Single nucleotide polymorphisms in Plasmodium falciparum V type H⁺ pyrophosphatase gene (pfvp2) and their associations with pfcrt and pfmdr1 polymorphisms

Highlights

• First report on pfvp2 SNPs and their possible associations with pfcrt and pfmdr1.

• Pfvp2 V405, K582 and P711 alleles were predominant in all eight countries studied.

• Pfvp2 405I, 582R and 711S were mainly seen in areas where CQ resistance was uncommon.

• There was an association between the pfvp2 VKP haplotype and CQ resistance pfcrt 76T.

Abstract

Background

Chloroquine resistance in Plasmodium falciparum malaria has been associated with pfcrt 76T (chloroquine resistance transporter gene) and pfmdr1 86Y (multidrug resistance gene 1) alleles. Pfcrt 76T enables transport of protonated chloroquine out of the parasites digestive vacuole resulting in a loss of hydrogen ions (H⁺). V type H⁺ pyrophosphatase (PfVP2) is thought to pump H⁺ into the digestive vacuole. This study aimed to describe the geographic distribution of single nucleotide polymorphisms in pfvp2 and their possible associations with pfcrt and pfmdr1 polymorphisms.

Methods

Blood samples from 384 patients collected (1981–2009) in Honduras (n = 35), Colombia (n = 50), Liberia (n = 50), Guinea Bissau (n = 50), Tanzania (n = 50), Iran (n = 50), Thailand (n = 49) and Vanuatu (n = 50) were analysed. The pfcrt 72–76 haplotype, pfmdr1 copy numbers, pfmdr1 N86Y and pfvp2 V405I, K582R and P711S alleles were identified using PCR based methods.

Results

Pfvp2 was amplified in 344 samples. The pfvp2 allele proportions were V405 (97%), 405I (3%), K582 (99%), 582R (1%), P711 (97%) and 711S (3%). The number of patients with any of pfvp2 405I, 582R and/or 711S were as follows: Honduras (2/30), Colombia (0/46), Liberia (7/48), Guinea-Bissau (4/50), Tanzania (3/48), Iran (3/50), Thailand (1/49) and Vanuatu (0/31). The alleles were most common in Liberia (P = 0.01) and Liberia + Guinea-Bissau (P = 0.01). The VKP haplotype was found in 189/194 (97%) and 131/145 (90%) samples harbouring pfcrt 76T and pfcrt K76 respectively (P = 0.007).

Conclusions

The VKP haplotype was dominant. Most pfvp2 405I, 582R and 711S SNPs were seen where CQ resistance was not highly prevalent at the time of blood sampling possibly due to greater genetic variation prior to the bottle neck event of spreading CQ resistance. The association between the pfvp2 VKP haplotype and pfcrt 76T, which may indicate that pfvp2 is involved in CQ resistance, should therefore be interpreted with caution.

Introduction

Plasmodium falciparum has persisted as a major cause of human suffering and death despite the deployment of antimalarial drugs. A contributing factor has been the development of resistance to antimalarial drugs such as chloroquine (CQ). For example, CQ resistance was associated with 2–6-fold increase in malaria attributed mortality (Trape, 2001). Thus, reports of developing tolerance to currently recommended artemisinin based combination therapies are of major concern (Dondorp et al., 2009, Noedl et al., 2008) and an understanding of the mechanisms of drug resistance in the malaria parasite is crucial.

Resistance to CQ appears to have developed independently in Colombia, Venezuela, Thai–Cambodian border, Papua New Guinea and the Philippines (Mita et al., 2009, Wootton et al., 2002). The P. falciparum chloroquine resistance transporter (pfcrt) gene appears to be the main determinant of CQ resistance. Specific haplotypes at positions 72–76 are linked to the regional evolution of CQ resistance (Awasthi and Das, 2013, Mita et al., 2009, Wootton et al., 2002) and the 76T allele is essential for resistance (Djimde et al., 2001, Plowe, 2003). Resistance has also been linked to the N86Y allele of the multidrug resistance gene 1 (pfmdr1) (Babiker et al., 2001). Resistance to CQ is associated with a loss of inherent fitness (Ord et al., 2007). It is therefore probable that compensatory mutations have evolved in P. falciparum over time as have been shown to occur in drug resistant bacteria (Jiang et al., 2008, Levin et al., 2000).

Pfcrt is located in the membrane of the digestive vacuole (DV) (Valderramos and Fidock, 2006) and transports protonated CQ down its electrochemical gradient out of the DV (Martin et al., 2009, Sanchez et al., 2007). This result in a loss of Hydrogen ions (H⁺) that must be replaced if the pH is to be maintained. Thus, the transport of H⁺ into the DV most probably increases when CQ is being removed. In line with this, a 10-fold increased transcription of a putative H⁺ pump located in the DV membrane has been observed in P. falciparum exposed to CQ and a 2-fold increase when exposed to lumefantrine (Jiang et al., 2008, Mwai et al., 2012). The putative pump is a V type H⁺ pyrophosphatase (PfVP2), which constitutes a novel class of H⁺ pump found in plants and some protozoa (Luo et al., 1999, McIntosh et al., 2001, McIntosh and Vaidya, 2002, Saliba et al., 2003). The aim of this study was to explore the role of the pfvp2 gene in antimalarial drug resistance by analysing single nucleotide polymorphisms (SNPs) in pfvp2 and their prevalence in eight different countries and possible association with polymorphisms in pfcrt and pfmdr1.