36 results found
Bailey AJ, Ukegbu CV, Giorgalli M, et al., 2023, Intracellular<i>Plasmodium</i>aquaporin 2 is required for sporozoite production in the mosquito vector and malaria transmission
<jats:title>Abstract</jats:title><jats:p>Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins which are channels facilitating the movement of water and other solutes across membranes. We identify a new aquaporin in malaria parasites and demonstrate that it is essential for disease transmission through mosquitoes. Disruption of AQP2 in the human parasite<jats:italic>Plasmodium falciparum</jats:italic>and the rodent parasite<jats:italic>Plasmodium berghei</jats:italic>blocks sporozoite production inside oocysts established on mosquito midguts, preventing parasite infection of salivary glands and transmission to a new host.<jats:italic>In vivo</jats:italic>epitope tagging of AQP2 in<jats:italic>P. berghei</jats:italic>, combined with immunofluorescence assays, reveals that the protein is localized in previously uncharacterized organelles found in the cytoplasm of gametocytes, ookinetes and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endolysosomal system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely related to other intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature not found in any aquaporins studied to date. This in conjunction with the requirement of AQP2 for malaria transmission suggests that AQP2 may be a fruitful new target of novel antimalarial interventions.</jats:p>
Ukegbu CV, Gomes AR, Giorgalli M, et al., 2023, Reverse genetic screen identifies malaria parasite genes required for gametocyte-to-sporozoite development in its mosquito host
<jats:title>Summary</jats:title><jats:p>Malaria remains one of the most devastating infectious diseases. Reverse genetic screens offer a powerful approach to identify genes and molecular processes governing malaria parasite biology. However, sexual reproduction and complex regulation of gene expression and genotype-phenotype associations in the mosquito have hampered the development of screens in this key part of the parasite lifecycle. We designed a genetic approach in the rodent parasite<jats:italic>Plasmodium berghei</jats:italic>, which in conjunction with barcode sequencing allowed us to overcome the fertilization roadblock and screen for gametocyte-expressed genes required for parasite infection of the mosquito<jats:italic>Anopheles coluzzii</jats:italic>. The results confirmed previous findings, validating our approach for scaling up, and identified new genes required for ookinete motility and mosquito midgut infection and for sporozoite development and oocyst egress and salivary gland infection. Our findings can assist efforts to study malaria transmission biology and develop new interventions to control disease transmission.</jats:p>
Ferdous Z, Fuchs S, Behrends V, et al., 2021, Anopheles coluzziistearoyl-CoA desaturase is essential for adult female survival and reproduction upon blood feeding, PLoS Pathogens, Vol: 17, ISSN: 1553-7366
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
Ukegbu CV, Christophides GK, Vlachou D, 2021, Identification of three novel plasmodium factors involved in ookinete to oocyst developmental transition, Frontiers in Cellular and Infection Microbiology, Vol: 11, Pages: 1-17, ISSN: 2235-2988
Plasmodium falciparum malaria remains a major cause of global morbidity and mortality, mainly in sub-Saharan Africa. The numbers of new malaria cases and deaths have been stable in the last years despite intense efforts for disease elimination, highlighting the need for new approaches to stop disease transmission. Further understanding of the parasite transmission biology could provide a framework for the development of such approaches. We phenotypically and functionally characterized three novel genes, PIMMS01, PIMMS57, and PIMMS22, using targeted disruption of their orthologs in the rodent parasite Plasmodium berghei. PIMMS01 and PIMMS57 are specifically and highly expressed in ookinetes, while PIMMS22 transcription starts already in gametocytes and peaks in sporozoites. All three genes show strong phenotypes associated with the ookinete to oocyst transition, as their disruption leads to very low numbers of oocysts and complete abolishment of transmission. PIMMS22 has a secondary essential function in the oocyst. Our results enrich the molecular understanding of the parasite-vector interactions and identify PIMMS01, PIMMS57, and PIMMS22 as new targets of transmission blocking interventions.
