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Invasion profiles of Brazilian field isolates of Plasmodium falciparum: phenotypic and genotypic analyses.

ABSTRACT: The invasion of red blood cells (RBCs) by Plasmodium falciparum is dependent on multiple molecular interactions between erythrocyte receptors and parasite ligands. Invasion studies using culture-adapted parasite strains have indicated significant receptor heterogeneity. It is not known whether this heterogeneity reflects the parasite invasion arsenal in the field. We have studied the invasion phenotypes of 14 distinct field isolates from the Legal Amazon areas of Brazil by using erythrocyte invasion assays to investigate invasion into normal, enzyme-treated, and clinical-mutant RBCs. Analysis of these isolates revealed four distinct invasion profiles. Using En(a-) cells to get an unequivocal estimate of the use of glycophorin A (GPA) as a receptor, we found that the 175-kDa erythrocyte-binding antigen (EBA-175)/GPA pathway was used by a minority of the parasite isolates studied. Although polymorphism of region II domains at specific amino acid positions in both EBA-140 and EBA-181 was found in these field isolates, this did not correlate with invasion profiles and thus receptor selectivity. These studies have further confirmed the existence of a significant diversity of invasion pathways in nature and suggest that additional parasite ligands will have to be targeted to devise global vaccines that will work in the field.

SUBMITTER: Lobo CA

PROVIDER: S-EPMC517604 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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Invasion profiles of Brazilian field isolates of Plasmodium falciparum: phenotypic and genotypic analyses.

Lobo Cheryl-Ann CA de Frazao Karla K Rodriguez Marilis M Reid Marion M Zalis Mariano M Lustigman Sara S

Infection and immunity 20041001 10

The invasion of red blood cells (RBCs) by Plasmodium falciparum is dependent on multiple molecular interactions between erythrocyte receptors and parasite ligands. Invasion studies using culture-adapted parasite strains have indicated significant receptor heterogeneity. It is not known whether this heterogeneity reflects the parasite invasion arsenal in the field. We have studied the invasion phenotypes of 14 distinct field isolates from the Legal Amazon areas of Brazil by using erythrocyte inva ...[more]

PMID: 15385490

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Project description:The Plasmodium falciparum in vitro culture system is critical for genotypic and phenotypic analyses of the parasites. For genotypic analysis, the genomic DNA can be obtained directly from the patient blood sample or from culture adapted parasites whereas for phenotypic analysis, immediate ex vivo or in vitro culture adapted parasites are used. However, parasite biology studies have not investigated whether culture adaptation process affects genotypic and/or phenotypic characteristics of the parasites in short- or long-term cultures. Here, we set out to study the dynamics and stability of parasite genetic and phenotypic profiles as field isolate parasites were adapted in continuous cultures. Parasites collected from three different patients presenting with uncomplicated malaria were adapted and maintained in drug-free continuous cultures. Aliquots from the continuous cultures were collected every 24-48 hours for analyses. Each aliquot was treated as a separate parasite sample. For genetic analysis, microsatellite (MS) typing and single nucleotide polymorphism (SNP) analyses of 23 drug resistance markers were done. The 50% inhibitory concentrations (IC50) for some of the samples were also established for four antimalarial drugs. Samples from each patient (parasite-line) were compared as they were passed through the continuous culture. Data revealed genotypic and phenotypic profiles for the three parasite-lines fluctuated from one generation to the next with no specific pattern or periodicity. With few exceptions, multilocus analysis revealed samples from each parasite-line had high genetic diversity with unique haplotypes. Interestingly, changes in MS and SNP profiles occurred simultaneously. The difference in the IC50s of samples in each parasite-line reached statistical significance. However, phenotypic changes did not correspond or correlate to genotypic changes. Our study revealed parasite genetic and phenotypic characteristics fluctuates in short- and long-term cultures, which indicates parasite genetic information obtained even in short cultures is likely to be different from the natural infection parasites.

Project description:BackgroundHigh rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 (pfpm2), exonuclease (pfexo) and chloroquine resistance transporter (pfcrt) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy.MethodsTo examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined.ResultsThe characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone-proguanil combinations revealed synergistic antimalarial activity.ConclusionsSurveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.

Project description:Studies of Plasmodium falciparum invasion pathways in field isolates have been limited. Red blood cell (RBC) invasion is a complex process involving two invasion protein families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins, which are polymorphic and not fully characterized in field isolates. To determine the various P. falciparum invasion pathways used by parasite isolates from South America, we studied the invasion phenotypes in three regions: Colombia, Peru and Brazil. Additionally, polymorphisms in three members of the EBL (EBA-181, EBA-175 and EBL-1) and five members of the PfRh (PfRh1, PfRh2a, PfRh2b, PfRh4, PfRh5) families were determined. We found that most P. falciparum field isolates from Colombia and Peru invade RBCs through an atypical invasion pathway phenotypically characterized as resistant to all enzyme treatments (NrTrCr). Moreover, the invasion pathways and the ligand polymorphisms differed substantially among the Colombian and Brazilian isolates while the Peruvian isolates represent an amalgam of those present in the Colombian and Brazilian field isolates. The NrTrCr invasion profile was associated with the presence of the PfRh2a pepC variant, the PfRh5 variant 1 and EBA-181 RVNKN variant. The ebl and Pfrh expression levels in a field isolate displaying the NrTrCr profile also pointed to PfRh2a, PfRh5 and EBA-181 as being possibly the major players in this invasion pathway. Notably, our studies demonstrate the uniqueness of the Peruvian P. falciparum field isolates in terms of their invasion profiles and ligand polymorphisms, and present a unique opportunity for studying the ability of P. falciparum parasites to expand their invasion repertoire after being reintroduced to human populations. The present study is directly relevant to asexual blood stage vaccine design focused on invasion pathway proteins, suggesting that regional invasion variants and global geographical variation are likely to preclude a simple one size fits all type of vaccine.

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Invasion profiles of Brazilian field isolates of Plasmodium falciparum: phenotypic and genotypic analyses.

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Invasion profiles of Brazilian field isolates of Plasmodium falciparum: phenotypic and genotypic analyses.

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