RESUMO
Despite the overall decline in malaria cases in Thailand, continuous surveillance in endemic areas remains crucial. This retrospective analysis examined Plasmodium falciparum samples from Tak province, Thailand, collected in 1998, 1999, and 2001, to investigate the prevalence and evolution of antimalarial genotypic drug resistance. The study revealed a high prevalence of drug-resistant P. falciparum, particularly to mefloquine and sulfadoxine/pyrimethamine, with significant mutations in genes associated with resistance. Notably, mutations indicative of artemisinin resistance, such as those in the kelch13 gene, were detected at low frequencies, suggesting an evolving resistance pattern. The underlying cause of these resistance mutations appears to be the historical and widespread use of these antimalarial drugs, which exerted selective pressure on the parasite population. These findings underscore the necessity of ongoing surveillance and adaptive control strategies to manage drug resistance, guide treatment policies, and prevent potential outbreaks, even as malaria cases decrease. Continuous monitoring and research are imperative to sustain malaria elimination efforts and address the dynamic challenges posed by evolving drug-resistant strains.
RESUMO
While the Plasmodium falciparum malaria parasite continues to cause severe disease globally, Mozambique is disproportionally represented in malaria case totals. Acquisition of copy number variations (CNVs) in the parasite genome contributes to antimalarial drug resistance through overexpression of drug targets. Of interest, piperaquine resistance is associated with plasmepsin 2 and 3 CNVs (pfpmp2 and pfpmp3, respectively), while CNVs in the multidrug efflux pump, multidrug resistance-1 (pfmdr1), increase resistance to amodiaquine and lumefantrine. These antimalarials are partner drugs in artemisinin combination therapies (ACTs) and therefore, CNV detection with accurate and efficient tools is necessary to track ACT resistance risk. Here, we evaluated ~300 clinically derived samples collected from three sites in Mozambique for resistance-associated CNVs. We developed a novel, medium-throughput, quadruplex droplet digital PCR (ddPCR) assay to simultaneously quantify the copy number of pfpmp3, pfpmp2, and pfmdr1 loci in these clinical samples. By using DNA from laboratory parasite lines, we show that this nanodroplet-based method is capable of detecting picogram levels of parasite DNA, which facilitates its application for low yield and human host-contaminated clinical surveillance samples. Following ddPCR and the application of quality control standards, we detected CNVs in 13 of 229 high-quality samples (prevalence of 5.7%). Overall, our study revealed a low number of resistance CNVs present in the parasite population across all three collection sites, including various combinations of pfmdr1, pfpmp2, and pfpmp3 CNVs. The potential for future ACT resistance across Mozambique emphasizes the need for continued molecular surveillance across the region.
Assuntos
Antimaláricos , Variações do Número de Cópias de DNA , Resistência a Medicamentos , Malária Falciparum , Plasmodium falciparum , Proteínas de Protozoários , Antimaláricos/farmacologia , Moçambique , Plasmodium falciparum/genética , Plasmodium falciparum/efeitos dos fármacos , Humanos , Resistência a Medicamentos/genética , Variações do Número de Cópias de DNA/genética , Malária Falciparum/parasitologia , Malária Falciparum/tratamento farmacológico , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase/métodos , Quinolinas/farmacologia , Amodiaquina/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Ácido Aspártico Endopeptidases/genética , Artemisininas/farmacologia , Lumefantrina/farmacologia , PiperazinasRESUMO
Protozoan parasites cause diverse diseases with large global impacts. Research on the pathogenesis and biology of these organisms is limited by economic and experimental constraints. Accordingly, studies of one parasite are frequently extrapolated to infer knowledge about another parasite, across and within genera. Model in vitro or in vivo systems are frequently used to enhance experimental manipulability, but these systems generally use species related to, yet distinct from, the clinically relevant causal pathogen. Characterization of functional differences among parasite species is confined to post hoc or single target studies, limiting the utility of this extrapolation approach. To address this challenge and to accelerate parasitology research broadly, we present a functional comparative analysis of 192 genomes, representing every high-quality, publicly-available protozoan parasite genome including Plasmodium, Toxoplasma, Cryptosporidium, Entamoeba, Trypanosoma, Leishmania, Giardia, and other species. We generated an automated metabolic network reconstruction pipeline optimized for eukaryotic organisms. These metabolic network reconstructions serve as biochemical knowledgebases for each parasite, enabling qualitative and quantitative comparisons of metabolic behavior across parasites. We identified putative differences in gene essentiality and pathway utilization to facilitate the comparison of experimental findings and discovered that phylogeny is not the sole predictor of metabolic similarity. This knowledgebase represents the largest collection of genome-scale metabolic models for both pathogens and eukaryotes; with this resource, we can predict species-specific functions, contextualize experimental results, and optimize selection of experimental systems for fastidious species.
