Plasmodium genes responsible for oocyst development and interaction with its Anopheline vector

The transmission of the malaria parasite Plasmodium is governed by a complex developmental cycle. This PhD thesis describes the transcriptional profiling of the rodent malaria parasite Plasmodium berghei developmental migration through its A. gambiae vector. The study was conducted in vivo, using a near complete P. berghei genome microarray platform. Emphasis was placed on the oocyst stage, as little is known about the genes implicated in the ookinete to oocyst transition, and oocyst maturation. The data presented here provide novel transcriptional information about Plasmodium transmission. The analysis revealed a large shift in gene utilisation as the parasite makes its transition from the motile ookinete to the sessile oocyst. Furthermore, this work has shown that different sets of co-regulated genes are important for early and late oocyst development. In addition, this PhD thesis outlines the characterisation of a novel Plasmodium formin-like protein essential for rodent malaria transmission named the male inherited sporulation factor important for transmission (misfit). MISFIT is expressed in the early mosquito stages, where the protein localises to the parasite nucleus. Misfit exhibits an absolute requirement for paternal inheritance, which is in accordance with an observed male-biased expression pattern. pbmisfitΔ ookinetes display significant ultrastructural and gene expression defects and fail to complete zygotic meiosis. However, pbmisfitΔ ookinetes retain functionality and can successfully cross the midgut epithelial barrier. In contrast, mosquito infections with pbmisfitΔ resulted in an arrest immediately upon ookinete-oocyst transformation, where defective oocysts fail to sporulate. An essential role in chromosome segregation during mitosis / meiosis is postulated for MISFIT. In conclusion, the work presented in this thesis has established the ookinete-oocyst transition as a major cell cycle check point during malaria transmission and identified misfit as the first male inherited Plasmodium gene known to affect development post-fertilisation

CRISPR/Cas9 and genetic screens in malaria parasites : small genomes, big impact

The ∼30 Mb genomes of the Plasmodium parasites that cause malaria each encode ∼5000 genes, but the functions of the majority remain unknown. This is due to a paucity of functional annotation from sequence homology, which is compounded by low genetic tractability compared with many model organisms. In recent years technical breakthroughs have made forward and reverse genome-scale screens in Plasmodium possible. Furthermore, the adaptation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein 9 (CRISPR/Cas9) technology has dramatically improved gene editing efficiency at the single gene level. Here, we review the arrival of genetic screens in malaria parasites to analyse parasite gene function at a genome-scale and their impact on understanding parasite biology. CRISPR/Cas9 screens, which have revolutionised human and model organism research, have not yet been implemented in malaria parasites due to the need for more complex CRISPR/Cas9 gene targeting vector libraries. We therefore introduce the reader to CRISPR-based screens in the related apicomplexan Toxoplasma gondii and discuss how these approaches could be adapted to develop CRISPR/Cas9 based genome-scale genetic screens in malaria parasites. Moreover, since more than half of Plasmodium genes are required for normal asexual blood-stage reproduction, and cannot be targeted using knockout methods, we discuss how CRISPR/Cas9 could be used to scale up conditional gene knockdown approaches to systematically assign function to essential genes.Instituto de BiotecnologíaFil: Ishizaki, Takahiro. Umeå University. Department of Molecular Biology; SueciaFil: Ishizaki, Takahiro. The Laboratory for Molecular Infection Medicine Sweden (MIMS); SueciaFil: Hernandez, Sophia. Umeå University. Department of Molecular Biology; SueciaFil: Hernandez, Sophia. The Laboratory for Molecular Infection Medicine Sweden (MIMS); SueciaFil: Paoletta, Martina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Paoletta, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Paoletta, Martina. Umeå University. Department of Molecular Biology; SueciaFil: Paoletta, Martina. The Laboratory for Molecular Infection Medicine Sweden (MIMS); SueciaFil: Sanderson, Theo. Francis Crick Institute; Reino UnidoFil: Bushell, Ellen S. C. Umeå University. Department of Molecular Biology; SueciaFil: Bushell, Ellen S. C. The Laboratory for Molecular Infection Medicine Sweden (MIMS); Sueci

An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi.

The rodent parasite Plasmodium chabaudi is an important in vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti- Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable in vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for P. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear PlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded P. chabaudi gene-deletion and -tagging vectors for transfection in our fluorescent P. chabaudi mother-lines. This produces a tool-kit of P. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community

Functional Profiling of a Plasmodium Genome Reveals an Abundance of Essential Genes

The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine

Regulators of male and female sexual development are critical for the transmission of a malaria parasite

Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite

Plasmodium genes responsible for oocyst development and interaction with its Anopheline vector

