the second antibody. The cells were washed twice with HEPES-NaCl (500 µl) and observed under a Nikon epifluorescence microscope.…”

Section: Methodsmentioning

confidence: 99%

Novel Features of a PIWI-Like Protein Homolog in the Parasitic Protozoan Leishmania

et al. 2012

View full text Add to dashboard Cite

In contrast to nearly all eukaryotes, the Old World Leishmania species L. infantum and L. major lack the bona fide RNAi machinery genes. Interestingly, both Leishmania genomes code for an atypical Argonaute-like protein that possesses a PIWI domain but lacks the PAZ domain found in Argonautes from RNAi proficient organisms. Using sub-cellular fractionation and confocal fluorescence microscopy, we show that unlike other eukaryotes, the PIWI-like protein is mainly localized in the single mitochondrion in Leishmania. To predict PIWI function, we generated a knockout mutant for the PIWI gene in both L. infantum (Lin) and L. major species by double-targeted gene replacement. Depletion of PIWI has no effect on the viability of insect promastigote forms but leads to an important growth defect of the mammalian amastigote lifestage in vitro and significantly delays disease pathology in mice, consistent with a higher expression of the PIWI transcript in amastigotes. Moreover, amastigotes lacking PIWI display a higher sensitivity to apoptosis inducing agents than wild type parasites, suggesting that PIWI may be a sensor for apoptotic stimuli. Furthermore, a whole-genome DNA microarray analysis revealed that loss of LinPIWI in Leishmania amastigotes affects mostly the expression of specific subsets of developmentally regulated genes. Several transcripts encoding surface and membrane-bound proteins were found downregulated in the LinPIWI(−/−) mutant whereas all histone transcripts were upregulated in the null mutant, supporting the possibility that PIWI plays a direct or indirect role in the stability of these transcripts. Although our data suggest that PIWI is not involved in the biogenesis or the stability of small noncoding RNAs, additional studies are required to gain further insights into the role of this protein on RNA regulation and amastigote development in Leishmania.

show abstract

Section: Methodsmentioning

confidence: 99%

Novel Features of a PIWI-Like Protein Homolog in the Parasitic Protozoan Leishmania

et al. 2012

View full text Add to dashboard Cite

show abstract

“…are digenetic pathogens where temperature shift takes place as part of their life-cycle. Phases of low temperatures in the insect vector and higher temperatures in the mammalian host are accompanied by a strong induction of heat-shock proteins [14, 37]. Increased expression of HSPs protect the cell by stabilizing unfolded proteins, giving the cell time to repair or re-synthesize damaged proteins during heat-shock transitions.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, in most organisms, ER and mitochondrial HSP70s are constituted by a sole protein, however an interrogation of the L. donovani genome revealed four HSPA9 genes (A-D) with minor sequence differences suggesting divergent evolution of this protein subfamily [10]. A previous report from 2008 demonstrated the existence of two distinct HSPA9B homologs in L. chagasi fortuitously isolated from an immunoscreening of a L. chagasi cDNA expression library [14] showing the same sequence arrangement.…”

Section: Discussionmentioning

confidence: 99%

“…All of these activities require HSP70 proteins to recognize and interact with hydrophobic peptide segments in an ATP-dependent mechanism. The HSP70 family is highly conserved and each protein contains an N-terminal ATPase domain, a substrate binding domain comprising of both a 25 kDa β sheet subdomain and a 10 kDa helical subdomain and lastly a C-terminal domain rich in alpha helical structure [11, 14]. Several biochemical studies carried out in both bacteria and yeast have demonstrated the essentiality of specific amino acids localized at the ATPase and substrate binding domains for proper protein functioning [15–18].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A mitochondrial HSP70 (HSPA9B) is linked to miltefosine resistance and stress response in Leishmania donovani

et al. 2016

View full text Add to dashboard Cite

BackgroundProtozoan parasites of the genus Leishmania are responsible for leishmaniasis, a neglected tropical disease affecting millions worldwide. Visceral leishmaniasis (VL), caused by Leishmania donovani, is the most severe form of leishmaniasis with high rates of mortality if left untreated. Current treatments include pentavalent antimonials and amphotericin B. However, high toxicity and emergence of resistance hinder the success of these options. Miltefosine (HePC) is the first oral treatment available for leishmaniasis. While treatment with HePC has proven effective, higher tolerance to the drug has been observed, and experimental resistance is easily developed in an in vitro environment. Several studies, including ours, have revealed that HePC resistance has a multi-factorial origin and this work aims to shed light on this complex mechanism.Methods2D-DIGE quantitative proteomics comparing the soluble proteomes of sensitive and HePC resistant L. donovani lines identified a protein of interest tentatively involved in drug resistance. To test this link, we employed a gain-of-function approach followed by mutagenesis analysis. Functional studies were complemented with flow cytometry to measure HePC incorporation and cell death.ResultsWe identified a mitochondrial HSP70 (HSPA9B) downregulated in HePC-resistant L. donovani promastigotes. The overexpression of HSPA9B in WT lines confers an increased sensitivity to HePC, regardless of whether the expression is ectopic or integrative. Moreover, the increased sensitivity to HePC is specific to the HSPA9B overexpression since dominant negative mutant lines were able to restore HePC susceptibility to WT values. Interestingly, the augmented susceptibility to HePC did not correlate with an increased HePC uptake. Leishmania donovani promastigotes overexpressing HSPA9B were subjected to different environmental stimuli. Our data suggest that HSPA9B is capable of protecting cells from stressful conditions such as low pH and high temperature. This phenotype was further corroborated in axenic amastigotes overexpressing HSPA9B.ConclusionsThe results from this study provide evidence to support the involvement of a mitochondrial HSP70 (HSPA9B) in experimental HePC resistance, a mechanism that is not yet fully understood, and reveal potential fundamental roles of HSPA9B in the biology of Leishmania. Overall, our findings are relevant for current and future antileishmanial chemotherapy strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1904-8) contains supplementary material, which is available to authorized users.

