Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor.

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.

Plasma endotoxin core antibody concentration and linear growth are unrelated in rural Malawian children aged 2-5 years.

BACKGROUND: Environmental enteropathy is subclinical inflammation of the upper gastrointestinal tract associated with reduced linear growth in developing countries. Usually investigators have used biopsy or a dual sugar absorption test to assess environmental enteropathy. Such tests are time and resource intensive, restricting their utility as screening methods. Serum endotoxin core antibody (EndoCab) concentration is a potential indicator of intestinal inflammation and integrity, and thus may be useful to predict environmental enteropathy. We analyzed the association of serum EndoCab levels versus linear growth and lactulose-mannitol assay results in 2-5 year old rural Malawian children. METHODS: This was an observational study of 388 rural, asymptomatic Malawian children who had anthropometric measurements taken at least every 3 months since birth. In June and July 2011, dual sugar permeability tests were performed and serum samples were drawn for EndoCab assays. Pearson correlation, Student's t test and multivariable linear regression were used to compare ln EndoCab concentrations with height-for-age z scores (HAZ) at time of sampling and 3 months later. Identical analysis was also performed for ln EndoCab versus measurements from dual sugar permeability testing performed in conjunction with serum sampling. In a subgroup of children with anthropometric data in the months prior to serum sampling, Pearson correlation was used to estimate the relationship between ln EndoCab and recent linear growth. RESULTS: Ln EndoCab concentrations were not correlated with HAZ at time of measurement (B = -0.078, P = 0.14) nor change in HAZ over the subsequent 3 months HAZ (B = -0.018, P = 0.27). EndoCab concentration was not associated with %lactulose excretion (B < 0.001, P = 0.98) nor the lactulose:mannitol ratio (B = 0.021, P = 0.62). Subgroup analysis also did not reveal any significant association between EndoCab and recent growth. CONCLUSION: EndoCab titers were not correlated with measurements of growth or intestinal permeability in rural pre-school aged Malawian children.

Mutations in the P-type cation-transporter ATPase 4, PfATP4, mediate resistance to both aminopyrazole and spiroindolone antimalarials.

Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic screen with potent activity against Plasmodium falciparum asexual and sexual stage parasites. To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance. Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell. Similarly to the spiroindolones, GNF-Pf4492 blocks parasite transmission to mosquitoes and disrupts intracellular sodium homeostasis. Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.

Drug resistance genomics of the antimalarial drug artemisinin.

Across the globe, over 200 million annual malaria infections result in up to 660,000 deaths, 77% of which occur in children under the age of five years. Although prevention is important, malaria deaths are typically prevented by using antimalarial drugs that eliminate symptoms and clear parasites from the blood. Artemisinins are one of the few remaining compound classes that can be used to cure multidrug-resistant Plasmodium falciparum infections. Unfortunately, clinical trials from Southeast Asia are showing that artemisinin-based treatments are beginning to lose their effectiveness, adding renewed urgency to the search for the genetic determinants of parasite resistance to this important drug class. We review the genetic and genomic approaches that have led to an improved understanding of artemisinin resistance, including the identification of resistance-conferring mutations in the P. falciparum kelch13 gene.

A high resolution case study of a patient with recurrent Plasmodium vivax infections shows that relapses were caused by meiotic siblings.

Plasmodium vivax infects a hundred million people annually and endangers 40% of the world's population. Unlike Plasmodium falciparum, P. vivax parasites can persist as a dormant stage in the liver, known as the hypnozoite, and these dormant forms can cause malaria relapses months or years after the initial mosquito bite. Here we analyze whole genome sequencing data from parasites in the blood of a patient who experienced consecutive P. vivax relapses over 33 months in a non-endemic country. By analyzing patterns of identity, read coverage, and the presence or absence of minor alleles in the initial polyclonal and subsequent monoclonal infections, we show that the parasites in the three infections are likely meiotic siblings. We infer that these siblings are descended from a single tetrad-like form that developed in the infecting mosquito midgut shortly after fertilization. In this natural cross we find the recombination rate for P. vivax to be 10 kb per centimorgan and we further observe areas of disequilibrium surrounding major drug resistance genes. Our data provide new strategies for studying multiclonal infections, which are common in all types of infectious diseases, and for distinguishing P. vivax relapses from reinfections in malaria endemic regions. This work provides a theoretical foundation for studies that aim to determine if new or existing drugs can provide a radical cure of P. vivax malaria.

