Plasmodium GPI-anchored micronemal antigen is essential for parasite transmission through the mosquito host.

Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function.

Epigenetic Silencing of RIPK3 in Hepatocytes Prevents MLKL-mediated Necroptosis From Contributing to Liver Pathologies.

BACKGROUND & AIMS: Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/ nonalcoholic steatohepatitis (NASH); however, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology. METHODS: Several animal models of human liver pathology, including diet-induced steatohepatitis in male mice and diverse infections in both male and female mice, were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. RESULTS: Identical infection kinetics and pathologic outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared with control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed a high-fat diet to induce NASH. Under all conditions tested, we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis. CONCLUSIONS: We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathologic conditions tested in this study.

Monitoring Plasmodium falciparum and Plasmodium vivax using microsatellite markers indicates limited changes in population structure after substantial transmission decline in Papua New Guinea.

Monitoring the genetic structure of pathogen populations may be an economical and sensitive approach to quantify the impact of control on transmission dynamics, highlighting the need for a better understanding of changes in population genetic parameters as transmission declines. Here we describe the first population genetic analysis of two major human malaria parasites, Plasmodium falciparum (Pf) and Plasmodium vivax (Pv), following nationwide distribution of long-lasting insecticide-treated nets (LLINs) in Papua New Guinea (PNG). Parasite isolates from pre- (2005-2006) and post-LLIN (2010-2014) were genotyped using microsatellite markers. Despite parasite prevalence declining substantially (East Sepik Province: Pf = 54.9%-8.5%, Pv = 35.7%-5.6%, Madang Province: Pf = 38.0%-9.0%, Pv: 31.8%-19.7%), genetically diverse and intermixing parasite populations remained. Pf diversity declined modestly post-LLIN relative to pre-LLIN (East Sepik: Rs  = 7.1-6.4, HE  = 0.77-0.71; Madang: Rs  = 8.2-6.1, HE  = 0.79-0.71). Unexpectedly, population structure present in pre-LLIN populations was lost post-LLIN, suggesting that more frequent human movement between provinces may have contributed to higher gene flow. Pv prevalence initially declined but increased again in one province, yet diversity remained high throughout the study period (East Sepik: Rs  = 11.4-9.3, HE  = 0.83-0.80; Madang: Rs  = 12.2-14.5, HE  = 0.85-0.88). Although genetic differentiation values increased between provinces over time, no significant population structure was observed at any time point. For both species, a decline in multiple infections and increasing clonal transmission and significant multilocus linkage disequilibrium post-LLIN were positive indicators of impact on the parasite population using microsatellite markers. These parameters may be useful adjuncts to traditional epidemiological tools in the early stages of transmission reduction.

Inhibition of Plasmepsin V Activity Blocks Plasmodium falciparum Gametocytogenesis and Transmission to Mosquitoes.

Plasmodium falciparum gametocytes infect mosquitoes and are responsible for malaria transmission. New interventions that block transmission could accelerate malaria elimination. Gametocytes develop within erythrocytes and activate protein export pathways that remodel the host cell. Plasmepsin V (PMV) is an aspartyl protease that is required for protein export in asexual parasites, but its function and essentiality in gametocytes has not been definitively proven, nor has PMV been assessed as a transmission-blocking drug target. Here, we show that PMV is expressed and can be inhibited specifically in P. falciparum stage I-II gametocytes. PMV inhibitors block processing and export of gametocyte effector proteins and inhibit development of stage II-V gametocytes. Gametocytogenesis in the presence of sublethal inhibitor concentrations results in stage V gametocytes that fail to infect mosquitoes. Therefore, PMV primes gametocyte effectors for export, which is essential for the development and fitness of gametocytes for transmission to mosquitoes.

Plasmodium falciparum subtilisin-like ookinete protein SOPT plays an important and conserved role during ookinete infection of the Anopheles stephensi midgut.