show abstract

“…Diversos genes de HSP70 foram descritos em tripanossomas e leishmanias no citoplasma (HSP70, HSP70.4, HSP70.c) (Folgueira, Requena, 2007;Glass et al, 1986;Lee et al, 1988Lee et al, , 1990Requena et al, 1988); retículo (Grp78) (Bangs et al, 1993;Jensen et al, 2001;Tibbetts et al, 1994); mitocôndria (mtp70 -Lc2.1 e Lc2.2) (Campos et al, 2008;Engman et al, 1989); e HSP110s (Grp170 e HSP110) (Folgueira, Requena, 2007). Além desses genes, a comparação entre os genomas de T. cruzi, T. b. brucei e L. major revelou a existência de dois parálogos divergentes, HSP70.a e HSP70.b, sendo o primeiro sem ortólogos conhecidos fora de tripanossomatídeos e o segundo, semelhante ao encontrado em algumas cianobactérias, uma possível aquisição por transferência horizontal (Folgueira, Requena, 2007).…”

Section: Proteínas De Choque Térmicounclassified

Genes codificadores de proteínas implicadas na relação de espécies do gênero <i>Trypanosoma</i> com seus hospedeiros: diversidade, transferência horizontal e relações filogenéticas.

Costa-Martins¹

View full text Add to dashboard Cite

discutir possíveis funções das proteínas ACD em tripanossomas, a arquitetura dos domínios e motivos funcionais foi comparada com os respectivos arquétipos com funções conhecidas em outros organismos. Este estudo inclui também análises de genes codificadores das enzimas prolina racemase (PRAC) e Catepsina L-like (CATL) de Trypanosoma spp. As enzimas CATL desempenham um papel importante nos processos de infecção, diferenciação celular, patogenicidade, virulência e evasão das defesas do hospedeiro em tripanossomas. Neste estudo, pela primeira vez, a principal CATL de T. congolense-congopaina, foi caracterizada em isolados dos três subgrupos genéticos (Savannah, Forest e Kilifi), que variam na virulência, patogenicidade e distribuição geográfica. O polimorfismo, organização estrutural e genealogia revelaram que o repertório de genes de congopaina divergiu de forma espécies-específica para cada subgrupo de T. congolense, com Savannah exibindo sequências altamente polimórficas, inter-e intra-isolados, enquanto os grupos Forest e Kilifi apresentaram diversidade moderada e limitada. Além disso, um PCR baseado nos genes de congopaina foi desenvolvido para um diagnóstico sensível e específico de todos os subgrupos de T. congolense. As descobertas deste estudo demonstram que os genes de congopaina são úteis para o diagnóstico, genotipagem e inferências filogenéticas e taxonômicas de T. congolense. A enzima PRAC é responsável pela interconversão entre L-e D-prolina livre, presente em um grupo restrito de bactérias agindo como um fator de virulência. O primeiro gene eucariótico de PRAC foi descrito em T. cruzi e, em seguida, em T. vivax, e estudos funcionais e imunológicos provaram que essa proteína promove ativação policlonal de células B em animais infectado, o que leva a um atraso na resposta imunológica específica do hospedeiro e garante a sobrevivência do parasita. Neste estudo, as análises genômicas de homólogos da PRAC revelaram uma única cópia em 12 das 15 espécies de tripanossomas investigadas: nos parasitas de mamíferos T. cruzi, T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli, T. conorhini e T. lewisi; e nos tripanossomas de anuros, serpentes, crocodilos, lagartos e pássaros. T. rangeli apresentou apenas pseudogenes; T. brucei, T. congolense e espécies relacionadas, exceto T. vivax que é filogeneticamente mais distante, perderam completamente o gene PRAC. A genealogia dos homólogos PRAC suportada uma história evolutiva congruente com a filogenia de Trypanosoma. Este dado, juntamente com dados de sintenia, das relações filogenéticas com PRAC procariotas, e a ausência de genes PRAC em tripanossomatídeos dos outros gêneros, bodonídeos e euglenídeos, sugerem que um ancestral comum de Trypanosoma adquiriu o gene PRAC, em um evento único de HGT, de uma bactéria Firmicutes ancestral, mais relacionada ao gênero Gemella e outros bacilos do que a Clostridium como previamente sugerido.

show abstract

Distinct mitochondrial HSP70 homologues conserved in various Leishmania species suggest novel biological functions

Cited by 15 publications

References 25 publications

Novel Features of a PIWI-Like Protein Homolog in the Parasitic Protozoan Leishmania

Novel Features of a PIWI-Like Protein Homolog in the Parasitic Protozoan Leishmania

A mitochondrial HSP70 (HSPA9B) is linked to miltefosine resistance and stress response in Leishmania donovani

Genes codificadores de proteínas implicadas na relação de espécies do gênero <i>Trypanosoma</i> com seus hospedeiros: diversidade, transferência horizontal e relações filogenéticas.

Contact Info

Product

Resources

About