PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector.

After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of malaria transmission blocking

Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites

Techniques for targeted genetic disruption in Plasmodium, the causative agent of malaria, are currently intractable for those genes that are essential for blood stage development. The ability to use RNA interference (RNAi) to silence gene expression would provide a powerful means to gain valuable insight into the pathogenic blood stages but its functionality in Plasmodium remains controversial. Here we have used various RNA-based gene silencing approaches to test the utility of RNAi in malaria parasites and have undertaken an extensive comparative genomics search using profile hidden Markov models to clarify whether RNAi machinery exists in malaria. These investigative approaches revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression and indeed no experimental evidence for RNAi was observed. In its absence, the most likely explanations for previously reported RNAi-mediated knockdown are either the general toxicity of introduced RNA (with global down-regulation of gene expression) or a specific antisense effect mechanistically distinct from RNAi, which will need systematic analysis if it is to be of use as a molecular genetic tool for malaria parasites

Conserved Mosquito/Parasite Interactions Affect Development of Plasmodium falciparum in Africa

In much of sub-Saharan Africa, the mosquito Anopheles gambiae is the main vector of the major human malaria parasite, Plasmodium falciparum. Convenient laboratory studies have identified mosquito genes that affect positively or negatively the developmental cycle of the model rodent parasite, P. berghei. Here, we use transcription profiling and reverse genetics to explore whether five disparate mosquito gene regulators of P. berghei development are also pertinent to A. gambiae/P. falciparum interactions in semi-natural conditions, using field isolates of this parasite and geographically related mosquitoes. We detected broadly similar albeit not identical transcriptional responses of these genes to the two parasite species. Gene silencing established that two genes affect similarly both parasites: infections are hindered by the intracellular local activator of actin cytoskeleton dynamics, WASP, but promoted by the hemolymph lipid transporter, ApoII/I. Since P. berghei is not a natural parasite of A. gambiae, these data suggest that the effects of these genes have not been drastically altered by constant interaction and co-evolution of A. gambiae and P. falciparum; this conclusion allowed us to investigate further the mode of action of these two genes in the laboratory model system using a suite of genetic tools and infection assays. We showed that both genes act at the level of midgut invasion during the parasite's developmental transition from ookinete to oocyst. ApoII/I also affects the early stages of oocyst development. These are the first mosquito genes whose significant effects on P. falciparum field isolates have been established by direct experimentation. Importantly, they validate for semi-field human malaria transmission the concept of parasite antagonists and agonists

Anopheles/Plasmodium interactions at the ookinete-to-oocyst developmental transition

The ookinete to oocyst developmental transition of the Plasmodium parasite represents amajor population bottleneck in the malaria life cycle. This suggests that it could be a target forintervention strategies, such as transmission blocking vaccines, provided essential parasite targetmolecules can be identified. A recent microarray analysis has identified a large number of transcriptsdifferentially expressed during the parasite?s developmental transitions. Genes differentiallyregulated during the ookinete-to-oocyst transition may determine the development of the parasitewithin the mosquito host, as well as, participating directly in parasite/mosquito interactions. Yet, thefunction of the majority of such molecules is largely unknown.This PhD thesis aims to identify and functionally characterise genes putatively involved inookinete development and/or the interactions between the parasite and the mosquito host in the modelsystem Plasmodium berghei. Thirty three proteins likely to be implicated in the parasite?s interactionwith the mosquito immune system and local epithelial response were identified based on theirexpression pattern and predicted structural features. Generation of knock-out mutants throughtargeted gene disruption by homologous recombination was the first step towards functionalcharacterization of these candidates. Successful mutants were assessed for their ability to completetheir sexual sporogonic development, as well as, their impact on mosquito immunity followinginfection of Anopheline mosquitoes of various immune backgrounds. Interestingly, two of thesuccessful mutants were hampered in their ability to undergo normal differentiation during ookinetedevelopment while the third one?s ability to invade the mosquito midgut epithelium was impaired.The inability to invade implies a potential interaction of this gene product with mosquito midgutligands. Eventually malaria transmission through Anopheline mosquitoes was affected in all threemutants. Moreover, challenging of a mosquito protein LRIM1, a major parasite antagonist, alsorevealed potential involvement of the three mutants in mosquito/parasite immune response pathways.Genetic crosses with parasite lines deficient in the production of either male or female fertile gametesdemonstrated in the case of two mutants that, this defect in ookinete development is sex dependent,thus underlining the critical importance of maternal and/or paternal control during the first few hoursof parasite development in the mosquito.EThOS - Electronic Theses Online ServiceA.G. Leventis FoundationGBUnited Kingdo

Anopheles coluzzii stearoyl-CoA desaturase is essential for adult female survival and reproduction upon blood feeding

Vitellogenesis and oocyte maturation require anautogenous female Anopheles mosquitoes to obtain a bloodmeal from a vertebrate host. The bloodmeal is rich in proteins that are readily broken down into amino acids in the midgut lumen and absorbed by the midgut epithelial cells where they are converted into lipids and then transported to other tissues including ovaries. The stearoyl-CoA desaturase (SCD) plays a pivotal role in this process by converting saturated (SFAs) to unsaturated (UFAs) fatty acids; the latter being essential for maintaining cell membrane fluidity amongst other housekeeping functions. Here, we report the functional and phenotypic characterization of SCD1 in the malaria vector mosquito Anopheles coluzzii. We show that RNA interference (RNAi) silencing of SCD1 and administration of sterculic acid (SA), a small molecule inhibitor of SCD1, significantly impact on the survival and reproduction of female mosquitoes following blood feeding. Microscopic observations reveal that the mosquito thorax is quickly filled with blood, a phenomenon likely caused by the collapse of midgut epithelial cell membranes, and that epithelial cells are depleted of lipid droplets and oocytes fail to mature. Transcriptional profiling shows that genes involved in protein, lipid and carbohydrate metabolism and immunity-related genes are the most affected by SCD1 knock down (KD) in blood-fed mosquitoes. Metabolic profiling reveals that these mosquitoes exhibit increased amounts of saturated fatty acids and TCA cycle intermediates, highlighting the biochemical framework by which the SCD1 KD phenotype manifests as a result of a detrimental metabolic syndrome. Accumulation of SFAs is also the likely cause of the potent immune response observed in the absence of infection, which resembles an auto-inflammatory condition. These data provide insights into mosquito bloodmeal metabolism and lipid homeostasis and could inform efforts to develop novel interventions against mosquito-borne diseases

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

PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector.

After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of malaria transmission blocking