RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: For millennia, Artemisia annua L. was used in Southeast Asia to treat "fever". This medicinal plant is effective against multiple pathogens and is used by many global communities as a source of artemisinin derivatives that are first-line drugs to treat malaria caused by Plasmodium parasites. AIM OF THE STUDY: The SARS-CoV-2 (Covid-19) global pandemic has killed millions and evolved numerous variants, with delta being the most transmissible to date and causing break-through infections of vaccinated individuals. We further queried the efficacy of A. annua cultivars against new variants. MATERIALS AND METHODS: Using Vero E6 cells, we measured anti-SARS-CoV-2 activity of dried-leaf hot-water A. annua L. extracts of four cultivars, A3, BUR, MED, and SAM, to determine their efficacy against five infectious variants of the virus: alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and kappa (B.1.617.1). RESULTS: In addition to being effective against the original wild type (WT) WA1, A. annua cultivars A3, BUR, MED, and SAM were also potent against all five variants. IC50 and IC90 values based on measured artemisinin content ranged from 0.3 to 8.4 µM and 1.4-25.0 µM, respectively. The IC50 and IC90 values based on dried leaf weight (DW) used to make the tea infusions ranged from 11.0 to 67.7 µg DW and 59.5-160.6 µg DW, respectively. Cell toxicity was insignificant at a leaf dry weight of ≤50 µg in the extract of any cultivar. CONCLUSIONS: Results suggest that oral consumption of A. annua hot-water extracts (tea infusions) could potentially provide a cost-effective therapy to help stave off the rapid global spread of these variants, buying time for broader implementation of vaccines.
Asunto(s)
Antivirales/farmacología , Artemisia annua/química , COVID-19/virología , Extractos Vegetales/farmacología , SARS-CoV-2/efectos de los fármacos , Animales , Antivirales/química , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Concentración 50 Inhibidora , Extractos Vegetales/química , Células VeroRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. has been used for millennia in Southeast Asia to treat "fever". Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria. AIM OF THE STUDY: SARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2. MATERIALS AND METHODS: Using Vero E6 and Calu-3 cells, we measured anti SARS-CoV-2 activity against fully infectious virus of dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as the concentrations that inhibited viral replication by 50%; CC50s were also calculated and defined as the concentrations that kill 50% of cells. RESULTS: Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 µM, 0.01-0.14 µg, and 23.4-57.4 µg, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet its hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 µM, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 µM. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values. CONCLUSIONS: A. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections.
Asunto(s)
Antivirales/farmacología , Artemisia annua/química , Extractos Vegetales/farmacología , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Artemisininas/farmacología , COVID-19/virología , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Flavonoides/farmacología , Humanos , Hojas de la Planta/química , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Tratamiento Farmacológico de COVID-19RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. has been used for millennia in Southeast Asia to treat "fever". Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria. AIM OF THE STUDY: SARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2. MATERIALS AND METHODS: Using Vero E6 and Calu-3 cells, we measured anti viral activity SARS-CoV-2 activity against fully infectious virusof dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as (the concentrations that inhibited viral replication by 50%.) and CC50s (the concentrations that kill 50% of cells) were calculated. RESULTS: Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 µM, 0.01-0.14 µg, and 23.4-57.4 µg, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet the hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 µM, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 µM. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values. CONCLUSIONS: A. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections.
RESUMEN
Malaria control and elimination are threatened by the emergence and spread of resistance to artemisinin-based combination therapies (ACTs). Experimental evidence suggests that when an artemisinin (ART)-sensitive (K13 wild-type) Plasmodium falciparum strain is exposed to ART derivatives such as dihydroartemisinin (DHA), a small population of the early ring-stage parasites can survive drug treatment by entering cell cycle arrest or dormancy. After drug removal, these parasites can resume growth. Dormancy has been hypothesized to be an adaptive physiological mechanism that has been linked to recrudescence of parasites after monotherapy with ART and, possibly contributes to ART resistance. Here, we evaluate the in vitro drug sensitivity profile of normally-developing P. falciparum ring stages and DHA-pretreated dormant rings (DP-rings) using a panel of antimalarial drugs, including the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. We report that while KDU691 shows no activity against rings, it is highly inhibitory against DP-rings; a drug effect opposite to that of ART. Moreover, we provide evidence that KDU691 also kills DP-rings of P. falciparum ART-resistant strains expressing mutant K13.