Transmission of the malaria parasite Plasmodium falciparum involves infection of Anopheles mosquitoes. Here we characterize SOPT, a protein expressed in P. falciparum ookinetes that facilitates infection of the mosquito midgut. SOPT was identified on the basis that it contains a signal peptide, a PEXEL-like sequence and is expressed in asexual, ookinete and sporozoite stages, suggesting it is involved in infecting the human or mosquito host. SOPT is predicted to contain a subtilisin-like fold with a non-canonical catalytic triad and is orthologous to P. berghei PIMMS2. Localization studies reveal that SOPT is not exported to the erythrocyte but is expressed in ookinetes at the parasite periphery. SOPT-deficient parasites develop normally through the asexual and sexual stages and produce equivalent numbers of ookinetes to NF54 controls, however, they form fewer oocysts and sporozoites in mosquitoes. SOPT-deficient parasites were also unable to activate the immune-responsive midgut invasion marker SRPN6 after mosquito ingestion, suggesting they are defective for entry into the midgut. Disruption of SOPT in P. berghei (PIMMS2) did not affect other lifecycle stages or ookinete development but again resulted in fewer oocysts and sporozoites in mosquitoes. Collectively, this study shows that SOPT/PIMMS2 plays a conserved role in ookinetes of different Plasmodium species.

Increasingly inbred and fragmented populations of Plasmodium vivax associated with the eastward decline in malaria transmission across the Southwest Pacific.

The human malaria parasite Plasmodium vivax is more resistant to malaria control strategies than Plasmodium falciparum, and maintains high genetic diversity even when transmission is low. To investigate whether declining P. vivax transmission leads to increasing population structure that would facilitate elimination, we genotyped samples from across the Southwest Pacific region, which experiences an eastward decline in malaria transmission, as well as samples from two time points at one site (Tetere, Solomon Islands) during intensified malaria control. Analysis of 887 P. vivax microsatellite haplotypes from hyperendemic Papua New Guinea (PNG, n = 443), meso-hyperendemic Solomon Islands (n = 420), and hypoendemic Vanuatu (n = 24) revealed increasing population structure and multilocus linkage disequilibrium yet a modest decline in diversity as transmission decreases over space and time. In Solomon Islands, which has had sustained control efforts for 20 years, and Vanuatu, which has experienced sustained low transmission for many years, significant population structure was observed at different spatial scales. We conclude that control efforts will eventually impact P. vivax population structure and with sustained pressure, populations may eventually fragment into a limited number of clustered foci that could be targeted for elimination.

Protein O-fucosylation in Plasmodium falciparum ensures efficient infection of mosquito and vertebrate hosts.

O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism.The role of O-glycosylation in the malaria life cycle is largely unknown. Here, the authors identify a Plasmodium protein O-fucosyltransferase and show that it is important for normal trafficking of a subset of surface proteins, particularly CSP and TRAP, and efficient infection of mosquito and vertebrate hosts.

Cell Traversal Activity Is Important for Plasmodium falciparum Liver Infection in Humanized Mice.

Malaria sporozoites are deposited into the skin by mosquitoes and infect hepatocytes. The molecular basis of how Plasmodium falciparum sporozoites migrate through host cells is poorly understood, and direct evidence of its importance in vivo is lacking. Here, we generated traversal-deficient sporozoites by genetic disruption of sporozoite microneme protein essential for cell traversal (PfSPECT) or perforin-like protein 1 (PfPLP1). Loss of either gene did not affect P. falciparum growth in erythrocytes, in contrast with a previous report that PfPLP1 is essential for merozoite egress. However, although traversal-deficient sporozoites could invade hepatocytes in vitro, they could not establish normal liver infection in humanized mice. This is in contrast with NF54 sporozoites, which infected the humanized mice and developed into exoerythrocytic forms. This study demonstrates that SPECT and perforin-like protein 1 (PLP1) are critical for transcellular migration by P. falciparum sporozoites and demonstrates the importance of cell traversal for liver infection by this human pathogen.

Role of the ER and Golgi in protein export by Apicomplexa.

Apicomplexan parasites cause diseases of medical and agricultural importance linked to dramatic changes they impart upon infected host cells. Following invasion, the malaria parasite Plasmodium falciparum renovates the host erythrocyte using mechanisms previously believed to be malaria-specific. This involves proteolytic cleavage of effectors in the endoplasmic reticulum that licences proteins for translocation into the host cell. Recently, it was demonstrated that the related parasite Toxoplasma gondii, responsible for disease in immunocompromised individuals and congenital birth defects, has an analogous pathway with some differences, including proteolytic processing in the Golgi. Here we review the similarities and distinctions in export mechanisms between these and other Apicomplexan parasites to reconcile how this group of pathogens modify their host cells to survive and proliferate.