Assuntos
Criptosporidiose , Cryptosporidium , Parasitos , Plasmodium , Animais , Criptosporidiose/genética , Cryptosporidium/genética , Eucariotos/genética , Genoma de Protozoário/genética , Parasitos/genética , Plasmodium/genéticaRESUMO
Extrachromosomal (ec) DNAs are genetic elements that exist separately from the genome. Since ecDNA can carry beneficial genes, they are a powerful adaptive mechanism in cancers and many pathogens. For the first time, we report ecDNA contributing to antimalarial resistance in Plasmodium falciparum, the most virulent human malaria parasite. Using pulse field gel electrophoresis combined with PCR-based copy number analysis, we detected two ecDNA elements that differ in migration and structure. Entrapment in the electrophoresis well and low susceptibility to exonucleases revealed that the biologically relevant ecDNA element is large and complex in structure. Using deep sequencing, we show that ecDNA originates from the chromosome and expansion of an ecDNA-specific sequence may improve its segregation or expression. We speculate that ecDNA is maintained using established mechanisms due to shared characteristics with the mitochondrial genome. Implications of ecDNA discovery in this organism are wide-reaching due to the potential for new strategies to target resistance development.
Assuntos
Resistência a Medicamentos/genética , Genoma de Protozoário , Malária Falciparum/prevenção & controle , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Adaptação Fisiológica , Antimaláricos/farmacologia , DNA de Protozoário , Amplificação de Genes , Humanos , Pirimidinas/farmacologiaRESUMO
BACKGROUND: Malaria remains a global health concern and is endemic in Limpopo, Mpumalanga and KwaZulu Natal Provinces of South Africa, which aims to eliminate malaria by 2025. Community engagement plays a significant role in improving the acceptability and effectiveness of programmes aimed at reducing malaria transmission. The success of such intervention efforts depends on the knowledge, attitudes and practices (KAP) of the community, and understanding the KAP of community residents may support malaria control efforts in the locality. In this context, a cross-sectional household survey to assess community KAP on malaria transmission and prevention in the Ha-Lambani village, Vhembe District, Limpopo Province was conducted. METHODS: Data were collected between November 2018 and May 2019 by questionnaire of 261 consenting adults (213 females and 48 males, aged between 18 and 95 years) selected from different households. Also, a focus group discussion among 13 randomly selected participants was conducted. Pearson's Chi Square test was used to determine statistical differences by village. RESULTS: Study participants (100%, 261/261) were aware of the presence of malaria in their community and 95% associated it with mosquito bites. The local health clinic was the most prominent source of malaria information (85%). Only 22% correctly identified headache, chills and fever as the three most common symptoms of malaria. The majority of participants (98%) knew that effective medication for malaria is available and had a positive treatment-seeking behaviour. Knowledge of malaria prevention measures was high (82%); contrarily, 97% of respondents did not sleep under a bed net the previous night. The focus group data concurred with these results and also revealed that poor bed net use resulted from lack of access to bed nets because community residents could not afford them. CONCLUSIONS: The study demonstrates that participants have appropriate knowledge about malaria transmission and a positive treatment-seeking behaviour. However, economic barriers are responsible for the inadequate use of bed nets. Therefore, distribution of bed nets to the community should be considered to improve practice of malaria prevention measures. Furthermore, knowledge of signs and symptoms and appropriate malaria treatment was limited, and initiatives to improve awareness on these topics should be continued.
