Genomic epidemiology of artemisinin resistant malaria.

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions

Tackling resistance: Emerging antimalarials and new parasite targets in the era of elimination [version 1; referees: 2 approved]

Malaria remains a significant contributor to global human mortality, and roughly half the world’s population is at risk for infection with Plasmodium spp. parasites. Aggressive control measures have reduced the global prevalence of malaria significantly over the past decade. However, resistance to available antimalarials continues to spread, including resistance to the widely used artemisinin-based combination therapies. Novel antimalarial compounds and therapeutic targets are greatly needed. This review will briefly discuss several promising current antimalarial development projects, including artefenomel, ferroquine, cipargamin, SJ733, KAF156, MMV048, and tafenoquine. In addition, we describe recent large-scale genetic and resistance screens that have been instrumental in target discovery. Finally, we highlight new antimalarial targets, which include essential transporters and proteases. These emerging antimalarial compounds and therapeutic targets have the potential to overcome multi-drug resistance in ongoing efforts toward malaria elimination

Recent advances in malaria genomics and epigenomics

Malaria continues to impose a significant disease burden on low- and middle-income countries in the tropics. However, revolutionary progress over the last 3 years in nucleic acid sequencing, reverse genetics, and post-genome analyses has generated step changes in our understanding of malaria parasite (Plasmodium spp.) biology and its interactions with its host and vector. Driven by the availability of vast amounts of genome sequence data from Plasmodium species strains, relevant human populations of different ethnicities, and mosquito vectors, researchers can consider any biological component of the malarial process in isolation or in the interactive setting that is infection. In particular, considerable progress has been made in the area of population genomics, with Plasmodium falciparum serving as a highly relevant model. Such studies have demonstrated that genome evolution under strong selective pressure can be detected. These data, combined with reverse genetics, have enabled the identification of the region of the P. falciparum genome that is under selective pressure and the confirmation of the functionality of the mutations in the kelch13 gene that accompany resistance to the major frontline antimalarial, artemisinin. Furthermore, the central role of epigenetic regulation of gene expression and antigenic variation and developmental fate in P. falciparum is becoming ever clearer. This review summarizes recent exciting discoveries that genome technologies have enabled in malaria research and highlights some of their applications to healthcare. The knowledge gained will help to develop surveillance approaches for the emergence or spread of drug resistance and to identify new targets for the development of antimalarial drugs and perhaps vaccines

Adaptation of translational machinery in malaria parasites to accommodate translation of poly-adenosine stretches throughout its life cycle

Malaria is caused by unicellular apicomplexan parasites of the genu

Artemether resistance in vitro is linked to mutations in PfATP6 that also interact with mutations in PfMDR1 in travellers returning with Plasmodium falciparum infections.

BACKGROUND: Monitoring resistance phenotypes for Plasmodium falciparum, using in vitro growth assays, and relating findings to parasite genotype has proved particularly challenging for the study of resistance to artemisinins. METHODS: Plasmodium falciparum isolates cultured from 28 returning travellers diagnosed with malaria were assessed for sensitivity to artemisinin, artemether, dihydroartemisinin and artesunate and findings related to mutations in pfatp6 and pfmdr1. RESULTS: Resistance to artemether in vitro was significantly associated with a pfatp6 haplotype encoding two amino acid substitutions (pfatp6 A623E and S769N; (mean IC50 (95% CI) values of 8.2 (5.7 - 10.7) for A623/S769 versus 623E/769 N 13.5 (9.8 - 17.3) nM with a mean increase of 65%; p = 0.012). Increased copy number of pfmdr1 was not itself associated with increased IC50 values for artemether, but when interactions between the pfatp6 haplotype and increased copy number of pfmdr1 were examined together, a highly significant association was noted with IC50 values for artemether (mean IC50 (95% CI) values of 8.7 (5.9 - 11.6) versus 16.3 (10.7 - 21.8) nM with a mean increase of 87%; p = 0.0068). Previously described SNPs in pfmdr1 are also associated with differences in sensitivity to some artemisinins. CONCLUSIONS: These findings were further explored in molecular modelling experiments that suggest mutations in pfatp6 are unlikely to affect differential binding of artemisinins at their proposed site, whereas there may be differences in such binding associated with mutations in pfmdr1. Implications for a hypothesis that artemisinin resistance may be exacerbated by interactions between PfATP6 and PfMDR1 and for epidemiological studies to monitor emerging resistance are discussed

Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance

Background: Plasmodium falciparum exhibits a high degree of inter-isolate genetic diversity in its variant surface antigen (VSA) families: P. falciparum erythrocyte membrane protein 1, repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR). The role of recombination for the generation of this diversity is a subject of ongoing research. Here the genome of E5, a sibling of the 3D7 genome strain is presented. Short and long read whole genome sequencing (WGS) techniques (Ilumina, Pacific Bioscience) and a set of 84 microsatellites (MS) were employed to characterize the 3D7 and non-3D7 parts of the E5 genome. This is the first time that VSA genes in sibling parasites were analysed with long read sequencing technology. Results: Of the 5733 E5 genes only 278 genes, mostly var and rifin/stevor genes, had no orthologues in the 3D7 genome. WGS and MS analysis revealed that chromosomal crossovers occurred at a rate of 0–3 per chromosome. var, stevor and rifin genes were inherited within the respective non-3D7 or 3D7 chromosomal context. 54 of the 84 MS PCR fragments correctly identified the respective MS as 3D7- or non-3D7 and this correlated with var and rifin/stevor gene inheritance in the adjacent chromosomal regions. E5 had 61 var and 189 rifin/stevor genes. One large non-chromosomal recombination event resulted in a new var gene on chromosome 14. The remainder of the E5 3D7-type subtelomeric and central regions were identical to 3D7. Conclusions: The data show that the rifin/stevor and var gene families represent the most diverse compartments of the P. falciparum genome but that the majority of var genes are inherited without alterations within their respective parental chromosomal context. Furthermore, MS genotyping with 54 MS can successfully distinguish between two sibling progeny of a natural P. falciparum cross and thus can be used to investigate identity by descent in field isolates

