Endoplasmic Reticulum ; genetics ; metabolism ; Plasmodium berghei ; genetics ; metabolism ; Plasmodium falciparum ; genetics ; metabolism ; Protozoan Proteins ; genetics ; metabolism

Hemoglobinopathy and malaria are commonly found worldwide particularly in malaria endemic areas. Thalassemia, the alteration of globin chain synthesis, has been reported to confer resistance against malaria. The prevalence of thalassemia was investigated in 101 malaria patients with Plasmodium falciparum and Plasmodium vivax along the Thai-Myanmar border to examine protective effect of thalassemia against severe malaria. Hemoglobin typing was performed using low pressure liquid chromatography (LPLC) and alpha-thalassemia was confirmed by multiplex PCR. Five types of thalassemia were observed in malaria patients. The 2 major types of thalassemia were Hb E (18.8%) and alpha-thalassemia-2 (11.9%). There was no association between thalassemia hemoglobinopathy and malaria parasitemia, an indicator of malaria disease severity. Thalassemia had no significant association with P. vivax infection, but the parasitemia in patients with coexistence of P. vivax and thalassemia was about 2-3 times lower than those with coexistence of P. falciparum and thalassemia and malaria without thalassemia. Furthermore, the parasitemia of P. vivax in patients with coexistence of Hb E showed lower value than coexistence with other types of thalassemia and malaria without coexistence. Parasitemia, hemoglobin, and hematocrit values in patients with coexistence of thalassemia other than Hb E were significantly lower than those without coexistence of thalassemia. Furthermore, parasitemia with coexistence of Hb E were 2 times lower than those with coexistence of thalassemia other than Hb E. In conclusion, the results may, at least in part, support the protective effect of thalassemia on the development of hyperparasitemia and severe anemia in malaria patients.
Female ; Hemoglobins/genetics/metabolism ; Humans ; Malaria, Falciparum/blood/complications/*genetics/parasitology ; Malaria, Vivax/blood/complications/*genetics/parasitology ; Male ; Middle Aged ; Plasmodium falciparum/physiology ; Plasmodium vivax/physiology ; Thailand/epidemiology ; Thalassemia/blood/complications/epidemiology/*genetics

BACKGROUNDDuring the blood stage of malaria infection, parasites internalize in the host red blood cells and degrade massive amounts of hemoglobin for their development. Although the morphology of the parasite's hemoglobin uptake pathway has been clearly observed, little has been known about its molecular mechanisms.

METHODSThe recombinant proteins from Plasmodium falciparum, dynamin like protein 1 (PfDYN1) and 2 (PfDYN2) GTPase domain, were expressed in E.coli and showed GTPase activity. By using a dynamin inhibitor, dynasore, we demonstrated the involvement of PfDYN1 in the hemoglobin uptake pathway.

RESULTSThe GTPase activity of the two recombinant proteins was inhibited by dynasore in vitro. Treatment of parasite cultures with 80 micromol/L dynasore at the ring and early trophozoite stage resulted in substantial inhibition of parasite growth and in an obvious decline of hemoglobin quantum. Furthermore, reduced intracellular hemozoin accumulation and decreased uptake of the FITC-dextran were also observed, together with distinctive changes in the ultrastructure of parasites after the dynasore treatment.

CONCLUSIONSOur results show that PfDYN1 plays an important role in the hemoglobin uptake pathway of P. falciparum and suggest its possibility of being a novel target for malaria chemotherapy.

Animals ; Antimalarials ; pharmacology ; Dynamins ; antagonists & inhibitors ; GTP Phosphohydrolases ; genetics ; metabolism ; Hemoglobins ; metabolism ; Hydrazones ; pharmacology ; Malaria, Falciparum ; metabolism ; Microscopy, Electron, Transmission ; Plasmodium falciparum ; drug effects ; metabolism ; ultrastructure ; Protozoan Proteins ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

After invasion of red blood cells, malaria matures within the cell by degrading hemoglobin avidly. For enormous protein breakdown in trophozoite stage, many efficient and ordered proteolysis networks have been postulated and exploited. In this study, a potential interaction of a 60-kDa Plasmodium falciparum (Pf)-heat shock protein (Hsp60) and Pf-calpain, a cysteine protease, was explored. Pf-infected RBC was isolated and the endogenous Pf-Hsp60 and Pf-calpain were determined by western blot analysis and similar antigenicity of GroEL and Pf-Hsp60 was determined with anti-Pf-Hsp60. Potential interaction of Pf-calpain and Pf-Hsp60 was determined by immunoprecipitation and immunofluorescence assay. Mizoribine, a well-known inhibitor of Hsp60, attenuated both Pf-calpain enzyme activity as well as P. falciparum growth. The presented data suggest that the Pf-Hsp60 may function on Pf-calpain in a part of networks during malaria growth.
Amino Acid Sequence ; Calpain/genetics/*metabolism ; Chaperonin 60/chemistry/genetics/*metabolism ; Erythrocytes/parasitology ; Humans ; Malaria, Falciparum/parasitology ; Molecular Sequence Data ; Plasmodium falciparum/chemistry/enzymology/genetics/*metabolism ; Protein Binding ; Protozoan Proteins/chemistry/genetics/*metabolism ; Sequence Alignment

