Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.     

Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria

Background

Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness.

Methods

Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits.

Results

Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions.

Conclusions

These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic ‘supermales’ could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.     

Cloning and characterization of Plasmodium vivax serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), which catalyzes the reversible reaction of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate, is one of the three enzymes in dTMP synthesis pathway that is highly active during cell division and has been proposed as a potential chemotherapeutic target in infectious diseases and cancer. This is the first study to describe nucleotide and amino acid sequences of SHMT from the malaria parasite Plasmodium vivax. Sequencing of 12 P. vivax isolates revealed limited polymorphisms in 3 noncoding regions. Its biological function is also reported.     

Diagnosing Severe Falciparum Malaria in Parasitaemic African Children: A Prospective Evaluation of Plasma PfHRP2 Measurement

Background

In African children, distinguishing severe falciparum malaria from other severe febrile illnesses with coincidental Plasmodium falciparum parasitaemia is a major challenge. P. falciparum histidine-rich protein 2 (PfHRP2) is released by mature sequestered parasites and can be used to estimate the total parasite burden. We investigated the prognostic significance of plasma PfHRP2 and used it to estimate the malaria-attributable fraction in African children diagnosed with severe malaria.

Methods and Findings

Admission plasma PfHRP2 was measured prospectively in African children (from Mozambique, The Gambia, Kenya, Tanzania, Uganda, Rwanda, and the Democratic Republic of the Congo) aged 1 month to 15 years with severe febrile illness and a positive P. falciparum lactate dehydrogenase (pLDH)-based rapid test in a clinical trial comparing parenteral artesunate versus quinine (the AQUAMAT trial, ISRCTN 50258054). In 3,826 severely ill children, Plasmadium falciparum PfHRP2 was higher in patients with coma (p = 0.0209), acidosis (p<0.0001), and severe anaemia (p<0.0001). Admission geometric mean (95%CI) plasma PfHRP2 was 1,611 (1,350–1,922) ng/mL in fatal cases (n = 381) versus 1,046 (991–1,104) ng/mL in survivors (n = 3,445, p<0.0001), without differences in parasitaemia as assessed by microscopy. There was a U-shaped association between log₁₀ plasma PfHRP2 and risk of death. Mortality increased 20% per log₁₀ increase in PfHRP2 above 174 ng/mL (adjusted odds ratio [AOR] 1.21, 95%CI 1.05–1.39, p = 0.009). A mechanistic model assuming a PfHRP2-independent risk of death in non-malaria illness closely fitted the observed data and showed malaria-attributable mortality less than 50% with plasma PfHRP2≤174 ng/mL. The odds ratio (OR) for death in artesunate versus quinine-treated patients was 0.61 (95%CI 0.44–0.83, p = 0.0018) in the highest PfHRP2 tertile, whereas there was no difference in the lowest tertile (OR 1.05; 95%CI 0.69–1.61; p = 0.82). A limitation of the study is that some conclusions are drawn from a mechanistic model, which is inherently dependent on certain assumptions. However, a sensitivity analysis of the model indicated that the results were robust to a plausible range of parameter estimates. Further studies are needed to validate our findings.

Conclusions

Plasma PfHRP2 has prognostic significance in African children with severe falciparum malaria and provides a tool to stratify the risk of “true” severe malaria-attributable disease as opposed to other severe illnesses in parasitaemic African children. Please see later in the article for the Editors'' Summary.     

Molecular characterization of the porcine testis-specificphosphoglycerate kinase 2 (<Emphasis Type="Italic">PGK2</Emphasis>) gene and its association with male fertility

