The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum,and their transmission by Anopheles stephensi,Anopheles gambiae and Aedes aegypti

Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.     

Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes

We present a detailed analysis of the interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei ookinetes during invasion of the mosquito by the parasite. In this mosquito, P. berghei ookinetes invade polarized columnar epithelial cells with microvilli, which do not express high levels of vesicular ATPase. The invaded cells are damaged, protrude towards the midgut lumen and suffer other characteristic changes, including induction of nitric oxide synthase (NOS) expression, a substantial loss of microvilli and genomic DNA fragmentation. Our results indicate that the parasite inflicts extensive damage leading to subsequent death of the invaded cell. Ookinetes were found to be remarkably plastic, to secrete a subtilisin-like serine protease and the GPI-anchored surface protein Pbs21 into the cytoplasm of invaded cells, and to be capable of extensive lateral movement between cells. The epithelial damage inflicted is repaired efficiently by an actin purse-string-mediated restitution mechanism, which allows the epithelium to 'bud off' the damaged cells without losing its integrity. A new model, the time bomb theory of ookinete invasion, is proposed and its implications are discussed.     

Intracellular calcium levels in the Plasmodium berghei ookinete

Ookinetes are the motile and invasive stages of Plasmodium parasites in the mosquito host. Here we explore the role of intracellular Ca2+ in ookinete survival and motility as well as in the formation of oocysts in vitro in the rodent malaria parasite Plasmodium berghei. Treatment with the Ca2+ ionophore A23187 induced death of the parasite, an effect that could be prevented if the ookinetes were co-incubated with insect cells before incubation with the ionophore. Treatment with the intracellular calcium chelator BAPTA/AM resulted in increased formation of oocysts in vitro. Calcium imaging in the ookinete using fluorescent calcium indicators revealed that the purified ookinetes have an intracellular calcium concentration in the range of 100 nm. Intracellular calcium levels decreased substantially when the ookinetes were incubated with insect cells and their motility was concomitantly increased. Our results suggest a pleiotropic role for intracellular calcium in the ookinete.     

Progression of Plasmodium berghei through Anopheles stephensi is density-dependent

It is well documented that the density of Plasmodium in its vertebrate host modulates the physiological response induced; this in turn regulates parasite survival and transmission. It is less clear that parasite density in the mosquito regulates survival and transmission of this important pathogen. Numerous studies have described conversion rates of Plasmodium from one life stage to the next within the mosquito, yet few have considered that these rates might vary with parasite density. Here we establish infections with defined numbers of the rodent malaria parasite Plasmodium berghei to examine how parasite density at each stage of development (gametocytes; ookinetes; oocysts and sporozoites) influences development to the ensuing stage in Anopheles stephensi, and thus the delivery of infectious sporozoites to the vertebrate host. We show that every developmental transition exhibits strong density dependence, with numbers of the ensuing stages saturating at high density. We further show that when fed ookinetes at very low densities, oocyst development is facilitated by increasing ookinete number (i.e., the efficiency of ookinete-oocyst transformation follows a sigmoid relationship). We discuss how observations on this model system generate important hypotheses for the understanding of malaria biology, and how these might guide the rational analysis of interventions against the transmission of the malaria parasites of humans by their diverse vector species.     

Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae

Anopheles mosquitoes are major vectors of human malaria in Africa. Large variation exists in the ability of mosquitoes to serve as vectors and to transmit malaria parasites, but the molecular mechanisms that determine vectorial capacity remain poorly understood. We report that the hemocyte-specific complement-like protein TEP1 from the mosquito Anopheles gambiae binds to and mediates killing of midgut stages of the rodent malaria parasite Plasmodium berghei. The dsRNA knockdown of TEP1 in adults completely abolishes melanotic refractoriness in a genetically selected refractory strain. Moreover, in susceptible mosquitoes this knockdown increases the number of developing parasites. Our results suggest that the TEP1-dependent parasite killing is followed by a TEP1-independent clearance of dead parasites by lysis and/or melanization. Further elucidation of the molecular mechanisms of TEP1-mediated parasite killing will be of great importance for our understanding of the principles of vectorial capacity in insects.     

Anopheles gambiae SRPN2 facilitates midgut invasion by the malaria parasite Plasmodium berghei

We report on a phylogenetic and functional analysis of genes encoding three mosquito serpins (SRPN1, SRPN2 and SRPN3), which resemble known inhibitors of prophenoloxidase-activating enzymes in other insects. Following RNA interference induction by double-stranded RNA injection, knockdown of SRPN2 in adult Anopheles gambiae produced a notable phenotype: the appearance of melanotic pseudotumours, which increased in size and number with time, indicating spontaneous melanization and association with an observed lifespan reduction. Furthermore, knockdown of SRPN2 strongly interfered with the invasion of A. gambiae midguts by the rodent malaria parasite Plasmodium berghei. It did not affect ookinete formation, but markedly reduced oocyst numbers, by 97%, as a result of increased ookinete lysis and melanization.     

