A G358S mutation in the Plasmodium falciparum Na⁺ pump PfATP4 confers clinically-relevant resistance to cipargamin

Diverse compounds target the Plasmodium falciparum Na(+) pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4(G358S) parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na(+) regulation. The G358S mutation reduces the affinity of PfATP4 for Na(+) and is associated with an increase in the parasite’s resting cytosolic [Na(+)]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4(G358S) parasites, and that their combination with unrelated antimalarials may mitigate against resistance development

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website

High Sporozoite Antibody Titers in Conjunction with Microscopically Detectable Blood Infection Display Signatures of Protection from Clinical Malaria

Immunoepidemiological studies typically reveal slow, age-dependent acquisition of immune responses against Plasmodium falciparum sporozoites. Naturally acquired immunity against preerythrocytic stages is considered inadequate to confer protection against clinical malaria. To explore previously unrecognized antisporozoite responses, we measured serum levels of naturally acquired antibodies to whole Plasmodium falciparum sporozoites (Pfspz) and the immunodominant (NANP)5 repeats of the major sporozoite surface protein, circumsporozoite protein, in a well-characterized Kenyan cohort. Sera were sampled at the start of the malaria transmission season, and all subjects were prospectively monitored for uncomplicated clinical malaria in the ensuing 6 months. We used Kaplan-Meier analysis and multivariable regression to investigate the association of antisporozoite immunity with incidence of clinical malaria. Although naturally acquired humoral responses against Pfspz and (NANP)5 were strongly correlated (p < 0.0001), 37% of Pfspz responders did not recognize (NANP)5. The prevalence and magnitude of antisporozoite responses increased with age, although some high Pfspz responders were identified among children. Survival analysis revealed a reduced risk of and increased time to first or only episode of clinical malaria among Pfspz or (NANP)5 responders carrying microscopically detectable Plasmodium falciparum (Pf) parasitemia at the start of the transmission season (p < 0.03). Our Cox regression interaction models indicated a potentially protective interaction between high anti-Pfspz (p = 0.002) or anti-(NANP)5 (p = 0.001) antibody levels and microscopically detectable Pf parasitemia on the risk of subsequent clinical malaria. Our findings indicate that robust antisporozoite immune responses can be naturally acquired already at an early age. A potentially protective role of high levels of anti-Pfspz antibodies against clinical episodes of uncomplicated malaria was detected, suggesting that antibody-mediated preerythrocytic immunity might indeed contribute to protection in nature

Levels of circumsporozoite protein in the Plasmodium oocyst determine sporozoite morphology

The sporozoite stage of the Plasmodium parasite is formed by budding from a multinucleate oocyst in the mosquito midgut. During their life, sporozoites must infect the salivary glands of the mosquito vector and the liver of the mammalian host; both events depend on the major sporozoite surface protein, the circumsporozoite protein (CS). We previously reported that Plasmodium berghei oocysts in which the CS gene is inactivated do not form sporozoites. Here, we analyzed the ultrastructure of P.berghei oocyst differentiation in the wild type, recombinants that do not produce or produce reduced amounts of CS, and corresponding complemented clones. The results indicate that CS is essential for establishing polarity in the oocyst. The amounts of CS protein correlate with the extent of development of the inner membranes and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites. This is a first example of a protein controlling both morphogenesis and infectivity of a parasite stage

TgSUB2 is a Toxoplasma gondii rhoptry organelle processing proteinase.

All parasites in the phylum Apicomplexa, including Toxoplasma gondii and Plasmodium falciparum, contain rhoptries, specialized secretory organelles whose contents are thought to be essential for successful invasion of host cells. Serine proteinase inhibitors have been reported to block host cell invasion by both T. gondii and P. falciparum. We describe the cloning and characterization of TgSUB2, a subtilisin-like serine proteinase, from T. gondii. Like its closest homologue P. falciparum PfSUB-2, TgSUB2 is predicted to be a type I transmembrane protein. Disruption of TgSUB2 was unsuccessful implying that TgSUB2 is an essential gene. TgSUB2 undergoes autocatalytic processing as it traffics through the secretory pathway. TgSUB2 localizes to rhoptries and associates with rhoptry protein ROP1, a potential substrate. A sequence within TgSUB2 with homology to the ROP1 cleavage site (after Glu) was identified and mutated by site-directed mutagenesis. This mutation abolished TgSUB2 autoprocessing suggesting that TgSUB2 is a rhoptry protein maturase with similar specificity to the ROP1 maturase. Processing of secretory organelle contents appears to be ubiquitous among the Apicomplexa. As subtilases are present in genomes of all the Apicomplexa sequenced to date, subtilases may represent a novel chemotherapeutic target

High Sporozoite Antibody Titers in Conjunction with Microscopically Detectable Blood Infection Display Signatures of Protection from Clinical Malaria

Immunoepidemiological studies typically reveal slow, age-dependent acquisition of immune responses against Plasmodium falciparum sporozoites. Naturally acquired immunity against preerythrocytic stages is considered inadequate to confer protection against clinical malaria. To explore previously unrecognized antisporozoite responses, we measured serum levels of naturally acquired antibodies to whole Plasmodium falciparum sporozoites (Pfspz) and the immunodominant (NANP)5 repeats of the major sporozoite surface protein, circumsporozoite protein, in a well-characterized Kenyan cohort. Sera were sampled at the start of the malaria transmission season, and all subjects were prospectively monitored for uncomplicated clinical malaria in the ensuing 6 months. We used Kaplan–Meier analysis and multivariable regression to investigate the association of antisporozoite immunity with incidence of clinical malaria. Although naturally acquired humoral responses against Pfspz and (NANP)5 were strongly correlated (p &lt; 0.0001), 37% of Pfspz responders did not recognize (NANP)5. The prevalence and magnitude of antisporozoite responses increased with age, although some high Pfspz responders were identified among children. Survival analysis revealed a reduced risk of and increased time to first or only episode of clinical malaria among Pfspz or (NANP)5 responders carrying microscopically detectable Plasmodium falciparum (Pf) parasitemia at the start of the transmission season (p &lt; 0.03). Our Cox regression interaction models indicated a potentially protective interaction between high anti-Pfspz (p = 0.002) or anti-(NANP)5 (p = 0.001) antibody levels and microscopically detectable Pf parasitemia on the risk of subsequent clinical malaria. Our findings indicate that robust antisporozoite immune responses can be naturally acquired already at an early age. A potentially protective role of high levels of anti-Pfspz antibodies against clinical episodes of uncomplicated malaria was detected, suggesting that antibody-mediated preerythrocytic immunity might indeed contribute to protection in nature