Genetic polymorphism of the extracellular region in surface associated interspersed 1.1 gene of Plasmodium falciparum field isolates from Thailand.

BACKGROUND: A novel variable surface antigens (VSAs), Surface-associated interspersed proteins (SUFRINs), is a protein that is modified on the surface of infected red blood cell (iRBC). Modified proteins on the iRBC surface cause severe malaria, which can lead to death throughout the life cycle of a malaria parasite. Previous study suggested that SURFIN1.1 is an immunogenic membrane-associated protein which was encoded by using the surf1.1 gene expressed during the trophozoite and schizont stages. This study aimed to identify the regions of SURFIN1.1 and investigate the genetic diversity of the extracellular region of the surf1.1 gene. METHODS: A total of 32 blood samples from falciparum malaria cases that were diagnosed in Si Sa Ket Province, Thailand were collected. Plasmodium genomic DNA was extracted, and the extracellular region of surf1.1 gene was amplified using the polymerase chain reaction (PCR). A sequence analysis was then performed to obtain the number of haplotypes (H), the haplotype diversity (Hd), and the segregating sites (S), while the average number of nucleotide differences between two sequences (Pi); in addition, neutrality testing, Tajima's D test, Fu and Li's D* and F* statistics was also performed. RESULTS: From a total of 32 patient-isolated samples, 31 DNA sequences were obtained and analysed for surf1.1 gene extracellular region polymorphism. Researchers observed six distinct haplotypes in the current research area. Haplotype frequencies were 61.3%, 16.2%, and 12.9% for H1, H2, and H3, respectively. The remaining haplotype (H4-H6) frequency was 3.2% for each haplotype. Hd was 0.598 ± 0.089 with the Pi of 0.00381, and S was 15. The most common amino acid polymorphic site was E251Q; other sites included N48D, I49V, E228D, E235S, L265F, K267T, E276Q, and S288F. Fu and Li's D* test value was - 1.24255, Fu and Li's F* test value was - 1.10175, indicating a tendency toward negative balancing selection acting on the surf1.1 N-terminal region. The most polymorphic region was variable 2 (Var2) while cysteine-rich domain (CRD) was conserved in both the amino acid and nucleotide extracellular region of surf1.1 gene. CONCLUSIONS: The Thai surf1.1 N-terminal region was well-conserved with only a few polymorphic sites remaining. In this study, the data regarding current bearing on the polymorphism of extracellular region of surf1.1 gene were reported, which might impact the biological roles of P. falciparum. In addition, may possibly serve as a suitable candidate for future development of SURFIN-based vaccines regarding malaria control.

A Novel Nested Multiplex Polymerase Chain Reaction Assay for Malaria Diagnosis Using the Hydroxymethyl Dihydropterin Pyrophosphokinase-Dihydropteroate Synthase (hppk-dhps) Gene.

There are many techniques for malaria diagnosis. Currently, the nested polymerase chain reaction (PCR) method based on a small subunit ribosomal RNA gene (18S rRNA) has been used as a confirmatory method. However, this method is time-consuming, laborious, and costly. Therefore, the objective of this study was to develop nested multiplex PCR for Plasmodium species identification using the dihydropterin pyrophosphokinase-dihydropteroate synthase (hppk-dhps) gene. Genus- and species-specific primers for the hppk-dhps gene were designed. The performance of the novel nested multiplex PCR was compared with 18S rRNA nested PCR. A total of 115 blood samples were used in this study, including 84 infected samples and 31 uninfected samples. Analysis of the blood samples by nested multiplex PCR targeting the hppk-dhps gene identified 81 infected cases. The level of agreement between this novel method and 18S rRNA nested PCR was 97.4%. Further, the novel method successfully detected all human malaria parasites except Plasmodium ovale and detected mixed Plasmodium falciparum/Plasmodium vivax infections. The sensitivity and specificity obtained from this novel method were 96.4% and 100%, respectively. The limit of detection of the hppk-dhps nested multiplex PCR for P. falciparum and P. vivax was 500 parasites/µL and 4 parasites/µL, respectively. The lowest parasite gDNA detected by this method was 0.5 ng/µL for P. falciparum and 0.1 ng/µL for P. vivax. These results corroborate that the hppk-dhps gene is a novel amplification target for the detection of human malaria. This novel target PCR-based method is a beneficial approach for malaria diagnosis, as well as species identification and differentiation.

Humoral immune responses to Plasmodium vivax subtelomeric transmembrane proteins in Thailand.

Plasmodium vivax subtelomeric transmembrane protein (PvSTP) is a homolog of P. falciparum SURFIN4.2', a protein exposed on the parasite-infected erythrocyte (iE) surface, and is thus considered to be exposed on P. vivax-iE. Because antibodies targeting antigens located on the surface of P. falciparum-iE, such as P. falciparum erythrocyte membrane protein 1, play an important role in regulating the course of disease, we evaluated the presence of antibodies in P. vivax-infected patients against two PvSTP paralogs, PvSTP1 and PvSTP2. Recombinant proteins corresponding to cysteine-rich domain (CRD) of the PvSTP extracellular region and the cytoplasmic region (CYT) were generated and used for the enzyme-linked immunosorbent assay. Plasma samples (n = 70) reacted positively with recombinant PvSTP1-CRD (40%), PvSTP1-CYT (31%), PvSTP2-CRD (27%), and PvSTP2-CYT (56%), suggesting that PvSTP1 and -2 are naturally immunogenic. Specific response against either PvSTP1 or PvSTP2 indicates the existence of specific antibodies for either PvSTP1 or -2.

Antimalarial effect of cell penetrating peptides derived from the junctional region of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase.

Dihydrofolate reductase-thymidylate synthase of Plasmodium falciparum (PfDHFR-TS) is an important target of antifolate antimalarial drugs. However, drug resistant parasites are widespread in malaria endemic regions. The unique bifunctional property of PfDHFR-TS could be exploited for the design of allosteric inhibitors that interfere with the active dimer conformation. In this study, peptides were derived from the junctional region (JR) of PfDHFR-TS amino acid sequence in the αj1 helix (JR-helix) and the DHFR domain that is necessary for interaction with αj1 helix (JR21). Five peptides were synthesized and tested for inhibition of PfDHFR-TS enzyme by Bacterial inhibition assay (BIA) based on the growth of an E. coli DHFR and TS knockout complemented with a recombinant plasmid expressing PfDHFR-TS enzyme. Significant inhibition was observed for JR21 and JR21 conjugated to cell-penetrating octa-arginine peptide (rR8-JR21) with 50 % inhibitory concentration (IC50) of 3.87 and 1.53 µM, respectively. The JR-helix and rR8-JR-helix peptides were inactive. JR21 and rR8-JR21 peptides showed similar growth inhibitory effects on P. falciparum NF54 parasites cultured in vitro. Treatment with rR8-JR21 delayed parasite development, in which an accumulation of ring stage parasites was observed after 12 h of culture. Minimal red blood cell (RBC) hemolysis was observed at the highest dose of peptide tested. The most potent peptide rR8-JR21 not only compromised the development of the P. falciparum, but also inhibited the parasite growth and has low hemolytic effect on human RBCs.