Assuntos
Conhecimentos, Atitudes e Prática em Saúde , Malária/psicologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Feminino , Humanos , Malária/prevenção & controle , Malária/transmissão , Masculino , Pessoa de Meia-Idade , Fatores Socioeconômicos , África do Sul , Adulto JovemRESUMO
BACKGROUND: Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries. METHODS: Quantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-Gene Q instrument was utilized to assess the impact of different PCR-HRM reagent mixes and the detection of mixed haplotypes present in the clinical samples. Finally, the prevalence of the wild type (CVMNK) and resistant pfcrt haplotypes (CVIET and SVMNT) was evaluated in this understudied Southwestern region of Uganda. RESULTS: The sample source (i.e. Giemsa-stained slides or blood spots) and type of LCGreen-based reagent mixes did not impact the success of PCR-HRM. The detection limit of 10- 5 ng and the ability to identify mixed haplotypes as low as 10 % was similar to other HRM platforms. The CVIET haplotype predominated in the clinical samples (66 %, 162/244); however, there was a large regional variation between the sample groups (94 % CVIET in Group 1 and 44 % CVIET in Group 2). CONCLUSIONS: The HRM-based method exhibits the flexibility required to conduct reliable assessment of resistance alleles from various sample types generated during the clinical management of malaria. Large regional variations in CQ resistance haplotypes across Southwestern Uganda emphasizes the need for continued local parasite genotype assessment to inform anti-malarial treatment policies.
Assuntos
Antimaláricos/farmacologia , Haplótipos , Malária Falciparum/prevenção & controle , Proteínas de Membrana Transportadoras/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Pré-Escolar , Resistência a Medicamentos/genética , Genótipo , Humanos , Lactente , Desnaturação de Ácido Nucleico , Plasmodium falciparum/efeitos dos fármacos , UgandaRESUMO
Antimalarial resistance is a major obstacle in the eradication of the human malaria parasite, Plasmodium falciparum. Genome amplifications, a type of DNA copy number variation (CNV), facilitate overexpression of drug targets and contribute to parasite survival. Long monomeric A/T tracks are found at the breakpoints of many Plasmodium resistance-conferring CNVs. We hypothesize that other proximal sequence features, such as DNA hairpins, act with A/T tracks to trigger CNV formation. By adapting a sequence analysis pipeline to investigate previously reported CNVs, we identified breakpoints in 35 parasite clones with near single base-pair resolution. Using parental genome sequence, we predicted the formation of stable hairpins within close proximity to all future breakpoint locations. Especially stable hairpins were predicted to form near five shared breakpoints, establishing that the initiating event could have occurred at these sites. Further in-depth analyses defined characteristics of these 'trigger sites' across the genome and detected signatures of error-prone repair pathways at the breakpoints. We propose that these two genomic signals form the initial lesion (hairpins) and facilitate microhomology-mediated repair (A/T tracks) that lead to CNV formation across this highly repetitive genome. Targeting these repair pathways in P. falciparum may be used to block adaptation to antimalarial drugs.
Assuntos
DNA/genética , Genômica , Plasmodium falciparum/genética , Análise de Sequência de DNA/métodos , DNA/química , Variações do Número de Cópias de DNA , Genoma de Protozoário/genética , Humanos , Malária Falciparum/parasitologia , Conformação de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
BACKGROUND: Malaria is a major global health problem, with the Plasmodium falciparum protozoan parasite causing the most severe form of the disease. Prevalence of drug-resistant P. falciparum highlights the need to understand the biology of resistance and to identify novel combination therapies that are effective against resistant parasites. Resistance has compromised the therapeutic use of many antimalarial drugs, including chloroquine, and limited our ability to treat malaria across the world. Fortunately, chloroquine resistance comes at a fitness cost to the parasite; this can be leveraged in developing combination therapies or to reinstate use of chloroquine. RESULTS: To understand biological changes induced by chloroquine treatment, we compared transcriptomics data from chloroquine-resistant parasites in the presence or absence of the drug. Using both linear models and a genome-scale metabolic network reconstruction of the parasite to interpret the expression data, we identified targetable pathways in resistant parasites. This study identified an increased importance of lipid synthesis, glutathione production/cycling, isoprenoids biosynthesis, and folate metabolism in response to chloroquine. CONCLUSIONS: We identified potential drug targets for chloroquine combination therapies. Significantly, our analysis predicts that the combination of chloroquine and sulfadoxine-pyrimethamine or fosmidomycin may be more effective against chloroquine-resistant parasites than either drug alone; further studies will explore the use of these drugs as chloroquine resistance blockers. Additional metabolic weaknesses were found in glutathione generation and lipid synthesis during chloroquine treatment. These processes could be targeted with novel inhibitors to reduce parasite growth and reduce the burden of malaria infections. Thus, we identified metabolic weaknesses of chloroquine-resistant parasites and propose targeted chloroquine combination therapies.