The parasite Plasmodium falciparum causes severe malaria and is the most dangerous to humans. However, it exhibits resistance to their drugs. Farnesyltransferase has been identified in pathogenic protozoa of the genera Plasmodium and the target of farnesyltransferase includes Ras family. Therefore, the inhibition of farnesyltransferase has been suggested as a new strategy for the treatment of malaria. However, the exact functional mechanism of this agent is still unknown. In addition, the effect of farnesyltransferase inhibitor (FTIs) on mitochondrial level of malaria parasites is not fully understood. In this study, therefore, the effect of a FTI R115777 on the function of mitochondria of P. falciparum was investigated experimentally. As a result, FTI R115777 was found to suppress the infection rate of malaria parasites under in vitro condition. It also reduces the copy number of mtDNA-encoded cytochrome c oxidase III. In addition, the mitochondrial membrane potential (DeltaPsim) and the green fluorescence intensity of MitoTracker were decreased by FTI R115777. Chloroquine and atovaquone were measured by the mtDNA copy number as mitochondrial non-specific or specific inhibitor, respectively. Chloroquine did not affect the copy number of mtDNA-encoded cytochrome c oxidase III, while atovaquone induced to change the mtDNA copy number. These results suggest that FTI R115777 has strong influence on the mitochondrial function of P. falciparum. It may have therapeutic potential for malaria by targeting the mitochondria of parasites.
Antimalarials/*pharmacology ; Enzyme Inhibitors/*pharmacology ; Farnesyltranstransferase/*antagonists & inhibitors/genetics/*metabolism ; Humans ; Malaria, Falciparum/drug therapy/*parasitology ; Mitochondria/*drug effects/metabolism ; Plasmodium falciparum/drug effects/*enzymology/genetics ; Protozoan Proteins/*antagonists & inhibitors/genetics/metabolism ; Quinolones/*pharmacology

The present study determined and compared the genetic diversity of Plasmodium falciparum strains infecting children living in 2 areas from Gabon with different malaria endemicity. Blood samples were collected from febrile children from 2008 to 2009 in 2 health centres from rural (Oyem) and urban (Owendo) areas. Genetic diversity was determined in P. falciparum isolates by analyzing the merozoite surface protein-1 (msp1) gene polymorphism using nested-PCR. Overall, 168 children with mild falciparum malaria were included. K1, Ro33, and Mad20 alleles were found in 110 (65.5%), 94 (55.9%), and 35 (20.8%) isolates, respectively, without difference according to the site (P>0.05). Allelic families' frequencies were comparable between children less than 5 years old from the 2 sites; while among the older children the proportions of Ro33 and Mad20 alleles were 1.7 to 2.0 fold higher at Oyem. Thirty-three different alleles were detected, 16 (48.5%) were common to both sites, and 10 out of the 17 specific alleles were found at Oyem. Furthermore, multiple infection carriers were frequent at Oyem (57.7% vs 42.2% at Owendo; P=0.04) where the complexity of infection was of 1.88 (+/-0.95) higher compared to that found at Owendo (1.55+/-0.75). Extended genetic diversity of P. falciparum strains infecting Gabonese symptomatic children and high multiplicity of infections were observed in rural area. Alleles common to the 2 sites were frequent; the site-specific alleles predominated in the rural area. Such distribution of the alleles should be taken into accounts when designing MSP1 or MSP2 malaria vaccine.
Child ; Child, Preschool ; Female ; Gabon ; *Gene Frequency ; Genetic Variation ; Genotype ; Humans ; Infant ; Malaria, Falciparum/*parasitology ; Male ; Merozoite Surface Protein 1/*genetics/metabolism ; Plasmodium falciparum/*genetics/metabolism ; Protozoan Proteins/*genetics/metabolism ; Rural Population ; Urban Population

To establish a platform for high throughput screening and in vitro evaluating novel metabolic enzyme-targeted inhibitors towards anti-malarial drugs, a lactate dehydrogenase gene of Plasmodium falciparum (PfLDH) was amplified from the Hainan isolate FCC1/HN. The fusion expression vectors, pGEX-2TK and pET-29a( + ), were utilized to introduce the PfLDH gene into strains of Escherichia coli, BL21 and BL21 (DE3), for over-expression. Consequently, the enzymatic activity of PfLDH was successfully detected in the suspension of lytic bacteria. The PfLDH gene cloned in pGEX-2TK was mainly expressed as inclusion bodies, while the same gene cloned in pET-29a( + ) was nearly expressed in a soluble form of PfLDH, demonstrating the latter vehicle might be more suitable for the large-scale preparation of recombinant PfLDH. Furthermore, according to the electrophoregram of SDS-PAGE and the sequencing data, a series of truncated PfLDH sequences generated randomly from gene amplification were screened and cloned, from which four pre-matured genes with a terminator mutation, PfLDH-delta271, -delta236, -delta167 and -delta53 coding for 45, 80, 149 and 263 amino acid residues, were individually recovered. Through the gene expression and enzymatic activity measurement, the effect of pre-matured terminator mutation on the activity of PfLDH was evaluated, which should pave the way for probing the relationship between structure and function of PfLDH.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Inclusion Bodies ; genetics ; metabolism ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Plasmodium falciparum ; enzymology ; genetics ; Protozoan Proteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Solubility