Witmer K, Fraschka S, Vlachou D, et al., 2020, An epigenetic map of malaria parasite development from host to vector, Scientific Reports, Vol: 10, ISSN: 2045-2322
The malaria parasite replicates asexually in the red blood cells of its vertebrate host employing epigenetic mechanisms to regulate gene expression in response to changes in its environment. We used chromatin immunoprecipitation followed by sequencing in conjunction with RNA sequencing to create an epigenomic and transcriptomic map of the developmental transition from asexual blood stages to male and female gametocytes and to ookinetes in the rodent malaria parasite Plasmodium berghei. Across the developmental stages examined, heterochromatin protein 1 associates with variantly expressed gene families localised at subtelomeric regions and variant gene expression based on heterochromatic silencing is observed only in some genes. Conversely, the euchromatin mark histone 3 lysine 9 acetylation (H3K9ac) is abundant in non-heterochromatic regions across all developmental stages. H3K9ac presents a distinct pattern of enrichment around the start codon of ribosomal protein genes in all stages but male gametocytes. Additionally, H3K9ac occupancy positively correlates with transcript abundance in all stages but female gametocytes suggesting that transcription in this stage is independent of H3K9ac levels. This finding together with known mRNA repression in female gametocytes suggests a multilayered mechanism operating in female gametocytes in preparation for fertilization and zygote development, coinciding with parasite transition from host to vector.
Ukegbu CV, Giorgalli M, Tapanelli S, et al., 2020, PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 7363-7373, ISSN: 0027-8424
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.
Ukegbu CV, Giorgalli M, Tapanelli S, et al., 2019, <i>Plasmodium</i> PIMMS43 is required for ookinete evasion of the mosquito complement-like response and sporogonic development in the oocyst
<jats:title>Abstract</jats:title><jats:p>Malaria transmission requires <jats:italic>Plasmodium</jats:italic> parasites to successfully infect a female <jats:italic>Anopheles</jats:italic> mosquito, surviving a series of robust innate immune responses. Understanding how parasites evade these responses can highlight new ways to block malaria transmission. We show that ookinete and sporozoite surface protein PIMMS43 is required for <jats:italic>Plasmodium</jats:italic> ookinete evasion of the <jats:italic>Anopheles coluzzii</jats:italic> complement-like system and for sporogonic development in the oocyst. Disruption of <jats:italic>P. berghei</jats:italic> PIMMS43 triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing the complement-like system restores ookinete-to-oocyst transition. 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 <jats:italic>P. falciparum</jats:italic> populations indicates allelic adaptation to sympatric vector populations. These data significantly add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of interventions aiming at malaria transmission blocking.</jats:p><jats:sec><jats:title>Author summary</jats:title><jats:p>Malaria is a devastating disease transmitted among humans through mosquito bites. Mosquito control has significantly reduced clinical malaria cases and deaths in the last decades. However, as mosquito resistance to insecticides is becoming widespread impacting on current control tools, such as insecticide impregnated bed nets and indoor spraying, new interventions are urgently needed, especially those that target disease transmission. Here, we characterize
Witmer K, Fraschka SAK, Vlachou D, et al., 2019, Epigenetic regulation underlying Plasmodium berghei gene expression during its developmental transition from host to vector, bioRxiv, ISSN: 2045-2322
ABSTRACT Epigenetic regulation of gene expression is an important attribute in the survival and adaptation of the malaria parasite Plasmodium in its human host. Our understanding of epigenetic regulation of gene expression in Plasmodium developmental stages beyond asexual replication in the mammalian host is sparse. We used chromatin immune-precipitation (ChIP) and RNA sequencing to create an epigenetic and transcriptomic map of the murine parasite Plasmodium berghei development from asexual blood stages to male and female gametocytes, and finally, to ookinetes. We show that heterochromatin 1 (HP1) almost exclusively associates with variantly expressed gene families at subtelomeric regions and remains stable across stages and various parasite lines. Variant expression based on heterochromatic silencing is observed only in very few genes. In contrast, the active histone mark histone 3 Lysine 9 acetylation (H3K9ac) is found between heterochromatin boundaries and occurs as a sharp peak around the start codon for ribosomal protein genes. H3K9ac occupancy positively correlates with gene transcripts in asexual blood stages, male gametocytes and ookinetes. Interestingly, H3K9ac occupancy does not correlate with transcript abundance in female gametocytes. Finally, we identify novel DNA motifs upstream of ookinete-specific genes thought to be involved in transcriptional activation upon fertilization.