The transmission of the malaria parasite Plasmodium is governed by a complex developmental cycle. This PhD thesis describes the transcriptional profiling of the rodent malaria parasite Plasmodium berghei developmental migration through its A. gambiae vector. The study was conducted in vivo, using a near complete P. berghei genome microarray platform. Emphasis was placed on the oocyst stage, as little is known about the genes implicated in the ookinete to oocyst transition, and oocyst maturation. The data presented here provide novel transcriptional information about Plasmodium transmission. The analysis revealed a large shift in gene utilisation as the parasite makes its transition from the motile ookinete to the sessile oocyst. Furthermore, this work has shown that different sets of co-regulated genes are important for early and late oocyst development. In addition, this PhD thesis outlines the characterisation of a novel Plasmodium formin-like protein essential for rodent malaria transmission named the male inherited sporulation factor important for transmission (misfit). MISFIT is expressed in the early mosquito stages, where the protein localises to the parasite nucleus. Misfit exhibits an absolute requirement for paternal inheritance, which is in accordance with an observed male-biased expression pattern. pbmisfitΔ ookinetes display significant ultrastructural and gene expression defects and fail to complete zygotic meiosis. However, pbmisfitΔ ookinetes retain functionality and can successfully cross the midgut epithelial barrier. In contrast, mosquito infections with pbmisfitΔ resulted in an arrest immediately upon ookinete-oocyst transformation, where defective oocysts fail to sporulate. An essential role in chromosome segregation during mitosis / meiosis is postulated for MISFIT. In conclusion, the work presented in this thesis has established the ookinete-oocyst transition as a major cell cycle check point during malaria transmission and identified misfit as the first male inherited Plasmodium gene known to affect development post-fertilisation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Toward the identification of cellular mechanisms behind the lethal phenotypes in Plasmodium berghei blood stages with PlasmoGEM and metabolic modeling

Recent advances in cell genome editing techniques enable the generation of high-throughput gene knockout data in the malaria parasites in vivo. Integrative analysis of this data can lead to the identification of biological mechanisms that explain the observed phenotypes and that provide testable hypotheses for further discoveries. Metabolic modeling can cope with the tangled and versatile metabolism of the malaria parasites, and hence is a valuable approach for understanding the parasites physiology. In this study, we present a combined experimental and computational study that suggests cellular mechanisms behind experimentally determined lethal phenotypes. We perform computational analyses on a newly developed genome-scale model of the malaria parasite Plasmodium berghei (iPbe), and we use high-throughput gene knockout data generated in the PlasmoGEM project. The comparison between data and gene essentiality predictions allow the understanding of the parasite’s physiology in the blood stages. When we simulate in iPbe the hypothesized physiology, we achieve an 80% consistency between the prediction of essential genes and the experimental data. We further predict in silico and test in vivo synthetic lethal pairs in the blood stages of the malaria infection. We identify the thermodynamic bottlenecks, genetic interactions, and the accessibility to nutrients behind the phenotypes. This result indicates that our model iPbe is a valuable framework for the generation of testable hypothesis. Overall, the knowledge generated in this experimental and computational framework will serve to tackle more efficiently the malaria parasites’ metabolism during infection

Toward the identification of cellular mechanisms behind the lethal phenotypes in malaria parasites blood stages with PlasmoGEM and metabolic modeling

Please contact the authors for the abstract

A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite

The genome-wide identification of gene functions in malaria parasites is hampered by a lack of reverse genetic screening methods. We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome. Cotransfecting dozens of vectors into the haploid blood stages creates complex pools of barcoded mutants, whose competitive fitness can be measured during infection of a single mouse using barcode sequencing (barseq). To validate the utility of this resource, we rescreen the P. berghei kinome, using published kinome screens for comparison. We find that several protein kinases function redundantly in asexual blood stages and confirm the targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 by genotyping cloned mutants. Thus, parallel phenotyping of barcoded mutants unlocks the power of reverse genetic screening for a malaria parasite and will enable the systematic identification of genes essential for in vivo parasite growth and transmission

Identifying and targeting key cellular mechanisms for proliferation in Plasmodium parasites: a combined experimental and computational strategy

Recent advances in cell genome editing techniques enable the generation of high-throughput gene knockout data in the malaria parasites in vivo. Integrative analysis of this data can lead to the identification of biological mechanisms that explain the observed phenotypes and that provide testable hypotheses for further discoveries. Metabolic modelling can cope with the tangled and versatile metabolism of the malaria parasites, and hence is a compelling approach for understanding the parasites physiology. In this study, we present a combined experimental and computational approach that suggests cellular mechanisms for targeting the malaria parasites. We predict in silico and test in vivo lethal knockouts and synthetic lethal pairs in the blood and liver stages of the malaria infection. We perform computational analyses on a newly developed genome-scale model of the malaria parasite Plasmodium berghei (iPbe), and we use high-throughput gene knockout data generated in the PlasmoGEM project. The comparison between data and gene essentiality predictions allow the understanding of the parasite’s physiology in the blood and liver stages. We identify the thermodynamic bottlenecks, genetic interactions, and the accessibility to nutrients behind the phenotypes. When we simulate in iPbe the hypothesised physiology, we achieve an 80% consistency between the prediction of essential genes and the experimental data. This result indicates that our model iPbe is a valuable framework for the generation of testable hypothesis on antimalarial targets. Overall, the knowledge generated in this experimental and computational framework will serve to tackle more efficiently the malaria parasites’ metabolism during infection