Abstract:All eukaryotic genomes encode multiple members of the heat shock protein 70 (HSP70) family, which evolved distinctive structural and functional features in response to specific environmental constraints. Phylogenetic analysis of this protein family thus can inform on genetic and molecular mechanisms that drive species-specific environmental adaptation. Here we use the eukaryotic pathogen Leishmania spp. as a model system to investigate the evolution of the HSP70 protein family in an early-branching eukaryote t… Show more
“…Another gene—today annotated as cysteine leucine-rich protein (CLrP, LinJ.34.0570)—was revealed causing antimony resistance in L. tarentolae transfected with a cosmid library prepared from arsenite and SbIII resistant parasites (Brochu et al, 2004 ), and in L. infantum axenic amastigotes (Genest et al, 2008 ). Brochu et al also reported members of the HSP70 protein family as important genes contributing to antimony tolerance, supporting recent phylogenetic evidence that HSP70 family members may allow parasite environmental adaptation with potential important consequences for drug susceptibility (Drini et al, 2016 ).…”
Section: Cosmid-based Functional Genetic Screening In
Leishmentioning
Leishmania biomarker discovery remains an important challenge that needs to be revisited in light of our increasing knowledge on parasite-specific biology, notably its genome instability. In the absence of classical transcriptional regulation in these early-branching eukaryotes, fluctuations in transcript abundance can be generated by gene and chromosome amplifications, which have been linked to parasite phenotypic variability with respect to virulence, tissue tropism, and drug resistance. Conducting in vitro evolutionary experiments to study mechanisms of Leishmania environmental adaptation, we recently validated the link between parasite genetic amplification and fitness gain, thus defining gene and chromosome copy number variations (CNVs) as important Leishmania biomarkers. These experiments also demonstrated that long-term Leishmania culture adaptation can strongly interfere with epidemiologically relevant, genetic signals, which challenges current protocols for biomarker discovery, all of which rely on in vitro expansion of clinical isolates. Here we propose an experimental framework independent of long-term culture termed “reverse” epidemiology, which applies established protocols for functional genetic screening of cosmid-transfected parasites in animal models for the identification of clinically relevant genetic loci that then inform targeted field studies for their validation as Leishmania biomarkers.
“…Another gene—today annotated as cysteine leucine-rich protein (CLrP, LinJ.34.0570)—was revealed causing antimony resistance in L. tarentolae transfected with a cosmid library prepared from arsenite and SbIII resistant parasites (Brochu et al, 2004 ), and in L. infantum axenic amastigotes (Genest et al, 2008 ). Brochu et al also reported members of the HSP70 protein family as important genes contributing to antimony tolerance, supporting recent phylogenetic evidence that HSP70 family members may allow parasite environmental adaptation with potential important consequences for drug susceptibility (Drini et al, 2016 ).…”
Section: Cosmid-based Functional Genetic Screening In
Leishmentioning
Leishmania biomarker discovery remains an important challenge that needs to be revisited in light of our increasing knowledge on parasite-specific biology, notably its genome instability. In the absence of classical transcriptional regulation in these early-branching eukaryotes, fluctuations in transcript abundance can be generated by gene and chromosome amplifications, which have been linked to parasite phenotypic variability with respect to virulence, tissue tropism, and drug resistance. Conducting in vitro evolutionary experiments to study mechanisms of Leishmania environmental adaptation, we recently validated the link between parasite genetic amplification and fitness gain, thus defining gene and chromosome copy number variations (CNVs) as important Leishmania biomarkers. These experiments also demonstrated that long-term Leishmania culture adaptation can strongly interfere with epidemiologically relevant, genetic signals, which challenges current protocols for biomarker discovery, all of which rely on in vitro expansion of clinical isolates. Here we propose an experimental framework independent of long-term culture termed “reverse” epidemiology, which applies established protocols for functional genetic screening of cosmid-transfected parasites in animal models for the identification of clinically relevant genetic loci that then inform targeted field studies for their validation as Leishmania biomarkers.
“…Both spots were identified as LinJ30.2480 (Tritryp gene ID). This gene is a putative heat-shock 70-related protein 1 (mitochondrial precursor) with a predicted MW of 71.6 kDa (designated as HSPA9B [10]).Fig. 12D-DIGE quantitative analysis of miltefosine-resistant L. donovani .…”
Section: Resultsmentioning
confidence: 99%
“…Members of the HSP70 family localized in the mitochondria, such as HSPA9B, are most similar to the bacterial DnaK [13]. 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%
“…Whole protein extracts from L. donovani HePC resistant promastigotes were analyzed via 2D-DIGE and a mitochondrial isoform of HSP70 (LinJ.30.2480), referred to as HSPA9B [10], was identified. Heat-shock proteins (HSP) are highly conserved and constitutively expressed molecules whose chief functions are to facilitate protein synthesis and folding.…”
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.
“…Several heat-shock protein (HSP) chaperones and co-chaperones have been described in kinetoplastids and classified according to molecular mass as sHSP (small HSP), p23, Hsp40, Hsp60, Hsp70, Hsp90 and Hsp110 [ 10 – 15 ]. The sHSP family consists of heterogeneous members characterized by a molecular signature, the conserved α-crystallin domain (ACD), flanked by a variable N and C-terminal.…”
Small Heat-Shock Proteins (sHSPs) and other proteins bearing alpha-crystallin domains (ACD) participate in defense against heat and oxidative stress and play important roles in cell cycle, cytoskeleton dynamics, and immunological and pathological mechanisms in eukaryotes. However, little is known about these proteins in early-diverging lineages of protists such as the kinetoplastids. Here, ACD-like proteins (ACDp) were investigated in genomes of 61 species of 12 kinetoplastid genera, including Trypanosoma spp. (23 species of mammals, reptiles and frogs), Leishmania spp. (mammals and lizards), trypanosomatids of insects, Phytomonas spp. of plants, and bodonids. Comparison of ACDps based on domain architecture, predicted tertiary structure, phylogeny and genome organization reveals a kinetoplastid evolutionarily conserved repertoire, which diversified prior to trypanosomatid adaptation to parasitic life. We identified 9 ACDp orthologs classified in 8 families of TryACD: four previously recognized (HSP20, Tryp23A, Tryp23B and ATOM69), and four characterized for the first time in kinetoplastids (TryACDP, TrySGT1, TryDYX1C1 and TryNudC). A single copy of each ortholog was identified in each genome alongside TryNudC1/TrypNudC2 homologs and, overall, ACDPs were under strong selection pressures at main phylogenetic lineages. Transcripts of all ACDPs were identified across the life stages of T. cruzi, T. brucei and Leishmania spp., but proteomic profiles suggested that most ACDPs may be species- and stage-regulated. Our findings establish the basis for functional studies, and provided evolutionary and structural support for an underestimated repertoire of ACDps in the kinetoplastids.
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