Ukegbu CV, Giogalli M, Yassine H, et al., 2017, Plasmodium berghei P47 is essential for ookinete protection from the Anopheles gambiae complement-like response, Scientific Reports, Vol: 7, ISSN: 2045-2322
Malaria is a mosquito-borne disease affecting millions of people every year. The rodent parasite Plasmodium berghei has served as a model for human malaria transmission studies and played a pivotal role in dissecting the mosquito immune response against infection. The 6-cysteine protein P47, known to be important for P. berghei female gamete fertility, is shown to serve a different function in Plasmodium falciparum, protecting ookinetes from the mosquito immune response. Here, we investigate the function of P. berghei P47 in Anopheles gambiae mosquito infections. We show that P47 is expressed on the surface of both female gametocytes and ookinetes where it serves distinct functions in promoting gametocyte-to-ookinete development and protecting ookinetes from the mosquito complement-like response, respectively. The latter function is essential, as ookinetes lacking P47 are targeted for killing while traversing the mosquito midgut cells and eliminated upon exposure to hemolymph proteins of the complement-like system. Silencing key factors of the complement-like system restores oocyst development and disease transmission to rodent hosts. Our data establish a dual role of P. berghei P47 in vivo and reinforce the use of this parasite to study the impact of the mosquito immune response on human malaria transmission.
Ukegbu CV, Akinosoglou KA, Christophides GKC, et al., 2017, Plasmodium berghei PIMMS2 promotes ookinete invasion of the Anopheles gambiae mosquito midgut, Infection and Immunity, Vol: 85, ISSN: 1098-5522
Mosquito midgut stages of the malaria parasite present an attractive biological system to study host-parasite interactions and develop interventions to block disease transmission. Mosquito infection ensues upon oocyst development that follows ookinete invasion and traversal of the mosquito midgut epithelium. Here, we report the characterization of PIMMS2 (Plasmodium Invasion of Mosquito Midgut Screen candidate 2), a Plasmodium berghei protein with structural similarities to subtilisin-like proteins. PIMMS2 orthologs are present in the genomes of all plasmodia and are mapped between the subtilisin-encoding genes SUB1 and SUB3. P. berghei PIMMS2 is specifically expressed in zygotes and ookinetes and is localized on the ookinete surface. Loss of PIMMS2 function through gene disruption by homologous recombination leads to normal development of motile ookinetes that exhibit severely impaired capacity to traverse the mosquito midgut and transform to oocysts. Genetic complementation of the disrupted locus with a mutated PIMMS2 allele reveals that amino acid residues corresponding to the putative subtilisin-like catalytic triad are important but not essential for the protein function. Our data demonstrate that PIMMS2 is a novel ookinete-specific protein that promotes parasite traversal of the mosquito midgut epithelium and establishment of mosquito infection.