Assuntos
Cloroquina/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Malária Falciparum/parasitologia , Parasitos/efeitos dos fármacos , Animais , Antimaláricos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Quimioterapia Combinada , Ácido Fólico/metabolismo , Humanos , Plasmodium falciparum/efeitos dos fármacos , Terpenos/metabolismoRESUMO
Microscopic diagnosis of malaria using Giemsa-stained blood smears is the standard of care in resource-limited settings. These smears represent a potential source of DNA for PCR testing to confirm Plasmodium infections or for epidemiological studies of archived samples. Therefore, we assessed the use of DNA extracts from stained blood smears for the detection of Plasmodium species using real-time PCR. We extracted DNA from archived blood smears and corresponding red blood cell pellets collected from asymptomatic children in southwestern Uganda in 2010. We then performed real-time PCR followed by high-resolution melting (HRM) to identify Plasmodium species, and we compared our results to those of microscopy. We analyzed a total of 367 blood smears and corresponding red blood cell pellets, including 185 smears (50.4%) that were positive by microscopy. Compared to microscopy, PCR-HRM analysis of smear DNA had a sensitivity of 93.0% (95% confidence interval [CI], 88.2 to 96.2%) and a specificity of 96.7% (95% CI, 93.0 to 98.8%), and PCR-HRM analysis of pellet DNA had a sensitivity of 100.0% (95% CI, 98.0 to 100.0%) and a specificity of 94.0% (95% CI, 89.4 to 96.9%). Identification of positive PCR-HRM results to the species level revealed Plasmodium falciparum (92.0%), Plasmodium ovale (5.6%), and Plasmodium malariae (2.4%). PCR-HRM analysis of DNA extracts from Giemsa-stained thick blood smears or corresponding blood pellets had high sensitivity and specificity for malaria diagnosis, compared to microscopy. Therefore, blood smears can provide an adequate source of DNA for confirmation of Plasmodium species infections and can be used for retrospective genetic studies.
Assuntos
Malária/sangue , Malária/parasitologia , Tipagem Molecular/métodos , Plasmodium/classificação , Plasmodium/genética , DNA de Protozoário/genética , Técnicas Genéticas , Malária/diagnóstico , Técnicas de Amplificação de Ácido Nucleico , Plasmodium/isolamento & purificação , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Sensibilidade e Especificidade , UgandaRESUMO
BACKGROUND: Malaria remains a major public health burden and resistance has emerged to every antimalarial on the market, including the frontline drug, artemisinin. Our limited understanding of Plasmodium biology hinders the elucidation of resistance mechanisms. In this regard, systems biology approaches can facilitate the integration of existing experimental knowledge and further understanding of these mechanisms. RESULTS: Here, we developed a novel genome-scale metabolic network reconstruction, iPfal17, of the asexual blood-stage P. falciparum parasite to expand our understanding of metabolic changes that support resistance. We identified 11 metabolic tasks to evaluate iPfal17 performance. Flux balance analysis and simulation of gene knockouts and enzyme inhibition predict candidate drug targets unique to resistant parasites. Moreover, integration of clinical parasite transcriptomes into the iPfal17 reconstruction reveals patterns associated with antimalarial resistance. These results predict that artemisinin sensitive and resistant parasites differentially utilize scavenging and biosynthetic pathways for multiple essential metabolites, including folate and polyamines. Our findings are consistent with experimental literature, while generating novel hypotheses about artemisinin resistance and parasite biology. We detect evidence that resistant parasites maintain greater metabolic flexibility, perhaps representing an incomplete transition to the metabolic state most appropriate for nutrient-rich blood. CONCLUSION: Using this systems biology approach, we identify metabolic shifts that arise with or in support of the resistant phenotype. This perspective allows us to more productively analyze and interpret clinical expression data for the identification of candidate drug targets for the treatment of resistant parasites.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos , Análise do Fluxo Metabólico , Redes e Vias Metabólicas , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Biologia de Sistemas , Biomassa , Resistência a Medicamentos/genética , Perfilação da Expressão Gênica , Metabolômica , Plasmodium falciparum/genéticaRESUMO
Antimalarial drug resistance exacerbates the global disease burden and complicates eradication efforts. To facilitate the surveillance of resistance markers in countries of malaria endemicity, we developed a suite of TaqMan assays for known resistance markers and compartmentalized them into a single array card (TaqMan array card, TAC). We included 87 assays for species identification, for the detection of Plasmodium falciparum mutations associated with chloroquine, atovaquone, pyrimethamine, sulfadoxine, and artemisinin resistance, and for neutral single nucleotide polymorphism (SNP) genotyping. Assay performance was first optimized using DNA from common laboratory parasite lines and plasmid controls. The limit of detection was 0.1 to 10 pg of DNA and yielded 100% accuracy compared to sequencing. The tool was then evaluated on 87 clinical blood samples from around the world, and the malaria TAC once again achieved 100% accuracy compared to sequencing and in addition detected the presence of mixed infections in clinical samples. With its streamlined protocol and high accuracy, this malaria TAC should be a useful tool for large-scale antimalarial resistance surveillance.
