PCR-amplification, sequencing, and comparison of the var/PfEMP-1 gene from the blood of patients with falciparum malaria in the Philippines.
The adhesion of Plasmodium falciparum-infected erythrocytes to vascular endothelium and to uninfected red blood cells (RBCs) plays a key role in the pathology of severe malaria. Adhesion is known to be mediated in part by the antigenically-variant erythrocyte membrane protein-1 (PfEMP-1), which is encoded by the var-gene family of P. falciparum. It has recently been reported that in vitro a single parasite simultaneously transcribes multiple var-genes but that, through a developmentally regulated process, the parasite selects only one PfEMP-1 that will to reach the surface of the host RBC. Were this to be true in vivo, one would expect a correlation between the type of var/PfEMP-1 that is expressed on the parasite-infected RBC and the severity of clinical disease. In order to test this assumption, we determined the sequence of the var-gene that was expressed by the parasites in patients' blood samples. Seven blood samples were collected from patients with or without severe clinical symptoms (cerebral malaria): two samples were from patients diagnosed as having imported falciparum malaria at the International Medical Center of Japan (IMCJ); the five others were from patients of the Davao Regional Hospital in Davao, the Philippines. The parasites (ring stage) in the blood samples were cultured for 24 hours; the matured trophozoites, in which the var-gene selection had taken place, served as material for mRNA isolation. The cDNA corresponding to the Duffy-binding-like (DBL)-1 domain of the var-gene was amplified by RT-PCR, using a region-specific primer set. The amplified cDNAs were cloned into the plasmid vector; the resultant clones (32) were sequenced on both strands. The results indicated that there was considerable diversity in the sequence of the DBL-1 domain among the clones, even among those from a single patient. In conclusion, it was difficult to demonstrate the correlation between the type of var-gene transcripts found in the RBCs of malaria patients and the severity of their symptoms.
Ikenoue
Nozomu
N
Gunma University School of Health Sciences, Gunma, Japan.
Kawazu
Shin-ichiro
S
Hatabu
Toshimitsu
T
Villacorte
Elena A
EA
Rivera
Pilarita T
PT
Kano
Shigeyuki
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D016778:Malaria, Falciparum / Q000097:blood / Q000235:genetics*; D008969:Molecular Sequence Data; D010679:Philippines; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D015800:Protozoan Proteins; D012333:RNA, Messenger; C085731:erythrocyte membrane protein 1, Plasmodium falciparum