Ukegbu CV, Cho J-S, Christophides GK, et al., 2015, Transcriptional silencing and activation of paternal DNA during Plasmodium berghei zygotic development and transformation to oocyst, Cellular Microbiology, Vol: 17, Pages: 1230-1240, ISSN: 1462-5822
The malaria parasite develops sexually in the mosquito midgut upon entry with the ingested blood meal before it can invade the midgut epithelium and embark on sporogony. Recent data have identified a number of distinct transcriptional programmes operating during this critical phase of the parasite life cycle. We aimed at characterizing the parental contribution to these transcriptional programmes and establish the genetic framework that would guide further studies of Plasmodium zygotic development and ookinete-to-oocyst transition. To achieve this we used in vitro and in vivo cross-fertilization experiments of various parasite lines expressing fluorescent reporters under the control of constitutive and stage-specific promoters. The results revealed that the zygote/ookinete stage exhibits a maternal phenotype with respect to constitutively expressed reporters, which is derived from either maternal mRNA inheritance or transcription of the maternal allele. The respective paternal alleles are silenced in the zygote/ookinete but reactivated after midgut invasion and transformation to oocyst. Transcripts specifically produced in the zygote/ookinete are synthesized de novo by both parental alleles. These findings highlight a putative role of epigenetic regulation of Plasmodium zygotic development and add substantially to the emerging picture of the molecular mechanisms regulating this important stage of malaria transmission.
Akinosoglou KA, Bushell ESC, Ukegbu CV, et al., 2015, Characterization of Plasmodium developmental transcriptomes in Anopheles gambiae midgut reveals novel regulators of malaria transmission, CELLULAR MICROBIOLOGY, Vol: 17, Pages: 254-268, ISSN: 1462-5814
- Author Web Link
- Open Access Link
- Citations: 21
Ukegbu VC, Bushell E, Trasanidis N, et al., 2014, Molecular characterization of the Anopheles-Plasmodium interactions in the mosquito midgut” Pathogens and Global Health 107, no. 8 (2013): 402
Akinosoglou KA, Cho JS, Sala K, Redmond S
Ukegbu VC, Bushell E, Trasanidis N, et al., 2013, MOLECULAR CHARACTERIZATION OF THE ANOPHELES PLASMODIUM INTERACTIONS IN THE MOSQUITO MIDGUT, PATHOGENS AND GLOBAL HEALTH, Vol: 107, Pages: 402-402, ISSN: 2047-7724
Schlegelmilch T, Vlachou D, 2013, Cell biological analysis of mosquito midgut invasion: the defensive role of the actin-based ookinete hood, PATHOGENS AND GLOBAL HEALTH, Vol: 107, Pages: 480-492, ISSN: 2047-7724
- Author Web Link
- Citations: 5
Vlachou D, 2011, Differences in An. gambiae gene regulation in response to ingestion of local and geographically distant isolates of P. falciparum., 60th Annual Meeting, American Society of Tropical Medicine and Hygiene (ASTMH), Philadelphia, PA, USA, December 2011.
Mendes AM, Awono-Ambene PH, Nsango SE, et al., 2011, Infection Intensity-Dependent Responses of Anopheles gambiae to the African Malaria Parasite Plasmodium falciparum, INFECTION AND IMMUNITY, Vol: 79, Pages: 4708-4715, ISSN: 0019-9567
- Author Web Link
- Citations: 40
Churcher TS, Bousema TJ, Drakeley CJ, et al., 2010, LOW GAMETOCYTE DENSITIES RESTRICT THE DEVELOPMENT OF PLASMODIUM FALCIPARUM WITHIN ANOPHELES GAMBIAE WITH IMPLICATIONS FOR THE HUMAN RESERVOIR OF INFECTION AND PARASITE ELIMINATION, 59th Annual Meeting of the American-Society-of-Tropical-Medicine-and-Hygiene (ASTMH), Publisher: AMER SOC TROP MED & HYGIENE, Pages: 169-169, ISSN: 0002-9637
Bushell ESC, Ecker A, Schlegelmilch T, et al., 2009, Paternal effect of the nuclear formin-like protein MISFIT on plasmodium development in then mosquito vector, PLoS Pathogens, Vol: 5, Pages: 1-13, ISSN: 1553-7366
Malaria parasites must undergo sexual and sporogonic development in mosquitoes before they can infect their vertebrate hosts. We report the discovery and characterization of MISFIT, the first protein with paternal effect on the development of the rodent malaria parasite Plasmodium berghei in Anopheles mosquitoes. MISFIT is expressed in male gametocytes and localizes to the nuclei of male gametocytes, zygotes and ookinetes. Gene disruption results in mutant ookinetes with reduced genome content, microneme defects and altered transcriptional profiles of putative cell cycle regulators, which yet successfully invade the mosquito midgut. However, developmental arrest ensues during the ookinete transformation to oocysts leading to malaria transmission blockade. Genetic crosses between misfit mutant parasites and parasites that are either male or female gamete deficient reveal a strict requirement for a male misfit allele. MISFIT belongs to the family of formin-like proteins, which are known regulators of the dynamic remodeling of actin and microtubule networks. Our data identify the ookinete-to-oocyst transition as a critical cell cycle checkpoint in Plasmodium development and lead us to hypothesize that MISFIT may be a regulator of cell cycle progression. This study offers a new perspective for understanding the male contribution to malaria parasite development in the mosquito vector.