Identification of pathogen genomic variants through an integrated pipeline.

BACKGROUND: Whole-genome sequencing represents a powerful experimental tool for pathogen research. We present methods for the analysis of small eukaryotic genomes, including a streamlined system (called Platypus) for finding single nucleotide and copy number variants as well as recombination events. RESULTS: We have validated our pipeline using four sets of Plasmodium falciparum drug resistant data containing 26 clones from 3D7 and Dd2 background strains, identifying an average of 11 single nucleotide variants per clone. We also identify 8 copy number variants with contributions to resistance, and report for the first time that all analyzed amplification events are in tandem. CONCLUSIONS: The Platypus pipeline provides malaria researchers with a powerful tool to analyze short read sequencing data. It provides an accurate way to detect SNVs using known software packages, and a novel methodology for detection of CNVs, though it does not currently support detection of small indels. We have validated that the pipeline detects known SNVs in a variety of samples while filtering out spurious data. We bundle the methods into a freely available package.

Targeting Plasmodium PI(4)K to eliminate malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Direct transfer of whole genomes from bacteria to yeast.

Transfer of genomes into yeast facilitates genome engineering for genetically intractable organisms, but this process has been hampered by the need for cumbersome isolation of intact genomes before transfer. Here we demonstrate direct cell-to-cell transfer of bacterial genomes as large as 1.8 megabases (Mb) into yeast under conditions that promote cell fusion. Moreover, we discovered that removal of restriction endonucleases from donor bacteria resulted in the enhancement of genome transfer.

A key role for lipoic acid synthesis during Plasmodium liver stage development.

The successful navigation of malaria parasites through their life cycle, which alternates between vertebrate hosts and mosquito vectors, requires a complex interplay of metabolite synthesis and salvage pathways. Using the rodent parasite Plasmodium berghei, we have explored the synthesis and scavenging pathways for lipoic acid, a short-chain fatty acid derivative that regulates the activity of α-ketoacid dehydrogenases including pyruvate dehydrogenase. In Plasmodium, lipoic acid is either synthesized de novo in the apicoplast or is scavenged from the host into the mitochondrion. Our data show that sporozoites lacking the apicoplast lipoic acid protein ligase LipB are markedly attenuated in their infectivity for mice, and in vitro studies document a very late liver stage arrest shortly before the final phase of intra-hepaticparasite maturation. LipB-deficient asexual blood stage parasites show unimpaired rates of growth in normal in vitro or in vivo conditions. However, these parasites showed reduced growth in lipid-restricted conditions induced by treatment with the lipoic acid analogue 8-bromo-octanoate or with the lipid-reducing agent clofibrate. This finding has implications for understanding Plasmodium pathogenesis in malnourished children that bear the brunt of malarial disease. This study also highlights the potential of exploiting lipid metabolism pathways for the design of genetically attenuated sporozoite vaccines.

Mitotic evolution of Plasmodium falciparum shows a stable core genome but recombination in antigen families.

Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases) revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone). In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1) on chromosome 1. We observed 18 large-scale (>1 kb on average) deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10(-6) structural variants per base pair per generation). Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0-9.7×10(-9) mutations per base pair per generation), we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum evolves to evade control efforts within both the individual hosts and large populations.

Detection of low-concentration host mRNA transcripts in Malawian children at risk for environmental enteropathy.

BACKGROUND AND OBJECTIVE: Transcriptomic analysis of fecal samples is an emerging method for the diagnosis of gastrointestinal pathology because it is noninvasive and requires minute volumes of analyte; however, detection of mRNA in low copy numbers in human stool is challenging. Our objective was to develop a method for detecting human mRNA suggestive of environmental enteropathy (EE) in feces. METHODS: Stool samples from 70 Malawian children, 34 without EE and 36 with EE, as defined by dual sugar absorption, were used to develop the methodology for mRNA detection. Multiple RNA isolation techniques and polymerase chain reaction formats were tested to detect 38 potential mRNA biomarkers suggestive of EE, and the results compared. RESULTS: RNA isolation using magnetic bead extraction best recovered host mRNA in stool, and digital droplet polymerase chain reaction was the most sensitive format to detect low copy numbers of mRNA. In all of the 70 samples, >20 copies of glyceraldehyde-3-phosphate dehydrogenase/200 mg of stool were detected. Copy numbers of potential biomarkers were normalized to glyceraldehyde-3-phosphate dehydrogenase, to account for interspecimen differences in concentration of human mRNA. Of the 38 transcripts chosen for initial evaluation, 24 had copy numbers >10 in all of the samples tested. Of the 6 potential markers measured in all of the 70 samples, REG4 best differentiated children with and without EE. CONCLUSIONS: A reproducible and reliable method to quantify human mRNA in stool present in low copy numbers has been developed, and may prove useful in investigations of EE and possibly other inflammatory gut conditions.