Asunto(s)
Antimaláricos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Pirazinas/farmacología , Animales , Artemisininas/farmacología , Resistencia a Medicamentos/efectos de los fármacos , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/patogenicidadRESUMEN
The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72-76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Antimaláricos/farmacología , Cloroquina/farmacología , Proteínas de la Membrana/genética , Plasmodium falciparum/efectos de los fármacos , Polimorfismo Genético , Animales , ADN Protozoario/química , Resistencia a Medicamentos , Genotipo , Humanos , Proteínas de Transporte de Membrana , Papúa Nueva Guinea , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , América del SurRESUMEN
Chloroquine resistance in Plasmodium falciparum has recently been shown to result from mutations in the novel vacuolar transporter, PfCRT. Field studies have demonstrated the importance of these mutations in clinical resistance. Although a pfcrt ortholog has been identified in Plasmodiumvivax, there is no association between in vivo chloroquine resistance and codon mutations in the P. vivax gene. This is consistent with lines of evidence that suggest alternative mechanisms of chloroquine resistance among various malaria parasite species.
Asunto(s)
Antimaláricos/farmacología , Cloroquina/farmacología , Proteínas de la Membrana/genética , Plasmodium falciparum/efectos de los fármacos , Animales , Antimaláricos/uso terapéutico , Cloroquina/uso terapéutico , Resistencia a Medicamentos , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Mutación , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas ProtozoariasRESUMEN
To initiate invasion of the mosquito midgut, Plasmodium ookinetes secrete chitinolytic activity to penetrate the peritrophic matrix surrounding the blood meal. While ookinetes of the avian malaria parasite Plasmodium gallinaceum appear to secrete products of two chitinase genes, to date only one chitinase gene, PfCHT1, has been identified in the nearly completed Plasmodium falciparum strain 3D7 genome database. To test the hypothesis that the single identified chitinase of P. falciparum is necessary for ookinete invasion, the PfCHT1 gene was disrupted 39 bp upstream of the stop codon. PfCHT1-disrupted parasites had normal gametocytogenesis, exflagellation, and ookinete formation but were markedly impaired in their ability to form oocysts in Anopheles freeborni midguts. Confocal microscopy demonstrated that the truncated PfCHT1 protein was present in mutant ookinetes but that the concentration of mutant PfCHT1 within the apical end of the ookinetes was substantially reduced. These data suggest that full-length PfCHT1 is essential for intracellular trafficking and secretion and that the PfCHT1 gene product is necessary for ookinetes to invade the mosquito midgut.
Asunto(s)
Anopheles/parasitología , Eliminación de Gen , Plasmodium falciparum/enzimología , Plasmodium falciparum/patogenicidad , Estómago/parasitología , Animales , Humanos , Malaria Falciparum/parasitología , Microscopía Confocal , Plásmidos , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , VirulenciaRESUMEN
Chloroquine (CQ)-resistant Plasmodium vivax malaria was first reported 12 years ago, nearly 30 years after the recognition of CQ-resistant P. falciparum. Loss of CQ efficacy now poses a severe problem for the prevention and treatment of both diseases. Mutations in a digestive vacuole protein encoded by a 13-exon gene, pfcrt, were shown recently to have a central role in the CQ resistance (CQR) of P. falciparum. Whether mutations in pfcrt orthologues of other Plasmodium species are involved in CQR remains an open question. This report describes pfcrt homologues from P. vivax, P. knowlesi, P. berghei, and Dictyostelium discoideum. Synteny between the P. falciparum and P. vivax genes is demonstrated. However, a survey of patient isolates and monkey-adapted lines has shown no association between in vivo CQR and codon mutations in the P. vivax gene. This is evidence that the molecular events underlying P. vivax CQR differ from those in P. falciparum.