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors as well as by individual host responses to these determinants. In both humans and mice, liver injury follows parasite entry, persisting to the erythrocytic stage in the case of infection with the fatal strain of Plasmodium falciparum. Hepatic nuclear factor (HNF)-1alpha is a master regulator of not only the liver damage and adaptive responses but also diverse metabolic functions. In this study, we analyzed the expression of host HNF-1alpha in relation to malaria infection and evaluated its interaction with the 5'-untranslated region of subtilisin-like protease 2 (subtilase, Sub2). Recombinant human HNF-1alpha expressed by a lentiviral vector (LV HNF-1alpha) was introduced into mice. Interestingly, differences in the activity of the 5'-untranslated region of the Pf-Sub2 promoter were detected in 293T cells, and LV HNF-1alpha was observed to influence promoter activity, suggesting that host HNF-1alpha interacts with the Sub2 gene.
5' Untranslated Regions/*genetics ; Animals ; Cell Line ; DNA, Protozoan/genetics ; Gene Expression Regulation/*genetics ; Genetic Vectors ; Hepatocyte Nuclear Factor 1-alpha/administration & dosage/genetics/*metabolism ; Host-Parasite Interactions ; Humans ; Injections, Intravenous ; Lentivirus/genetics ; Malaria, Falciparum/metabolism/*parasitology/pathology ; Mice ; Plasmodium falciparum/drug effects/*genetics ; Promoter Regions, Genetic/genetics ; RNA, Messenger/genetics ; RNA, Protozoan/genetics ; Recombinant Proteins ; Signal Transduction ; Subtilisins/*genetics/metabolism

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors and individual host responses to these determinants. In both humans and mice, liver injury is involved after parasite entry, which persists until the erythrocyte stage after infection with the fatal strain Plasmodium falciparum (Pf). Hepatocyte growth factor (HGF) has strong anti-apoptotic effects in various kinds of cells, and also has diverse metabolic functions. In this work, Pf-subtilisin-like protease 2 (Pf-Sub2) 5'untranslated region (UTR) was analyzed and its transcriptional activity was estimated by luciferase expression. Fourteen TATA boxes were observed but only one Oct-1 and c-Myb were done. In addition, host HGF interaction with Pf-Sub2 was evaluated by co-transfection of HGF- and Pf-Sub2-cloned vector. Interestingly, -1,422/+12 UTR exhibited the strongest luciferase activity but -329 to +12 UTR did not exhibit luciferase activity. Moreover, as compared with the control of unexpressed HGF, the HGF protein suppressed luciferase expression driven by the 5'untranslated region of the Pf-Sub2 promoter. Taken together, it is suggested that HGF controls and interacts with the promoter region of the Pf-Sub2 gene.
*5' Untranslated Regions ; Artificial Gene Fusion ; Cell Line ; Genes, Reporter ; Hepatocyte Growth Factor/*metabolism ; Hepatocytes/parasitology ; *Host-Parasite Interactions ; Humans ; Luciferases/genetics/metabolism ; Plasmodium falciparum/*pathogenicity ; Protein Binding ; Subtilisins ; *Transcription, Genetic

OBJECTIVETo compare the protein patterns from the extracts of the mutant clone T9/94-M1-1(b3) induced by pyrimethamine, and the original parent clone T9/94 following separation of parasite extracts by two-dimensional electrophoresis (2-DE).

METHODSProteins were solubilized and separated according to their charges and sizes. The separated protein spots were then detected by silver staining and analyzed for protein density by the powerful image analysis software.

RESULTSDifferentially expressed protein patterns (up- or down-regulation) were separated from the extracts from the two clones. A total of 223 and 134 protein spots were detected from the extracts of T9/94 and T9/94-M1-1(b3) clones, respectively. Marked reduction in density of protein expression was observed with the extract from the mutant (resistant) clone compared with the parent (sensitive) clone. A total of 25 protein spots showed at least two-fold difference in density, some of which exhibited as high as ten-fold difference.

CONCLUSIONSThese proteins may be the molecular targets of resistance of Plasmodium falciparum to pyrimethamine. Further study to identify the chemical structures of these proteins by mass spectrometry is required.

Antimalarials ; metabolism ; Drug Resistance ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Image Processing, Computer-Assisted ; Mutation ; Plasmodium falciparum ; chemistry ; drug effects ; genetics ; Proteome ; analysis ; Protozoan Proteins ; analysis ; Pyrimethamine ; metabolism ; Staining and Labeling