The human malaria parasite Plasmodium vivax is responsible for 25-40% of the ~515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated in the laboratory except in non-human primates. We determined the genome sequence of P. vivax in order to shed light on its distinctive biologic features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternate invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance scientific investigation into this neglected species.

No abstract

Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc 1 (CYT bc 1 ) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25-to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYT b.The widespread resistance of malaria parasites to standard antimalarial drugs is a serious global health problem. The urgent need for new antimalarial drugs has led to the development of atovaquone (566C80) which, combined with proguanil, has been licensed as Malarone. There is some concern that parasites may develop resistance to Malarone. In one study, 33% of patients treated with atovaquone alone experienced a recrudescence of parasitemia after treatment. These parasites tolerated up to 1,000-fold higher concentrations of atovaquone than did the pretreated parasites (16). Atovaquone-resistant parasites have been readily selected in vitro. Up to 1 in 10 5 parasites became resistant to the drug after having been cultured in the presence of 10 Ϫ8 M atovaquone for 5 weeks (21, 23).Atovaquone has potent blood schizonticidal activity and is also effective against the preerythrocytic (2, 4, 5) and sexual stages (8, 9) of the malaria parasite. It acts by inhibiting mitochondrial electron transport (10) and collapsing mitochondrial membrane potential (25). From these observations and on the basis of its structural similarity to ubiquinol, it has been postulated that atovaquone binds to parasite cytochrome b (CYT b) (31). The inhibitors stigmatellin and 5-n-undecyl-4,7-dioxobenzoxythiazol (UHDBT), which are structurally similar to atovaquone, have been shown to bind at the ubihydroquinone (Q o ) site of CYT b and inhibit electron transport. Single point mutations within the Q o site confer resistance to these inhibitors in a variety of microorganisms (7). Two mutations in close proximity to the Q o site in Pneumocystis carinii are associated with atovaquone prophylaxis failure (33). Atovaquone-resistant Plasmodium yoelii lines have been derived from infected mice treated with suboptimal doses of atovaquone. All resistant lines ...

The most potent known naturally occurring BowmanBirk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine ␤-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI-1[6,5] is ϳ9:1 regardless of whether trypsin is incubated with SFTI-1[6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[6,5] revealed high heterogeneity, and multiple species of SFTI-1[6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a ␤-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an isoaspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.Over recent years there has been much interest in the discovery of circular proteins in higher organisms (1) and in the development in synthetic approaches to cyclize proteins (2). In general, backbone cyclic peptides have several advantages over their non-cyclic counterparts. They are resistant to attack by exopeptidases, making them less vulnerable to degradation and can have an increased thermal stability (3). Also, unfavorable entropic losses upon binding to target proteins are significantly reduced, resulting in a thermodynamically more efficient binding interaction (1). These biological advantages of backbone cyclized peptides may lead to their use as scaffolds for the design of stable pharmaceuticals and pesticides (4).The new generation of circular peptides/proteins discovered in the last few years differs from previously known cyclic peptides such as cyclosporins in that the latter are generally not direct gene products but are synthesized in bacteria by multifunctional enzymes and often contain non-conventional amino acids (5). By contrast, recently discovered circular miniproteins such as the plant cyclotides (6) are gene products that are post-translationally processed to cyclize their conventional peptide backbone (7). Although in vitro cyclization procedures are now being developed for the synthetic production of circular proteins, little is known about the mechanisms and driving force behind in vivo cyclization of naturally o...

Sulfadoxine is predominantly used in combination with pyrimethamine, commonly known as Fansidar, for the treatment of Plasmodium falciparum. This combination is usually less effective against Plasmodium vivax, probably due to the innate refractoriness of parasites to the sulfadoxine component. To investigate this mechanism of resistance by P. vivax to sulfadoxine, we cloned and sequenced the P. vivax dhps (pvdhps) gene. The protein sequence was determined, and three-dimensional homology models of dihydropteroate synthase (DHPS) from P. vivax as well as P. falciparum were created. The docking of sulfadoxine to the two DHPS models allowed us to compare contact residues in the putative sulfadoxine-binding site in both species. The predicted sulfadoxine-binding sites between the species differ by one residue, V585 in P. vivax, equivalent to A613 in P. falciparum. V585 in P. vivax is predicted by energy minimization to cause a reduction in binding of sulfadoxine to DHPS in P. vivax compared to P. falciparum. Sequencing dhps genes from a limited set of geographically different P. vivax isolates revealed that V585 was present in all of the samples, suggesting that V585 may be responsible for innate resistance of P. vivax to sulfadoxine. Additionally, amino acid mutations were observed in some P. vivax isolates in positions known to cause resistance in P. falciparum, suggesting that, as in P. falciparum, these mutations are responsible for acquired increases in resistance of P. vivax to sulfadoxine.