Plasmodial Hsp40s: New avenues for antimalarial drug discovery

Malaria, an infectious disease caused by Plasmodium spp, is one of the world\u27s most dangerous diseases, accounting for more than half a million deaths yearly. The long years of co-habitation between the parasite and its hosts (human and mosquito), is a testimony to the parasite’s ability to escape the immune system and develop drug resistance mechanisms. Currently, an important search area for improved pharmacotherapy are molecular chaperones of the heat shock protein family, abundant in Plasmodium falciparum and contributing to its continuous survival and development. Thus far, small molecule inhibitor studies on P. falciparum Hsp70s and Hsp90s have indicated that they are promising antimalarial targets. However, not much attention has been given to Hsp40s as potential antimalarial drug targets. Hsp40s are known to function as chaperones by preventing protein aggregation, and as co-chaperones, by regulating the chaperone activities of Hsp70s to ensure proper protein folding. There are only a limited number of reviews on Hsp40s as drug targets, and the few reviews on plasmodial Hsp40s tend to focus largely on the intra-erythrocytic stage of the parasite life cycle. Therefore, this review will summarize what is known about Hsp40s throughout the malaria parasite life cycle, and critically evaluate their potential to serve as new avenues for antimalarial drug discovery

Case-control approach to identify Plasmodium falciparum polymorphisms associated with severe malaria.

BACKGROUND: Studies to identify phenotypically-associated polymorphisms in the Plasmodium falciparum 23 Mb genome will require a dense array of marker loci. It was considered promising to undertake initial allelic association studies to prospect for virulence polymorphisms in Thailand, as the low endemicity would allow higher levels of linkage disequilibrium (LD) than would exist in more highly endemic areas. METHODOLOGY/PRINCIPAL FINDINGS: Assessment of LD was first made with 11 microsatellite loci widely dispersed in the parasite genome, and 16 microsatellite loci covering a approximately 140 kb region of chromosome 2 (an arbitrarily representative non-telomeric part of the genome), in a sample of 100 P. falciparum isolates. The dispersed loci showed minimal LD (Index of Association, I(S) (A) = 0.013, P = 0.10), while those on chromosome 2 showed significant LD values mostly between loci <5 kb apart. A disease association study was then performed comparing parasites in 113 severe malaria cases and 245 mild malaria controls. Genotyping was performed on almost all polymorphisms in the binding domains of three erythrocyte binding antigens (eba175, eba140 and eba181), and repeat sequence polymorphisms approximately 2 kb apart in each of three reticulocyte binding homologues (Rh1, Rh2a/b, and Rh4). Differences between cases and controls were seen for (i) codons 388-90 in eba175, and (ii) a repeat sequence centred on Rh1 codon 667. CONCLUSIONS/SIGNIFICANCE: Allelic association studies on P. falciparum require dense genotypic markers, even in a population of only moderate endemicity that has more extensive LD than highly endemic populations. Disease-associated polymorphisms in the eba175 and Rh1 genes encode differences in the middle of previously characterised erythrocyte binding domains, marking these for further investigation

Estimating novel potential drug targets of Plasmodium falciparum by analysing the metabolic network of knock-out strains in silico

Malaria is one of the world’s most common and serious diseases causing death of about 3 million people each year. Its most severe occurrence is caused by the protozoan Plasmodium falciparum. Biomedical research could enable treating the disease by effectively and specifically targeting essential enzymes of this parasite. However, the parasite has developed resistance to existing drugsmaking it indispensable to discover new drugs. We have established a simple computational tool which analyses the topology of the metabolic network of P. falciparum to identify essential enzymes as possible drug targets.Weinvestigated the essentiality of a reaction in the metabolic network by deleting (knocking-out) such a reaction in silico. The algorithmselected neighbouring compounds of the investigated reaction that had to be produced by alternative biochemical pathways. Using breadth first searches, we tested qualitatively if these products could be generated by reactions that serve as potential deviations of the metabolic flux. With this we identified 70 essential reactions. Our results were compared with a comprehensive list of 38 targets of approved malaria drugs. When combining our approach with an in silico analysis performed recently [Yeh, I., Hanekamp, T., Tsoka, S., Karp, P.D., Altman, R.B., 2004. Computational analysis of Plasmodium falciparum metabolism: organizing genomic information to facilitate drug discovery. Genome Res. 14, 917–924] we could improve the precision of the prediction results. Finally we present a refined list of 22 new potential candidate targets for P. falciparum, half of which have reasonable evidence to be valid targets against micro-organisms and cancer