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As malaria control programmes concentrate their efforts towards malaria elimination a better understanding of malaria transmission patterns at fine spatial resolution units becomes necessary. Defining spatial units that consider transmission heterogeneity, human movement and migration will help to set up achievable malaria elimination milestones and guide the creation of efficient operational administrative control units. Using a combination of genetic and epidemiological data we defined a malaria transmission unit as the area contributing 95% of malaria cases diagnosed at the catchment facility located in the town of Guapi in the South Pacific Coast of Colombia. We provide data showing that P. falciparum malaria transmission is heterogeneous in time and space and analysed, using topological data analysis, the spatial connectivity, at the micro epidemiological level, between parasite populations circulating within the unit. To illustrate the necessity to evaluate the efficacy of malaria control measures within the transmission unit in order to increase the efficiency of the malaria control effort, we provide information on the size of the asymptomatic reservoir, the nature of parasite genotypes associated with drug resistance as well as the frequency of the Pfhrp2/3 deletion associated with false negatives when using Rapid Diagnostic Tests.

The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in human malaria species such as P. falciparum and P. knowlesi, in part because the extent to which pooled transfections can be performed in these species remains to be evaluated. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vector acquisition allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution cloning of P. falciparum transfectants showed that individual clones possess as many as seven episomal barcodes, revealing that an intake of multiple vectors is a frequent event despite the inefficient transfection efficiency. Transfection of three spectrally-distinct fluorescent reporters allowed us to evaluate different transfection methods and revealed that schizont-stage transfection limited the tendency for parasites to take up multiple vectors. In contrast to P. falciparum, we observed that the higher transfection efficiency of P. knowlesi resulted in near complete representation of the library. These findings have important implications for how reverse genetics can be scaled in culturable Plasmodium species.

Plasmodium falciparum interacts with several human cell types during their complex life cycle, including erythrocytes and hepatocytes. The enuclated nature of erythrocytes makes them inaccessible to genetic tools, which in turn makes studying erythrocyte proteins involved in malaria invasion and development particularly difficult. Here we overcome this limitation using stem cell technology to develop a universal differentiation protocol for in vitro derivation of erythrocytes from a variety of stem cell lines of diverse origin. This allows manipulation of erythrocytic genes and examination of their impact on the parasite by flow cytometric detection of parasite haemozoin. Deletion of Basigin, the essential receptor for P. falciparum, abrogates invasion, while other less studied proteins such as ATP2B4 have a minor effect. Reprogramming of induced pluripotent stem cells from α-thalassemia primary samples shows reduced infection levels, demonstrating this approach is useful for understanding the effect of natural human polymorphisms on the disease.

Invasion of human erythrocytes is essential for Plasmodium falciparum parasite survival and pathogenesis, and is also a complex phenotype. While some later steps in invasion appear to be invariant and essential, the earlier steps of recognition are controlled by a series of redundant, and only partially understood, receptor-ligand interactions. Reverse genetic analysis of laboratory adapted strains has identified multiple genes that when deleted can alter invasion, but how the relative contributions of each gene translate to the phenotypes of clinical isolates is far from clear. We used a forward genetic approach to identify genes responsible for variable erythrocyte invasion by phenotyping the parents and progeny of previously generated experimental genetic crosses. Linkage analysis using whole genome sequencing data revealed a single major locus was responsible for the majority of phenotypic variation in two invasion pathways. This locus contained the PfRh2a and PfRh2b genes, members of one of the major invasion ligand gene families, but not widely thought to play such a prominent role in specifying invasion phenotypes. Variation in invasion pathways was linked to significant differences in PfRh2a and PfRh2b expression between parasite lines, and their role in specifying alternative invasion was confirmed by CRISPR-Cas9-mediated genome editing. Expansion of the analysis to a large set of clinical P. falciparum isolates revealed common deletions, suggesting that variation at this locus is a major cause of invasion phenotypic variation in the endemic setting. This work has implications for blood-stage vaccine development and will help inform the design and location of future large-scale studies of invasion in clinical isolates., Author summary Plasmodium parasites cause more than 200 million cases of malaria each year. All the symptoms of malaria are caused after Plasmodium parasites invade human red blood cells. Once inside, they grow, multiply and break open the red blood cells to release new parasites. This cycle is repeated every 48 hours, rapidly amplifying the number of parasites and causing severe anemia and other complications. Plasmodium falciparum, the parasite species responsible for almost all malaria deaths, can use multiple different pathways to invade human red blood cells, but the relative importance of each is not well understood. We tested the invasion pathways used by a collection of closely related parasites and compared their genome sequences to identify the genes responsible. This analysis revealed that expression differences in two neighboring genes of the Reticulocyte Binding Homologue family are responsible for most of the variation in two invasion pathways. P. falciparum may use variation in these genes to avoid the immune system or adapt to specific blood groups, which has important implications for vaccine development against malaria.

The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in P. falciparum, mainly because the extent to which pooled transfections can be performed in this species still remains unknown. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vectors in different transfections allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution cloning showed that single transfected parasites routinely carry as many as seven episomal barcodes, revealing an intake of multiple vectors in a highly non-uniform fashion. Transfection of non-overlapping fluorescent proteins, which allowed us to follow the dynamics of the process, confirmed the tendency for parasites to take up multiple vectors from the early stages of transfection. This finding has important implications for how reverse genetics can be scaled in P. falciparum.