Asunto(s)
Cloroquina/farmacología , Chaperonas Moleculares/genética , Plasmodium/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Codón , Dictyostelium/química , Dictyostelium/genética , Resistencia a Medicamentos , Humanos , Datos de Secuencia Molecular , Mutación , Pruebas de Sensibilidad Parasitaria , Plasmodium/química , Plasmodium/genética , Alineación de SecuenciaRESUMEN
A novel Plasmodium falciparum gene, MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P. falciparum-infected and irradiated mosquitoes. The single-exon, single-copy MB2 gene is predicted to encode a protein with an M(r) of 187,000. The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2. MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes. The MB2 protein is distributed as a approximately 120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a approximately 66-kDa species. Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of the MB2 protein. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target.
Asunto(s)
Genes Protozoarios , Plasmodium falciparum/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Alineación de SecuenciaRESUMEN
BACKGROUND: Chloroquine-resistant Plasmodium falciparum malaria is a major health problem, particularly in sub-Saharan Africa. Chloroquine resistance has been associated in vitro with point mutations in two genes, pfcrt and pfmdr 1, which encode the P. falciparum digestive-vacuole transmembrane proteins PfCRT and Pgh1, respectively. METHODS: To assess the value of these mutations as markers for clinical chloroquine resistance, we measured the association between the mutations and the response to chloroquine treatment in patients with uncomplicated falciparum malaria in Mali. The frequencies of the mutations in patients before and after treatment were compared for evidence of selection of resistance factors as a result of exposure to chloroquine. RESULTS: The pfcrt mutation resulting in the substitution of threonine (T76) for lysine at position 76 was present in all 60 samples from patients with chloroquine-resistant infections (those that persisted or recurred after treatment), as compared with a base-line prevalence of 41 percent in samples obtained before treatment from 116 randomly selected patients (P<0.001), indicating absolute selection for this mutation. The pfmdr 1 mutation resulting in the substitution of tyrosine for asparagine at position 86 was also selected for, since it was present in 48 of 56 post-treatment samples from patients with chloroquine-resistant infections (86 percent), as compared with a base-line prevalence of 50 percent in 115 samples obtained before treatment (P<0.001). The presence of pfcrt T76 was more strongly associated with the development of chloroquine resistance (odds ratio, 18.8; 95 percent confidence interval, 6.5 to 58.3) than was the presence of pfmdr 1 Y86 (odds ratio, 3.2; 95 percent confidence interval, 1.5 to 6.8) or the presence of both mutations (odds ratio, 9.8; 95 percent confidence interval, 4.4 to 22.1). CONCLUSIONS: This study shows an association between the pfcrt T76 mutation in P. falciparum and the development of chloroquine resistance during the treatment of malaria. This mutation can be used as a marker in surveillance for chloroquine-resistant falciparum malaria.
Asunto(s)
Cloroquina/uso terapéutico , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/genética , Mutación Puntual , Adulto , Factores de Edad , Animales , Niño , Cloroquina/farmacología , Análisis Mutacional de ADN , Resistencia a Medicamentos/genética , Marcadores Genéticos , Humanos , Modelos Logísticos , Malaria Falciparum/parasitología , Plasmodium falciparum/efectos de los fármacos , Valor Predictivo de las Pruebas , Prevalencia , Selección Genética , Resultado del TratamientoRESUMEN
The determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K761 mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.
Asunto(s)
Cloroquina/farmacología , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Vacuolas/fisiología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Sistema Digestivo/metabolismo , Resistencia a Medicamentos , Exones , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plasmodium falciparum/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Transfección , Verapamilo/farmacologíaRESUMEN
The determinant of chloroquine resistance (CQR) in a Plasmodium falciparum cross was previously mapped by linkage analysis to a 36 kb segment of chromosome 7. Candidate genes within this segment have been previously shown to include two genes, cg2 and cg1, that have complex polymorphisms linked to the CQR phenotype. Using DNA transfection and allelic exchange, we have replaced these polymorphisms in CQR parasites with cg2 and cg1 sequences from chloroquine sensitive parasites. Drug assays of the allelically-modified lines show no change in the degree of CQR, providing evidence against the hypothesis that these polymorphisms are important to the CQR phenotype. Similarly, no change was found in the degree to which verapamil or other chloroquine sensitizers reverse CQR in the transformants. These results and the high though not complete degree of association of CQR with cg2 and cg1 polymorphisms in field isolates suggest involvement of another nearby gene in the P. falciparum CQR mechanism.