Baum J, Papenfuss AT, Mair GR, et al., 2009, Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites, NUCLEIC ACIDS RESEARCH, Vol: 37, Pages: 3788-3798, ISSN: 0305-1048
- Author Web Link
- Citations: 149
Mendes AM, Schlegelmilch T, Cohuet A, et al., 2008, Conserved mosquito/parasite interactions affect development of Plasmodium falciparum in Africa, PLoS Pathogens, Vol: 4, Pages: 1-12, ISSN: 1553-7366
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.
Waterhouse RM, Kriventseva EV, Meister S, et al., 2007, Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes, SCIENCE, Vol: 316, Pages: 1738-1743, ISSN: 0036-8075
- Author Web Link
- Citations: 474
Vlachou D, Schlegelmilch T, Runn E, et al., 2006, The developmental migration of Plasmodium in mosquitoes, CURRENT OPINION IN GENETICS & DEVELOPMENT, Vol: 16, Pages: 384-391, ISSN: 0959-437X
- Author Web Link
- Citations: 55
Vlachou D, Kafatos FC, 2005, The complex interplay between mosquito positive and negative regulators of Plasmodium development, CURRENT OPINION IN MICROBIOLOGY, Vol: 8, Pages: 415-421, ISSN: 1369-5274
- Author Web Link
- Citations: 27
Vlachou D, Schlegelmilch T, Christophides GK, et al., 2005, Functional genomic analysis of midgut epithelial responses in Anopheles during Plasmodium invasion, CURRENT BIOLOGY, Vol: 15, Pages: 1185-1195, ISSN: 0960-9822
- Author Web Link
- Citations: 149
Christophides G, Meister S, Koutsos A, et al., 2005, Systems biology of the Anopheles gainbiae innate immunity [MIM-GC-22590], ACTA TROPICA, Vol: 95, Pages: S44-S45, ISSN: 0001-706X
Vlachou D, Zimmermann T, Cantera R, et al., 2004, Real-time, in vivo analysis of malaria ookinete locomotion and mosquito midgut invasion, CELLULAR MICROBIOLOGY, Vol: 6, Pages: 671-685, ISSN: 1462-5814
- Author Web Link
- Citations: 141
Osta MA, Christophides GK, Vlachou D, et al., 2004, Innate immunity in the malaria vector Anopheles gambiae: comparative and functional genomics, JOURNAL OF EXPERIMENTAL BIOLOGY, Vol: 207, Pages: 2551-2563, ISSN: 0022-0949
- Author Web Link
- Citations: 105
Christophides GK, Vlachou D, Kafatos FC, 2004, Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae, IMMUNOLOGICAL REVIEWS, Vol: 198, Pages: 127-148, ISSN: 0105-2896
- Author Web Link
- Citations: 209
Dessens JT, Siden-Kiamos I, Mendoza J, et al., 2003, SOAP, a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development, MOLECULAR MICROBIOLOGY, Vol: 49, Pages: 319-329, ISSN: 0950-382X
- Author Web Link
- Citations: 105
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