Nuclear repositioning precedes promoter accessibility and is linked to the switching frequency of a Plasmodium falciparum invasion gene.

Variation of surface adhesins, such as the Plasmodium falciparum erythrocyte invasion ligand PfRh4, is critical for virulence and immune evasion in many microbes. While phenotypic switching is linked to transcriptional changes and chromatin function, the determinants of switching frequency remain poorly defined. By expressing a prokaryotic DNA methylase in P. falciparum, we directly assayed accessibility of transcriptionally active and silent chromatin at the PfRh4 locus. Parasites selected for in vivo PfRh4 activation show a reversible increase in promoter accessibility and exhibit perinuclear repositioning of the locus from a silent to a conserved activation domain. Forced activation of a proximal gene results in a similar repositioning of the PfRh4 locus, with a concomitant increase in PfRh4 activation in a subpopulation of parasites and promoter accessibility correlating with actively transcribed loci. Thus, nuclear repositioning is associated with increased P. falciparum switching frequency, while promoter accessibility is tightly linked to clonally active PfRh4 promoters.

Abnormal gut integrity is associated with reduced linear growth in rural Malawian children.

The aim of the present study was to investigate the relation of environmental enteropathy, as measured by the dual sugar absorption test, to linear growth faltering in 2- to 5-year-old Malawian children. Dietary quality, food insecurity, anthropometry, and site-specific sugar testing were measured in 418 children, and anthropometry was reassessed 3 months later. A linear regression model predicting linear growth was created. Better growth was associated with less urinary lactulose excretion, more clean water usage, not sleeping with animals, and no previous history of malnutrition. Eighty-seven percent of children studied demonstrated evidence of environmental enteropathy. In conclusion, abnormal gut integrity is associated with reduced linear growth in a population of rural African preschool-age children.

Perturbed zinc homeostasis in rural 3-5-y-old Malawian children is associated with abnormalities in intestinal permeability attributed to tropical enteropathy.

Tropical enteropathy and zinc deficiency are major public health problems worldwide. Tropical enteropathy is characterized by reduced mannitol absorption with normal or increased lactulose absorption when a dual sugar absorption test is administered, the results of which are reported as the lactulose:mannitol ratio (L:M). Zinc homeostasis is quantified with a dual stable isotope test. This study tested the hypothesis that endogenous fecal zinc (EFZ) was correlated with the L:M. A dual sugar absorption test and dual stable isotope test were performed on 25 asymptomatic Malawian children aged 3-5 y at risk for tropical enteropathy and zinc deficiency. EFZ and net zinc retention were estimated and correlated with the L:M. Twenty-two children (88%) had an abnormal L:M (L:M>0.10), and the L:M was 0.24+/-0.10 (mean+/-SD). EFZ was 1.68+/-1.06 mg/d, a quantity greater than is seen in healthy populations from the developed world. EFZ was positively correlated with the L:M (r=0.62, p<0.001). Net zinc retention (0.67+/-1.6 mg/d) was negatively correlated with the L:M (r=-0.47, p=0.02). This suggests that perturbed zinc homeostasis is associated with subclinical enteropathy in these children.

Detection of bacterial spores with lanthanide-macrocycle binary complexes.