Asunto(s)
Alelos , Antimaláricos/farmacología , Cloroquina/farmacología , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/genética , Transformación Genética , Animales , Resistencia a Medicamentos/genética , Genes Protozoarios , Humanos , Malaria/parasitología , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/genética , Polimorfismo Genético , Recombinación Genética , TransfecciónRESUMEN
EBA-175 is a Plasmodium falciparum micronemal protein that binds to sialic acid in the context of the peptide backbone of glycophorin A and has been implicated in sialic acid-dependent invasion of erythrocytes. The existence of an alternative invasion pathway has been suggested by the finding that the P. falciparum clone Dd2/Nm can invade sialic acid-depleted erythrocytes. To study the role of EBA-175 in this alternative pathway, we have generated Dd2/Nm clones expressing a truncated form of EBA-175 that lacks region 6 and the cytoplasmic domain. The protein still appears to be localized to the apical end in the vicinity of the micronemes, suggesting that region 6 and the cytoplasmic domain are not involved in EBA-175 trafficking to the micronemes. In these genetically modified clones, the level of truncated EBA-175 protein expression was greatly reduced. EBA-175-disrupted clones displayed normal rates of invasion of untreated and enzyme-treated human and animal erythrocytes, suggesting a lack of involvement of EBA-175 in this alternative invasion pathway.
Asunto(s)
Antígenos de Protozoos , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Eritrocitos/parasitología , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Southern Blotting , Proteínas Portadoras/genética , Clonación Molecular , Eritrocitos/metabolismo , Humanos , Malaria Falciparum/parasitología , Microscopía Fluorescente , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico/metabolismo , Plásmidos/genética , Pruebas de Precipitina , Proteínas Protozoarias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Transformación GenéticaRESUMEN
We have adapted the "directional tag subtractive hybridization" technique as a means of investigating stage-specific gene expression in Plasmodium falciparum. This technique utilizes unidirectional cDNA libraries cloned into separate lambda vectors and involves hydroxyapatite chromatographic separation of target antisense cDNA and driver sense strand cRNA followed by PCR amplification of cDNA sequences specific to the target stage. This technique enabled efficient subtraction of asexual blood stage sequences from a P. falciparum sporozoite cDNA library and led to identification of novel sporozoite sequences. This technique can be applied to study gene expression in parasite stages that are difficult to obtain routinely.
Asunto(s)
ADN Complementario/química , ADN Protozoario/química , Biblioteca de Genes , Hibridación de Ácido Nucleico/métodos , Plasmodium falciparum/genética , Animales , Anopheles , Regulación de la Expresión Génica , Reacción en Cadena de la PolimerasaRESUMEN
Apicomplexan protozoa possess a family of micronemal and cell surface-associated proteins, each comprised a combination of cell-adhesive vertebrate von Willebrand factor (vWF)-like A domains and thrombospondin (TSP) type 1-like domains. The human malaria parasite Plasmodium falciparum has in the extracellular portion of the CS protein TRAP-related protein (CTRP) six tandemly arrayed A domains followed by seven TSP type 1-like domains, whereas a second member of this family, thrombospondin-related anonymous protein (TRAP), contains a single vWF-like A domain and a single TSP type 1-like domain. Here we show that CTRP transcripts are present within the infected mosquito midgut and that CTRP protein is expressed with a punctate distribution and a predominance at the apical end of mosquito midgut-stage ookinetes. This expression pattern is analogous to micronemal expression of TRAP in Plasmodium sporozoites. Disruption of the CTRP gene by homologous recombination in cultures of the human malaria parasite P. falciparum demonstrates that CTRP is essential for mosquito midgut development. Oocyst formation was never observed following membrane feeds of CTRP disruptant lines to Anopheline mosquitoes, despite the development of mature ookinetes. We propose that CTRP is involved in essential recognition or motility processes at the ookinete cell surface within the mosquito midgut.
Asunto(s)
Anopheles/parasitología , Genes Protozoarios , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/genética , Proteínas Protozoarias , Receptores de Superficie Celular/genética , Animales , Sistema Digestivo/parasitología , Técnica del Anticuerpo Fluorescente , Mutagénesis , Fenotipo , Receptores de Superficie Celular/aislamiento & purificaciónRESUMEN
Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite, Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (hDHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the hDHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of hDHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the hDHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite (CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of hDHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite.