Assuntos
Antimaláricos/farmacologia , Monitoramento Epidemiológico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico , Artemisininas/farmacologia , Atovaquona/farmacologia , Cloroquina/farmacologia , Resistência a Medicamentos , Técnicas de Genotipagem , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/classificação , Plasmodium falciparum/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , Pirimetamina/farmacologia , Sulfadoxina/farmacologiaRESUMO
BACKGROUND: Culture-adapted Plasmodium falciparum parasites can offer deeper understanding of geographic variations in drug resistance, pathogenesis and immune evasion. To help ground population-based calculations and inferences from culture-adapted parasites, the complete range of parasites from a study area must be well represented in any collection. To this end, standardized adaptation methods and determinants of successful in vitro adaption were sought. METHODS: Venous blood was collected from 33 P. falciparum-infected individuals at Goa Medical College and Hospital (Bambolim, Goa, India). Culture variables such as whole blood versus washed blood, heat-inactivated plasma versus Albumax, and different starting haematocrit levels were tested on fresh blood samples from patients. In vitro adaptation was considered successful when two four-fold or greater increases in parasitaemia were observed within, at most, 33 days of attempted culture. Subsequently, parasites from the same patients, which were originally cryopreserved following blood draw, were retested for adaptability for 45 days using identical host red blood cells (RBCs) and culture media. RESULTS: At a new endemic area research site, ~65% of tested patient samples, with varied patient history and clinical presentation, were successfully culture-adapted immediately after blood collection. Cultures set up at 1% haematocrit and 0.5% Albumax adapted most rapidly, but no single test condition was uniformly fatal to culture adaptation. Success was not limited by low patient parasitaemia nor by patient age. Some parasites emerged even after significant delays in sample processing and even after initiation of treatment with anti-malarials. When 'day 0' cryopreserved samples were retested in parallel many months later using identical host RBCs and media, speed to adaptation appeared to be an intrinsic property of the parasites collected from individual patients. CONCLUSIONS: Culture adaptation of P. falciparum in a field setting is formally shown to be robust. Parasites were found to have intrinsic variations in adaptability to culture conditions, with some lines requiring longer attempt periods for successful adaptation. Quantitative approaches described here can help describe phenotypic diversity of field parasite collections with precision. This is expected to improve population-based extrapolations of findings from field-derived fresh culture-adapted parasites to broader questions of public health importance.
Assuntos
Plasmodium falciparum/citologia , Células Cultivadas , Criopreservação , Eritrócitos/parasitologia , Técnicas de Genotipagem , Humanos , Plasmodium falciparum/genéticaRESUMO
Atovaquone is a component of Malarone, a widely prescribed antimalarial combination, that targets malaria respiration. Here we show that parasites with high-level resistance to an inhibitor of dihydroorotate dehydrogenase demonstrate unexpected atovaquone tolerance. Fortunately, the tolerance is diminished with proguanil, the second partner in Malarone. It is important to understand such "genetic cross talk" between respiration and pyrimidine biosynthesis since many antimalarial drug development programs target these two seemingly independent pathways.