Plasmodium vivax populations are more genetically diverse and less structured than sympatric Plasmodium falciparum populations.

INTRODUCTION: The human malaria parasite, Plasmodium vivax, is proving more difficult to control and eliminate than Plasmodium falciparum in areas of co-transmission. Comparisons of the genetic structure of sympatric parasite populations may provide insight into the mechanisms underlying the resilience of P. vivax and can help guide malaria control programs. METHODOLOGY/PRINCIPLE FINDINGS: P. vivax isolates representing the parasite populations of four areas on the north coast of Papua New Guinea (PNG) were genotyped using microsatellite markers and compared with previously published microsatellite data from sympatric P. falciparum isolates. The genetic diversity of P. vivax (He = 0.83-0.85) was higher than that of P. falciparum (He = 0.64-0.77) in all four populations. Moderate levels of genetic differentiation were found between P. falciparum populations, even over relatively short distances (less than 50 km), with 21-28% private alleles and clear geospatial genetic clustering. Conversely, very low population differentiation was found between P. vivax catchments, with less than 5% private alleles and no genetic clustering observed. In addition, the effective population size of P. vivax (30353; 13043-69142) was larger than that of P. falciparum (18871; 8109-42986). CONCLUSIONS/SIGNIFICANCE: Despite comparably high prevalence, P. vivax had higher diversity and a panmictic population structure compared to sympatric P. falciparum populations, which were fragmented into subpopulations. The results suggest that in comparison to P. falciparum, P. vivax has had a long-term large effective population size, consistent with more intense and stable transmission, and limited impact of past control and elimination efforts. This underlines suggestions that more intensive and sustained interventions will be needed to control and eventually eliminate P. vivax. This research clearly demonstrates how population genetic analyses can reveal deeper insight into transmission patterns than traditional surveillance methods.

Genetic diversity of VAR2CSA ID1-DBL2Xb in worldwide Plasmodium falciparum populations: impact on vaccine design for placental malaria.

In placental malaria (PM), sequestration of infected erythrocytes in the placenta is mediated by an interaction between VAR2CSA, a Plasmodium falciparum protein expressed on erythrocytes, and chondroitin sulfate A (CSA) on syncytiotrophoblasts. Recent works have identified ID1-DBL2Xb as the minimal CSA-binding region within VAR2CSA able to induce strong protective immunity, making it the leading candidate for the development of a vaccine against PM. Assessing the existence of population differences in the distribution of ID1-DBL2Xb polymorphisms is of paramount importance to determine whether geographic diversity must be considered when designing a candidate vaccine based on this fragment. In this study, we examined patterns of sequence variation of ID1-DBL2Xb in a large collection of P. falciparum field isolates (n=247) from different malaria-endemic areas, including Africa (Benin, Senegal, Cameroon and Madagascar), Asia (Cambodia), Oceania (Papua New Guinea), and Latin America (Peru). Detection of variants and estimation of their allele frequencies were performed using next-generation sequencing of DNA pools. A considerable amount of variation was detected along the whole gene segment, suggesting that several allelic variants may need to be included in a candidate vaccine to achieve broad population coverage. However, most sequence variants were common and extensively shared among worldwide parasite populations, demonstrating long term persistence of those polymorphisms, probably maintained through balancing selection. Therefore, a vaccine mixture including such stable antigen variants will be putatively applicable and efficacious in all world regions where malaria occurs. Despite similarity in ID1-DBL2Xb allele repertoire across geographic areas, several peaks of strong population differentiation were observed at specific polymorphic loci, pointing out putative targets of humoral immunity subject to positive immune selection.

Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally.

BACKGROUND: Malaria in humans is caused by apicomplexan parasites belonging to 5 species of the genus Plasmodium. Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease is not known. Dimorphism in defined genes has led to P. ovale parasites being divided into classic and variant types. We hypothesized that these dimorphs represent distinct parasite species. METHODS: Multilocus sequence analysis of 6 genetic characters was carried out among 55 isolates from 12 African and 3 Asia-Pacific countries. RESULTS: Each genetic character displayed complete dimorphism and segregated perfectly between the 2 types. Both types were identified in samples from Ghana, Nigeria, São Tomé, Sierra Leone, and Uganda and have been described previously in Myanmar. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts. CONCLUSIONS: We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.