The detection of bacterial spores via dipicolinate-triggered lanthanide luminescence has been improved in terms of detection limit, stability, and susceptibility to interferents by use of lanthanide-macrocycle binary complexes. Specifically, we compared the effectiveness of Sm, Eu, Tb, and Dy complexes with the macrocycle 1,4,7,10-tetraazacyclododecane-1,7-diacetate (DO2A) to the corresponding lanthanide aquo ions. The Ln(DO2A)(+) binary complexes bind dipicolinic acid (DPA), a major constituent of bacterial spores, with greater affinity and demonstrate significant improvement in bacterial spore detection. Of the four luminescent lanthanides studied, the terbium complex exhibits the greatest dipicolinate binding affinity (100-fold greater than Tb(3+) alone, and 10-fold greater than other Ln(DO2A)(+) complexes) and highest quantum yield. Moreover, the inclusion of DO2A extends the pH range over which Tb-DPA coordination is stable, reduces the interference of calcium ions nearly 5-fold, and mitigates phosphate interference 1000-fold compared to free terbium alone. In addition, detection of Bacillus atrophaeus bacterial spores was improved by the use of Tb(DO2A)(+), yielding a 3-fold increase in the signal-to-noise ratio over Tb(3+). Out of the eight cases investigated, the Tb(DO2A)(+) binary complex is best for the detection of bacterial spores.

Supplementary feeding with either ready-to-use fortified spread or corn-soy blend in wasted adults starting antiretroviral therapy in Malawi: randomised, investigator blinded, controlled trial.

OBJECTIVE: To investigate the effect of two different food supplements on body mass index (BMI) in wasted Malawian adults with HIV who were starting antiretroviral therapy. DESIGN: Randomised, investigator blinded, controlled trial. SETTING: Large, public clinic associated with a referral hospital in Blantyre, Malawi. PARTICIPANTS: 491 adults with BMI <18.5. INTERVENTIONS: Ready-to-use fortified spread (n=245) or corn-soy blend (n=246). PRIMARY OUTCOMES: changes in BMI and fat-free body mass after 3.5 months. SECONDARY OUTCOMES: survival, CD4 count, HIV viral load, quality of life, and adherence to antiretroviral therapy. RESULTS: The mean BMI at enrolment was 16.5. After 14 weeks, patients receiving fortified spread had a greater increase in BMI and fat-free body mass than those receiving corn-soy blend: 2.2 (SD 1.9) v 1.7 (SD 1.6) (difference 0.5, 95% confidence interval 0.2 to 0.8), and 2.9 (SD 3.2) v 2.2 (SD 3.0) kg (difference 0.7 kg, 0.2 to 1.2 kg), respectively. The mortality rate was 27% for those receiving fortified spread and 26% for those receiving corn-soy blend. No significant differences in the CD4 count, HIV viral load, assessment of quality of life, or adherence to antiretroviral therapy were noted between the two groups. CONCLUSION: Supplementary feeding with fortified spread resulted in a greater increase in BMI and lean body mass than feeding with corn-soy blend. TRIAL REGISTRATION: Current Controlled Trials ISRCTN67515515.

A large-scale operational study of home-based therapy with ready-to-use therapeutic food in childhood malnutrition in Malawi.

Home-based therapy with ready-to-use therapeutic food (RUTF) for the treatment of malnutrition has better outcomes in the research setting than standard therapy. This study examined outcomes of malnourished children aged 6-60 months enrolled in operational home-based therapy with RUTF. Children enrolled in 12 rural centres in southern Malawi were diagnosed with moderate or severe malnutrition according to the World Health Organization guidelines. They were treated with 733 kJ kg(-1) day(-1) of RUTF and followed fortnightly for up to 8 weeks. Staff at each centre followed one of three models: medical professionals administered treatment (5 centres), patients were referred by medical professionals and treated by community health aids (4 centres), or community health aids administered treatment (3 centres). The primary outcome of the study was clinical status, defined as recovered, failed, died or dropped out. Regression modelling was conducted to determine what aspects of the centre (formal training of staff, location along a main road) contributed to the outcome. Of 2131 severely malnourished children and 806 moderately malnourished, 89% and 85% recovered, respectively. Thirty-four (4%) of the moderately malnourished children failed, with 20 (2%) deaths, and 61 (3%) of the severely malnourished children failed, with 29 (1%) deaths. Centre location along a road was associated with a poor outcome. Outcomes for severely malnourished children were acceptable with respect to both the Sphere guidelines and the Prudhon case fatality index. Home-based therapy with RUTF yields acceptable results without requiring formally medically trained personnel; further implementation in comparable settings should be considered.