Assuntos
Antimaláricos/farmacologia , Atovaquona/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Combinação de Medicamentos , Resistência a Medicamentos , Testes de Sensibilidade Parasitária , Proguanil/farmacologiaRESUMO
Malaria drug resistance contributes to up to a million annual deaths. Judicious deployment of new antimalarials and vaccines could benefit from an understanding of early molecular events that promote the evolution of parasites. Continuous in vitro challenge of Plasmodium falciparum parasites with a novel dihydroorotate dehydrogenase (DHODH) inhibitor reproducibly selected for resistant parasites. Genome-wide analysis of independently-derived resistant clones revealed a two-step strategy to evolutionary success. Some haploid blood-stage parasites first survive antimalarial pressure through fortuitous DNA duplications that always included the DHODH gene. Independently-selected parasites had different sized amplification units but they were always flanked by distant A/T tracks. Higher level amplification and resistance was attained using a second, more efficient and more accurate, mechanism for head-to-tail expansion of the founder unit. This second homology-based process could faithfully tune DNA copy numbers in either direction, always retaining the unique DNA amplification sequence from the original A/T-mediated duplication for that parasite line. Pseudo-polyploidy at relevant genomic loci sets the stage for gaining additional mutations at the locus of interest. Overall, we reveal a population-based genomic strategy for mutagenesis that operates in human stages of P. falciparum to efficiently yield resistance-causing genetic changes at the correct locus in a successful parasite. Importantly, these founding events arise with precision; no other new amplifications are seen in the resistant haploid blood stage parasite. This minimizes the need for meiotic genetic cleansing that can only occur in sexual stage development of the parasite in mosquitoes.
Assuntos
DNA de Protozoário , Resistência a Medicamentos/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Plasmodium falciparum , Ploidias , Proteínas de Protozoários , Animais , Culicidae/parasitologia , DNA de Protozoário/biossíntese , DNA de Protozoário/genética , Di-Hidro-Orotato Desidrogenase , Loci Gênicos/genética , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoAssuntos
Malária , Administração Massiva de Medicamentos , África , Artemisininas , Gâmbia , Humanos , Estudos Prospectivos , QuinolinasRESUMO
Mounting evidence demonstrates that nutritional environment can alter pathogen drug sensitivity. While the rich media used for in vitro culture contains supraphysiological nutrient concentrations, pathogens encounter a relatively restrictive environment in vivo. We assessed the effect of nutrient limitation on the protozoan parasite that causes malaria and demonstrated that short-term growth under physiologically relevant mild nutrient stress (or "metabolic priming") triggers increased tolerance of a potent antimalarial drug. We observed beneficial effects using both short-term survival assays and longer-term proliferation studies, where metabolic priming increases parasite survival to a level previously defined as resistant (>1% survival). We performed these assessments by either decreasing single nutrients that have distinct roles in metabolism or using a media formulation that simulates the human plasma environment. We determined that priming-induced tolerance was restricted to parasites that had newly invaded the host red blood cell, but the effect was not dependent on genetic background. The molecular mechanisms of this intrinsic effect mimic aspects of genetic tolerance, including translational repression and protein export. This finding suggests that regardless of the impact on survival rates, environmental stress could stimulate changes that ultimately directly contribute to drug tolerance. Because metabolic stress is likely to occur more frequently in vivo compared to the stable in vitro environment, priming-induced drug tolerance has ramifications for how in vitro results translate to in vivo studies. Improving our understanding of how pathogens adjust their metabolism to impact survival of current and future drugs is an important avenue of research to slow the evolution of resistance. IMPORTANCE There is a dire need for effective treatments against microbial pathogens. Yet, the continuing emergence of drug resistance necessitates a deeper knowledge of how pathogens respond to treatments. We have long appreciated the contribution of genetic evolution to drug resistance, but transient metabolic changes that arise in response to environmental factors are less recognized. Here, we demonstrate that short-term growth of malaria parasites in a nutrient-limiting environment triggers cellular changes that lead to better survival of drug treatment. We found that these strategies are similar to those employed by drug-tolerant parasites, which suggests that starvation "primes" parasites to survive and potentially evolve resistance. Since the environment of the human host is relatively nutrient restrictive compared to growth conditions in standard laboratory culture, this discovery highlights the important connections among nutrient levels, protective cellular pathways, and resistance evolution.