Asunto(s)
Genoma de Protozoos , Plasmodium berghei/genética , Tetrahidrofolato Deshidrogenasa/genética , Animales , Animales Modificados Genéticamente , Antimaláricos/farmacología , Secuencia de Bases , Cartilla de ADN/genética , Resistencia a Medicamentos/genética , Antagonistas del Ácido Fólico/farmacología , Marcadores Genéticos , Humanos , Plásmidos/genética , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/enzimología , Proteínas Protozoarias/genética , Pirimetamina/farmacología , Transfección , Triazinas/farmacologíaRESUMEN
Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC(50) (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.
Asunto(s)
Quitina/metabolismo , Quitinasas/genética , Precursores Enzimáticos/genética , Plasmodium falciparum/enzimología , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Quitinasas/antagonistas & inhibidores , Quitinasas/química , Quitinasas/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/antagonistas & inhibidores , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Expresión Génica , Genes Protozoarios , Humanos , Concentración de Iones de Hidrógeno , Malaria/parasitología , Modelos Moleculares , Datos de Secuencia Molecular , Plasmodium falciparum/genética , Conformación Proteica , Proteínas Protozoarias , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Trisacáridos/farmacologíaRESUMEN
Genome analysis of the Plasmodium falciparum malaria parasite already is identifying genes relevant to therapeutic- and vaccine-related research. The genetic blueprint of P. falciparum will ultimately need to be understood at multiple levels of an integrated system and will provide a detailed account of the life processes of the parasite and of the devastating disease it causes.
Asunto(s)
Genoma de Protozoos , Plasmodium falciparum/genética , Animales , Humanos , Malaria Falciparum/parasitología , Malaria Falciparum/prevención & controlRESUMEN
In Plasmodium-infected mosquitoes, oocysts are preferentially located at the posterior half of the posterior midgut. Because mosquitoes rest vertically after feeding, the effect of gravity on the ingested blood has been proposed as the cause of such a biased distribution. In this paper, we examined the oocyst distribution on the midguts of mosquitoes that were continuously rotated to nullify the effect of gravity and found that the typical pattern of oocyst distribution did not change. Invasion of the midgut epithelium by ookinetes was similarly found to be biased toward the posterior part of the posterior midgut. We examined whether the distribution of oocysts depends on the distribution of vesicular ATPase (V-ATPase)-overexpressing cells that Plasmodium ookinetes preferentially use to cross the midgut epithelium. An antiserum raised against recombinant Aedes aegypti V-ATPase B subunit indicated that the majority of V-ATPase-overexpressing cells in Ae. aegypti and Anopheles gambiae are localized at the posterior part of the posterior midgut. We propose that the typical distribution of oocysts on the mosquito midgut is attributable to the presence and the spatial distribution of the V-ATPase-overexpressing cells in the midgut epithelium.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Aedes/parasitología , Plasmodium gallinaceum/aislamiento & purificación , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Aedes/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Intestinos/parasitología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plasmodium gallinaceum/crecimiento & desarrollo , Homología de Secuencia de AminoácidoRESUMEN
The lack of suitable antimalarial agents to replace chloroquine and pyrimethamine/sulfadoxine threatens efforts to control the spread of drug-resistant strains of the malaria parasite Plasmodium falciparum. Here we describe a transformation system, involving WR99210 selection of parasites transformed with either wild-type or methotrexate-resistant human dihydrofolate reductase (DHFR), that has application for the screening of P. falciparum-specific DHFR inhibitors that are active against drug-resistant parasites. Using this system, we have found that the prophylactic drug cycloguanil has a mode of pharmacological action distinct from the activity of its parent compound proguanil. Complementation assays demonstrate that cycloguanil acts specifically on P. falciparum DHFR and has no other significant target. The target of proguanil itself is separate from DHFR. We propose a strategy of combination chemotherapy incorporating the use of multiple parasite-specific inhibitors that act at the same molecular target and thereby maintain, in combination, their effectiveness against alternative forms of resistance that arise from different sets of point mutations in the target. This approach could be combined with traditional forms of combination chemotherapy in which two or more compounds are used against separate targets.