This month’s Genome Watch explores the genetic variability of the anti-malaria vaccine protein and discusses its significance for an efficacious intervention.

Intraerythrocytic development of the human malaria parasite Plasmodium falciparum appears as a continuous flow through growth and proliferation. To develop a greater understanding of the critical regulatory events, we utilized piggyBac insertional mutagenesis to randomly disrupt genes. Screening a collection of piggyBac mutants for slow growth, we isolated the attenuated parasite C9, which carried a single insertion disrupting the open reading frame (ORF) of PF3D7_1305500. This gene encodes a protein structurally similar to a mitogen-activated protein kinase (MAPK) phosphatase, except for two notable characteristics that alter the signature motif of the dual-specificity phosphatase domain, suggesting that it may be a low-activity phosphatase or pseudophosphatase. C9 parasites demonstrated a significantly lower growth rate with delayed entry into the S/M phase of the cell cycle, which follows the stage of maximum PF3D7_1305500 expression in intact parasites. Genetic complementation with the full-length PF3D7_1305500 rescued the wild-type phenotype of C9, validating the importance of the putative protein phosphatase PF3D7_1305500 as a regulator of pre-S-phase cell cycle progression in P. falciparum .

The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types.

This month’s Genome Watch highlights discoveries of high variability in the genes underlying cytoplasmic incompatibility among Wolbachia strains that help identify the best strains to use for the control of vector-borne diseases such as Zika virus and malaria.

Coordinated regulation of gene expression is a hallmark of the Plasmodium falciparum asexual blood-stage development cycle. We report that carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1) is critical in regulating more than 1,000 genes during malaria parasites' intraerythrocytic stages, especially egress and invasion proteins. CAF1 knockout results in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress, and invasion of new host cells, leading to premature release of predominantly half-finished merozoites, drastically reducing the intraerythrocytic growth rate of the parasite. This study demonstrates that CAF1 of the CCR4-Not complex is a significant gene regulatory mechanism needed for Plasmodium development within the human host.

The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in human malaria species such as P. falciparum and P. knowlesi, in part because the extent to which pooled transfections can be performed in these species remains to be evaluated. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vector acquisition allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution cloning of P. falciparum transfectants showed that individual clones possess as many as seven episomal barcodes, revealing that an intake of multiple vectors is a frequent event despite the inefficient transfection efficiency. Transfection of three spectrally-distinct fluorescent reporters allowed us to evaluate different transfection methods and revealed that schizont-stage transfection limited the tendency for parasites to take up multiple vectors. In contrast to P. falciparum, we observed that the higher transfection efficiency of P. knowlesi resulted in near complete representation of the library. These findings have important implications for how reverse genetics can be scaled in culturable Plasmodium species.

As malaria control programmes concentrate their efforts towards malaria elimination a better understanding of malaria transmission patterns at fine spatial resolution units becomes necessary. Defining spatial units that consider transmission heterogeneity, human movement and migration will help to set up achievable malaria elimination milestones and guide the creation of efficient operational administrative control units. Using a combination of genetic and epidemiological data we defined a malaria transmission unit as the area contributing 95% of malaria cases diagnosed at the catchment facility located in the town of Guapi in the South Pacific Coast of Colombia. We provide data showing that P. falciparum malaria transmission is heterogeneous in time and space and analysed, using topological data analysis, the spatial connectivity, at the micro epidemiological level, between parasite populations circulating within the unit. To illustrate the necessity to evaluate the efficacy of malaria control measures within the transmission unit in order to increase the efficiency of the malaria control effort, we provide information on the size of the asymptomatic reservoir, the nature of parasite genotypes associated with drug resistance as well as the frequency of the Pfhrp2/3 deletion associated with false negatives when using Rapid Diagnostic Tests.

Malaria antibody detection is valuable in providing retrospective confirmation of an attack of malaria. Blood bank screening is another area were malaria serology is potentially useful. In the present study, we tested the presence of antibodies to Plasmodium falciparum in sera from blood bank donors of non-endemic and malaria-endemic areas of Venezuela. Sera from 1,000 blood donors were tested by an indirect immunofluorescent antibody (IFA) assay and an IgG-ELISA for the presence of malaria antibodies using a synchronized in vitro-cultured Venezuelan isolate of P. falciparum as the antigen source. A selected group of positive and negative sera (n = 100) was also tested by a dot-IgG-ELISA. Positive results (reciprocal titer > or = 40) were found in 0.8% and 3.8% of blood donors when tested by the IFA assay and in 0.8% and 2% (optical density > or = 0.2) when tested by the IgG-ELISA in Caracas (non-endemic area) and Bolivar City (endemic area), respectively. The presence of anti-malarial antibodies in some sera from non-endemic areas such as Caracas reflects the increased potential risk of post-transfusional malaria in those areas due to the mobility of the blood donors. The data obtained indicate the need to implement new blood donor policy in blood banks in developing areas. Our results also indicate that the IFA assay is the most reliable test to use in malaria serodiagnosis.