We have isolated and characterized the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene, and 1665 bp of full-length PGK2 cDNA were also compiled using modified rapid amplification 5-RACE and 3-RACE information. The results of genomic and cDNA sequences of the porcine PGK2 gene demonstrated that it is a single-exon intronless gene with a complete open reading frame of 1251 bp encoding a PGK protein of 417 amino acids. Real-time quantitative PCR results showed that PGK2 mRNA was solely expressed in the testis. There was a lower amount of PGK2 expression in the testis of a 10-month-old herniated boar and a very small amount of PGK2 expression in the testis of an 8–week-old cryptorchid piglet compared to an adult boar. Two SNPs in the PGK2 gene (SNP-A: T427C; SNP-B: C914A) resulting in amino acid substitutions (SNP-A: Ser¹⁰²–Pro¹⁰²; SNP-B: Thr²⁶⁴–Lys²⁶⁴) were detected and genotyped among six pig breeds. The nucleotide C at SNP-A responsible for the amino acid exchange to proline could lead to the loss of a casein kinase II (CK2) phosphorylation site in the PGK2 peptide. Association analyses between PGK2 genotypes and several traits of sperm quantity and quality were performed. The results showed that SNP-B has a positive significant effect on semen volume in the breed Pietrain (p=0.08), i.e., boars carrying genotype CC revealed an increased volume of 49 ml compared with boars having the genotype AA.The nucleotide sequence data reported in this article have been submitted to GenBank and have been assigned the accession numbers AY500132 and AY486962.     

Short-term in vitro culture of field isolates of Plasmodium vivax using umbilical cord blood

Plasmodium vivax research has been hampered by the lack of technology for culturing this parasite. Culturing P. vivax is difficult because the parasite selectively invades reticulocytes. Here we describe a modified procedure to establish and maintain short-term cultures of freshly collected P. vivax parasites using reticulocyte-enriched cord blood. Using this method, parasites could be cultured for a month. Manipulation of the culture allowed procurement of synchronized stages of the parasite. This short-term culture method can be easily adapted to study various aspects of the parasite biology.     

Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2

BackgroundIn falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria.ConclusionPlasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes.     

Runx1 is involved in the fusion of the primary and the secondary palatal shelves

Runx1 is expressed in medial edge epithelial (MEE) cells of the palatal shelf. Conditionally rescued Runx1^−/− mice showed limited clefting in the anterior junction between the primary and the secondary palatal shelves, but not in the junction between the secondary palates. In wild type mice, the fusing epithelial surface exhibited a rounded cobblestone-like appearance, while such cellular prominence was less evident in the Runx1 mutants. We also found that Fgf18 was expressed in the mesenchyme underlying the MEE and that locally applied FGF18 induced ectopic Runx1 expression in the epithelium of the palatal explants, indicating that Runx1 was induced by mesenchymal Fgf18 signaling. On the other hand, unpaired palatal explant cultures revealed the presence of anterior-posterior (A-P) differences in the MEE fates and fusion mechanism. Interestingly, the location of anterior clefting in Runx1 mutants corresponded to the region with different MEE behavior. These data showed a novel function of Runx1 in morphological changes in the MEE cells in palatal fusion, which is, at least in part, regulated by the mesenchymal Fgf signaling via an epithelial-mesenchymal interaction.     

Sequence variation does not confound the measurement of plasma PfHRP2 concentration in African children presenting with severe malaria

ABSTRACT: BACKGROUND: Plasmodium falciparum histidine-rich protein PFHRP2 measurement is used widely for diagnosis, and more recently for severity assessment in falciparum malaria. The Pfhrp2 gene is highly polymorphic, with deletion of the entire gene reported in both laboratory and field isolates. These issues potentially confound the interpretation of PFHRP2 measurements. METHODS: Studies designed to detect deletion of Pfhrp2 and its paralog Pfhrp3 were undertaken with samples from patients in seven countries contributing to the largest hospital-based severe malaria trial (AQUAMAT). The quantitative relationship between sequence polymorphism and PFHRP2 plasma concentration was examined in samples from selected sites in Mozambique and Tanzania. RESULTS: There was no evidence for deletion of either Pfhrp2 or Pfhrp3 in the 77 samples with lowest PFHRP2 plasma concentrations across the seven countries. Pfhrp2 sequence diversity was very high with no haplotypes shared among 66 samples sequenced. There was no correlation between Pfhrp2 sequence length or repeat type and PFHRP2 plasma concentration. CONCLUSIONS: These findings indicate that sequence polymorphism is not a significant cause of variation in PFHRP2 concentration in plasma samples from African children. This justifies the further development of plasma PFHRP2 concentration as a method for assessing African children who may have severe falciparum malaria. The data also add to the existing evidence base supporting the use of rapid diagnostic tests based on PFHRP2 detection.