Implications of Time Bomb model of ookinete invasion of midgut cells

In this review, we describe the experimental observations that led us to propose the Time Bomb model of ookinete midgut invasion and discuss potential implications of this model when considering malaria transmission-blocking strategies aimed at arresting parasite development within midgut cells. A detailed analysis of the molecular interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei parasites, as they migrate through midgut cells, revealed that ookinetes induce nitric oxide synthase (NOS) expression, remodeling of the actin cytoskeleton and characteristic morphological changes in the invaded epithelial cells. Parasites inflict extensive damage that ultimately leads to genome fragmentation and cell death. During their migration through the cytoplasm, ookinetes release a subtilisin-like protease (PbSub2) and the surface protein (Pbs21). The model proposes that ookinetes must escape rapidly from the invaded cells, as the responses mediating cell death could be potentially lethal to the parasites. In other words, the physical and/or chemical damage triggered by the parasite can be thought of as a 'lethal bomb'. Once this cascade of events is initiated, the parasite must leave the cellular compartment within a limited time to escape unharmed from the 'bomb' it has activated. The midgut epithelium has the ability to heal rapidly by 'budding off' the damaged cells to the midgut lumen without losing its integrity.     

Analysis of the Plasmodium and Anopheles transcriptional repertoire during ookinete development and midgut invasion

Plasmodium, the causative agent of malaria, has to undergo sexual differentiation and development in anopheline mosquitoes for transmission to occur. To isolate genes specifically induced in both organisms during the early stages of Plasmodium differentiation in the mosquito, two cDNA libraries were constructed, one enriched for sequences expressed in differentiating Plasmodium berghei ookinetes and another enriched for sequences expressed in Anopheles stephensi guts containing invading ookinetes and early oocysts. Sequencing of 457 ookinete library clones and 652 early oocyst clones represented 175 and 346 unique expressed sequence tags, respectively. Nine of 13 Plasmodium and four of the five Anopheles novel expressed sequence tags analyzed on Northern blots were induced during ookinete differentiation and mosquito gut invasion. Ancaspase-7, an Anopheles effector caspase, is proteolytically activated during Plasmodium invasion of the midgut. WARP, a gene encoding a Plasmodium surface protein with a von Willebrand factor A-like adhesive domain, is expressed only in ookinetes and early oocysts. An anti-WARP polyclonal antibody strongly inhibits (70-92%) Plasmodium development in the mosquito, making it a candidate antigen for transmission blocking vaccines. The present results and those of an accompanying report (Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M. C., Dimopoulos G., Kafatos, F. C., Adams, J. H., and Jacobs-Lorena, M. (2004) J. Biol. Chem. 279, 5581-5587) provide the foundation for further analysis of Plasmodium differentiation in the mosquito and of mosquito responses to the parasite.     

The infectivity and purification of cultured Plasmodium berghei ookinetes

Plasmodium berghei ookinetes were cultured from hamster blood as described previously (Kurtti and Munderloh, 1986). An average of 7.3 X 10(6) ookinetes was harvested from each ml of blood. Ookinetes were purified by centrifugation on first a 40% and then a 36% Percoll gradient. The final preparation comprised 32.8% of the ookinetes initially obtained, and contained 3.3 other parasite stages or blood cells per ookinete. Unpurified and purified ookinetes were resuspended in hamster blood and fed to Anopheles stephensi. There was a strong linear correlation between the concentration of purified or unpurified ookinetes and the number of oocysts formed. With unpurified ookinetes, a maximum was reached when preparations containing 1 X 10(7) ookinetes/ml were fed, and feeding preparations containing a higher concentration did not produce more oocysts. Sporozoites were found in the salivary glands of mosquitoes fed ookinetes by days 14 (unpurified) or 15 (purified) PI. Approximately 5 times as many purified as unpurified ookinetes were required to produce each oocyst.     

A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell

Plasmodium parasites are fertilized in the mosquito midgut and develop into motile zygotes, called ookinetes, which invade the midgut epithelium. Here we show that a calcium-dependent protein kinase, CDPK3, of the rodent malarial parasite (Plasmodium berghei) is produced in the ookinete stage and has a critical role in parasite transmission to the mosquito vector. Targeted disruption of the CDPK3 gene decreased ookinete ability to infect the mosquito midgut by nearly two orders of magnitude. Electron microscopic analyses demonstrated that the disruptant ookinetes could not access midgut epithelial cells by traversing the layer covering the cell surface. An in vitro migration assay showed that these ookinetes lack the ability to migrate through an artificial gel, suggesting that this defect caused their failure to access the epithelium. In vitro migration assays also suggested that this motility is induced in the wild type by mobilization of intracellular stored calcium. These results indicate that a signalling pathway involving calcium and CDPK3 regulates ookinete penetration of the layer covering the midgut epithelium. Because humans do not possess CDPK family proteins, CDPK3 is a good target for blocking malarial transmission to the mosquito vector.     