Assuntos
Antimaláricos , Artemisininas , Malária , Humanos , Plasmodium falciparum/metabolismo , Artemisininas/metabolismo , Malária/tratamento farmacológico , Antimaláricos/farmacologia , Tolerância a Medicamentos , Resistência a Medicamentos , NutrientesRESUMO
Increases in the copy number of large genomic regions, termed genome amplification, are an important adaptive strategy for malaria parasites. Numerous amplifications across the Plasmodium falciparum genome contribute directly to drug resistance or impact the fitness of this protozoan parasite. During the characterization of parasite lines with amplifications of the dihydroorotate dehydrogenase (DHODH) gene, we detected increased copies of an additional genomic region that encompassed 3 genes (~5 kb) including GTP cyclohydrolase I (GCH1 amplicon). While this gene is reported to increase the fitness of antifolate resistant parasites, GCH1 amplicons had not previously been implicated in any other antimalarial resistance context. Here, we further explored the association between GCH1 and DHODH copy number. Using long read sequencing and single read visualization, we directly observed a higher number of tandem GCH1 amplicons in parasites with increased DHODH copies (up to 9 amplicons) compared to parental parasites (3 amplicons). While all GCH1 amplicons shared a consistent structure, expansions arose in 2-unit steps (from 3 to 5 to 7, etc copies). Adaptive evolution of DHODH and GCH1 loci was further bolstered when we evaluated prior selection experiments; DHODH amplification was only successful in parasite lines with pre-existing GCH1 amplicons. These observations, combined with the direct connection between metabolic pathways that contain these enzymes, lead us to propose that the GCH1 locus is beneficial for the fitness of parasites exposed to DHODH inhibitors. This finding highlights the importance of studying variation within individual parasite genomes as well as biochemical connections of drug targets as novel antimalarials move towards clinical approval.
RESUMO
Like other eukaryotes, trypanosomes have an essential type II fatty acid synthase in their mitochondrion. We have investigated the function of this synthase in bloodstream-form parasites by studying the effect of a conditional knockout of acyl carrier protein (ACP), a key player in this fatty acid synthase pathway. We found that ACP depletion not only caused small changes in cellular phospholipids but also, surprisingly, caused changes in the kinetoplast. This structure, which contains the mitochondrial genome in the form of a giant network of several thousand interlocked DNA rings (kinetoplast DNA [kDNA]), became larger in some cells and smaller or absent in others. We observed the same pattern in isolated networks viewed by either fluorescence or electron microscopy. We found that the changes in kDNA size were not due to the disruption of replication but, instead, to a defect in segregation. kDNA segregation is mediated by the tripartite attachment complex (TAC), and we hypothesize that one of the TAC components, a differentiated region of the mitochondrial double membrane, has an altered phospholipid composition when ACP is depleted. We further speculate that this compositional change affects TAC function, and thus kDNA segregation.
Assuntos
Proteína de Transporte de Acila/deficiência , DNA de Cinetoplasto/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/parasitologia , Proteína de Transporte de Acila/genética , Sangue/parasitologia , DNA de Cinetoplasto/metabolismo , Humanos , Proteínas Mitocondriais/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Single-cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. We present a single-cell sequencing pipeline for an intracellular parasite, Plasmodium falciparum, with a small genome of extreme base content. Through optimization of a quasi-linear amplification method, we target the parasite genome over contaminants and generate coverage levels allowing detection of minor genetic variants. This work, as well as efforts that build on these findings, will enable detection of parasite heterogeneity contributing to P. falciparum adaptation. Furthermore, this study provides a framework for optimizing single-cell amplification and variant analysis in challenging genomes.
Assuntos
Composição de Bases , Genoma de Protozoário , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Plasmodium/genética , Análise de Célula Única , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Eritrócitos/parasitologia , Genômica/métodos , Humanos , Malária/parasitologia , Plasmodium falciparum/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Análise de Célula Única/métodosRESUMO
Research on Plasmodium parasites has driven breakthroughs in reducing malaria morbidity and mortality. Experimental analysis of in vivo/ex vivo versus in vitro samples serve unique roles in Plasmodium research. However, these distinctly different environments lead to discordant biology between parasites in host circulation and those under laboratory cultivation. Here, we review how in vitro factors, such as nutrient levels and physical forces, differ from those in the human host and the resulting implications for parasite growth, survival, and virulence. Additionally, we discuss the current utility of direct-from-host methodologies, which avoid the potentially confounding effects of in vitro cultivation. Finally, we make the case for methodological improvements that will drive research progress of physiologically relevant phenotypes.