Prevalence of Drug Resistance-Associated Gene Mutations in Plasmodium vivax in Central China

Resistance of Plasmodium spp. to anti-malarial drugs is the primary obstacle in the fight against malaria, and molecular markers for the drug resistance have been applied as an adjunct in the surveillance of the resistance. In this study, we investigated the prevalence of mutations in pvmdr1, pvcrt-o, pvdhfr, and pvdhps genes in temperate-zone P. vivax parasites from central China. A total of 26 isolates were selected, including 8 which were previously shown to have a lower susceptibility to chloroquine in vitro. For pvmdr1, pvcrt-o, and pvdhps genes, no resistance-conferring mutations were discovered. However, a highly prevalent (69.2%), single-point mutation (S117N) was found in pvdhfr gene. In addition, tandem repeat polymorphisms existed in pvdhfr and pvdhps genes, which warranted further studies in relation to the parasite resistance to antifolate drugs. The study further suggests that P. vivax populations in central China may still be relatively susceptible to chloroquine and sulfadoxine-pyrimethamine.

Positive Diversifying Selection on the <i>Plasmodium falciparum</i> <i>surf_4.1</i> Gene in Thailand

<i>Plasmodium falciparum</i> SURFIN_4.1 is a type I transmembrane protein thought to locate on the merozoite surface and to be responsible for a reversible adherence to the erythrocyte before invasion. In this study, we evaluated <i>surf_4.1</i> gene segment encoding extracellular region for polymorphism, the signature of positive selection, the degree of linkage disequilibrium, and temporal change in allele frequency distribution in <i>P. falciparum</i> isolates from Thailand in 1988–89, 2003, and 2005. We found that SURFIN_4.1 is highly polymorphic, particularly at the C-terminal side of the variable region located just before a predicted transmembrane region. A signature of positive diversifying selection on the variable region was detected by multiple tests and, to a lesser extent, on conserved N-terminally located cysteine-rich domain by Tajima’s <i>D</i> test. Linkage disequilibrium between sites over a long distance (> 1.5 kb) was detected, and multiple SURFIN_4.1 haplotype sequences detected in 1988/89 still circulated in 2003. Few of the single amino acid polymorphism allele frequency distributions were significantly different between the 1988/89 and 2003 groups, suggesting that the frequency distribution of SURFIN_4.1 extracellular region remained stable over 14 years.<br>

Positive Diversifying Selection on the <i>Plasmodium falciparum</i> <i>surf_4.1</i> Gene in Thailand

<i>Plasmodium falciparum</i> SURFIN_4.1 is a type I transmembrane protein thought to locate on the merozoite surface and to be responsible for a reversible adherence to the erythrocyte before invasion. In this study, we evaluated <i>surf_4.1</i> gene segment encoding extracellular region for polymorphism, the signature of positive selection, the degree of linkage disequilibrium, and temporal change in allele frequency distribution in <i>P. falciparum</i> isolates from Thailand in 1988–89, 2003, and 2005. We found that SURFIN_4.1 is highly polymorphic, particularly at the C-terminal side of the variable region located just before a predicted transmembrane region. A signature of positive diversifying selection on the variable region was detected by multiple tests and, to a lesser extent, on conserved N-terminally located cysteine-rich domain by Tajima’s <i>D</i> test. Linkage disequilibrium between sites over a long distance (> 1.5 kb) was detected, and multiple SURFIN_4.1 haplotype sequences detected in 1988/89 still circulated in 2003. Few of the single amino acid polymorphism allele frequency distributions were significantly different between the 1988/89 and 2003 groups, suggesting that the frequency distribution of SURFIN_4.1 extracellular region remained stable over 14 years.

Molecular Cloning of Plasmodium vivax Calcium-Dependent Protein Kinase 4

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.

Selective pressure on the merozoite surface protein-1 genes of Plasmodium vivax, P. knowlesi and P. cynomolgi

Background: The merozoite surface protein 1 (msp-1) of malarial parasites is a promising target for malaria vaccine development. Because antigenic polymorphism of this protein occurs in both Plasmodium falciparum and P. vivax, it is important to know the extent of sequence variation of this gene in nonhuman primate malarias that can infect humans. Objective: To determine the complete nucleotide sequences of the msp-1 genes of Plasmodium knowlesi (Pkmsp-1) and P. cynomolgi (Pcymsp-1) and their evolutionary history comparing with an orthologous gene of P. vivax (Pvmsp-1). Method: The msp-1 genes of P. knowlesi and P. cynomolgi were amplified by the polymerase chain reaction and their nucleotide sequences were determined directly from the amplified products. Sequences were aligned using the Clustal X program. The extent of polymorphism is expressed in terms of nucleotide diversity (π) and the average number of nucleotide substitutions per site between populations (Dxy) in pair-wise comparison. The ratio of nucleotide substitutions at synonymous and nonsynonymous sites was computed by the McDonald and Kreitman test to detect evidence of departure from neutrality. The phylogenetic tree was constructed by the neighbor-joining method using the maximum composite likelihood with 1,000 bootstrap iterations. Results: The deduced amino acid sequences of the msp-1 of P. knowlesi, P. cynomolgi and P. vivax exhibit sequence similarity, ranging from 56.2 to 60.8 %. Variable blocks detected in Pkmsp-1 and Pcymsp-1 are located in regions corresponding to variable blocks of Pvmsp-1. Sliding window plots of Dxy along the entire coding regions of the msp-1 genes gave similar profiles for each pair of comparison. Pvmsp-1 is more closely related to Pcymsp-1 than that of Pkmsp-1 based on a phylogenetic analysis. However, the Tajima’s relative rate test suggests that the merozoite surface protein 1 (msp-1) of each species has not evolved at the same rate. The McDonald-Kreitman test detects positive selection on regions equivalent to blocks 1 and 9 of Pvmsp-1. Conclusion: The msp-1 genes of P. knowlesi, P. cynomolgi and P. vivax exhibit a similar pattern of sequence variation. Regions equivalent to conserved blocks 1 and 9 of Pvmsp-1 have evolved under positive selective pressure, suggestive of functional importance probably as targets for host immune responses.