Experimental cerebral malaria is suppressed by disruption of nucleoside transporter 1 but not purine nucleoside phosphorylase

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Abstract

Protozoan parasites rely on purine nucleosides supplied by the host because they are unable to synthesise purine rings de novo. Nucleoside transporter 1 (NT1) and purine nucleoside phosphorylase (PNP) play an essential role in purine salvage in Plasmodium. It is unclear whether severe pathology, such as cerebral malaria (CM), develops in hosts infected with Plasmodium parasites that lack activity of NT1 or PNP. Plasmodium berghei (Pb) ANKA-infected mice show features similar to human CM, such as cerebral paralysis and cerebral haemorrhage. Therefore, Pb ANKA infection in mice is a good experimental model of CM. In this study, we generated pbnt1-disrupted Pb ANKA (Δpbnt1 parasites) and pbpnp-disrupted Pb ANKA (Δpbpnp parasites), and investigated the effect of pbnt1 or pbpnp disruption on the outcome of infection with Pb ANKA. We showed that the rapid increase of wild-type Pb ANKA (WT parasites) in mice early in infection was significantly inhibited by disruption of pbnt1. Moreover, Δpbnt1 parasite-infected mice showed neither cerebral paralysis nor cerebral haemorrhage, and all mice spontaneously recovered from infection. By contrast, mice infected with Δpbpnp parasites showed features similar to those of mice infected with WT parasites. In this study, we demonstrated that the high virulence of Pb ANKA in the asexual phase is suppressed by disruption of pbnt1 but not pbpnp.

Highlights

► We generated pbnt1-disrupted Pb ANKA and pbpnp-disrupted Pb ANKA. ► ECM caused by Pb ANKA was suppressed by disruption of pbnt1 but not pbpnp. ► Mice infected with pbnt1-disrupted Pb ANKA spontaneously recovered from infection.

Introduction

Malaria, caused by protozoan parasites of the genus Plasmodium, is the major parasitic disease in tropical and subtropical regions, including parts of the Americas, Asia and Africa. An estimated 0.6–1 million malarial deaths per year have been reported [1]. Four species of Plasmodium can infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. Several kinds of drug are used to treat a Plasmodium-infected human. However, the increased incidence of drug-resistant parasites [1] has raised the importance of developing effective drugs or vaccines against Plasmodium infection.

It has long been recognised that protozoan parasites, including Plasmodium spp., are unable to synthesise purine rings de novo; instead, they rely on purine nucleosides from the host [2], [3]. Nucleosides and nucleobases are transported across the parasite plasma membrane by nucleoside transporters (NTs) of Plasmodium spp. [3]. The P. falciparum (Pf) genome sequencing project revealed four nucleoside transporters, PfNT1, PfNT2, PfNT3 and PfNT4 [4], [5]. PfNT1 has been cloned and expressed in Xenopus oocytes [6], [7] and was shown to transport hypoxanthine, in addition to adenosine and inosine [6], [7], [8], [9], [10].

During the asexual phase of malaria parasites, adenosine is converted to inosine by adenosine deaminase (ADA) in the purine salvage pathway. Hypoxanthine is produced from inosine by purine nucleoside phosphorylase (PNP). Hypoxanthine–guanine–xanthine phosphoribosyl transferase (HGXPRT) converts hypoxanthine to inosine monophosphate (IMP), guanine to guanosine monophosphate (GMP), and xanthine to xanthosine monophosphate (XMP). IMP, GMP and XMP are then converted to guanylate and adenylate nucleotides by the action of several other enzymes [3].

It was demonstrated that Plasmodium NT1 and PNP are essential for the viability of Plasmodium parasites in the host [11], [12]. Therefore, Plasmodium NT1 and PNP may be targets for anti-malarial drugs. On the other hand, it is unclear whether severe pathology, such as cerebral malaria (CM), is suppressed during infection with parasites lacking activity of Plasmodium NT1 or PNP.

Plasmodium berghei (Pb) ANKA is a lethal murine malaria parasite strain, and mice infected with Pb ANKA show features similar to human CM [13], [14], [15], [16]. Therefore, Pb ANKA infection in mice is a good experimental model of CM. In this study, we generated pbnt1-disrupted Pb ANKA (Δpbnt1 parasites) and pbpnp-disrupted Pb ANKA (Δpbpnp parasites), and investigated the effect of disruption of pbnt1 or pbpnp on the outcome of infection with Pb ANKA. We show that the high virulence of Pb ANKA in the asexual phase was suppressed by disruption of pbnt1 but not pbpnp.

Section snippets

Mice

Female C57BL/6J (B6) mice 5- to 6-weeks old were purchased from CLEA Japan INC (Tokyo, Japan). The experiments were approved by the Experimental Animal Ethics Committee of Kyorin University School of Medicine, Tokyo, and all experimental animals were kept at the animal facility in a specific-pathogen-free unit with sterile bedding, food and water.

Parasites and infections

Malaria parasites were stored as frozen stocks in liquid nitrogen. Wild-type Pb ANKA (WT parasites) is a high-virulence strain and the parasites,

Effect of disruption of pbnt1 or pbpnp on the outcome of infection with Pb ANKA

To investigate the effect of pbnt1 or pbpnp disruption on the outcome of infection with Pb ANKA, mice were infected with wild-type Pb ANKA (WT parasites), Δpbnt1 parasites or Δpbpnp parasites. Mice infected with WT parasites showed high levels of parasitaemia and neurological signs, such as a cerebral paralysis, and all mice died within 10 days post-infection (Fig. 1A and B). In mice infected with Δpbnt1 parasites, parasitaemia on days 6 and 8 post-infection was significantly milder than that in

Discussion

We aimed to investigate the effect of disruption of pbnt1 or pbpnp on the outcome of infection with lethal Pb ANKA. In P. falciparum, disruption of pfnt1 resulted in blockage of growth at the ring stage [8]. A recent study using murine malaria parasites demonstrated that mice infected with pynt1-disrupted nonlethal Plasmodium yoelii 17X showed significantly lower levels of parasitaemia than mice infected with wild-type nonlethal P. yoelii 17X [11]. We showed here that the growth of Pb ANKA

Author contributions

M.N. designed research; M.N. and Y.Y. performed research; I.K., S.I. and M.Y. contributed reagents/materials/analysis tools; M.N., S.-I.I., S.M., and F.K. analyzed data; and M.N. and F.K. wrote the paper.

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS) to M.N. (No. 24790405) and the Incentive Award from Kyorin University School of Medicine to M.N. This work was also supported in part by a Grant-in-Aid for Young Scientists (B) from JSPS to S.–I.I. (No. 23790462), and a Grant-in-Aid for Scientific Research (C) from JSPS to F.K. (No. 23590493)

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