PSOP1, putative secreted ookinete protein 1, is localized to the micronemes of Plasmodium yoelii and P. berghei ookinetes

Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Activated gametocytes in the mosquito midgut egress from erythrocytes followed by fertilization and zygote formation. Zygotes differentiate into motile invasive ookinetes, which penetrate the midgut epithelium before forming oocysts beneath the basal lamina. Ookinete development and traversal across the mosquito midgut wall are major bottlenecks in the parasite life cycle. In ookinetes, surface proteins and proteins stored in apical organelles have been shown to be involved in parasite-host interactions. A group of ookinete proteins that are predicted to have such functions are named PSOPs (putative secreted ookinete protein). PSOP1 is possibly involved in migration through the midgut wall, and here its subcellular localization was examined in ookinetes by immunoelectron microscopy. PSOP1 localizes to the micronemes of Plasmodium yoelii and Plasmodium berghei ookinetes, indicating that it is stored and possibly apically secreted during ookinete penetration through the mosquito midgut wall.     

A genetic module regulates the melanization response of Anopheles to Plasmodium

Two modes of refractoriness to Plasmodium, ookinete lysis and melanization, are known in the malaria vector, Anopheles gambiae. Melanization, a potent insect immune response, is manifested in a genetically selected refractory strain and in susceptible mosquitoes that are depleted of specific C-type lectins (CTLs). Here we use a systematic in vivo RNA interference-mediated reverse genetic screen and other recent results to define a melanization-regulating genetic module or network. It encompasses at least 14 genes, including those that encode five Easter-like clip domain serine proteases and four Masquerade-like serine protease homologues of the mosquito CLIPB and CLIPA subfamilies respectively. We show that several but not all CLIPB genes promote Plasmodium melanization, exhibiting partial functional overlap and synergy. We also report that several CLIPA genes have contrasting roles: CLIPA8 is essential for parasite melanization, while three other CLIPAs are novel synergistic inhibitors of this response. Importantly, the roles of certain CLIPAs and CLIPBs are strain specific, indicating that this network may differ between strains. Finally, we provide evidence that in susceptible mosquitoes melanization induced by knockdown of either CTL4 or CLIPA2/CLIPA5 directly kills ookinetes, in contrast to refractory mosquitoes where it merely disposes of dead parasites.     

Hemolytic C-type lectin CEL-III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development

The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50) of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions.     

Isonicotinic acid hydrazide: an anti-tuberculosis drug inhibits malarial transmission in the mosquito gut

We studied the transmission-blocking effect of isonicotinic acid hydrazide (INH), a widely used anti-tuberculosis drug, against Plasmodium gallinaceum and Plasmodium berghei. INH-treatment of infected animals did not inhibit parasite development in the blood of the vertebrate host, but did inhibit exflagellation, ookinete formation, and oocyst development in the mosquito. Oocyst development was inhibited in a dose-dependent manner. The ED(50) in the P. gallinaceum/chicken/Aedes aegypti model and P. berghei/mouse/Anopheles stephensi model was 72 and 109 mg/kg, respectively. In marked contrast, in vitro exflagellation and ookinete development were not directly affected by physiological concentrations of INH. We suggest that INH exerts its inhibitory effects on the mosquito stages of the malaria parasite by an indirect, and at present undefined mechanism. Further elucidation of the mechanism how INH inhibits parasite development specifically on mosquito stages may allow us to identify new targets for malaria control strategy.     

PbGCbeta is essential for Plasmodium ookinete motility to invade midgut cell and for successful completion of parasite life cycle in mosquitoes

When malaria parasites enter to mosquitoes, they fertilize and differentiate to zygotes and ookinetes. The motile ookinetes cross the midgut cells and arrive to the basement membranes where they differentiate into oocysts. The midgut epithelium is thus a barrier for ookinetes to complete their life cycle in the mosquitoes. The ookinetes develop gliding motility to invade midgut cells successfully, but the molecular mechanisms behind are poorly understood. Here, we identified a single molecule with guanylate cyclase domain and N-terminal P-type ATPase like domain in the rodent malaria parasite Plasmodium berghei and named it PbGCbeta. We demonstrated that transgenic parasites in which the PbGCbeta gene was disrupted formed normal ookinetes but failed to produce oocyst. Confocal microscopic analysis showed that the disruptant ookinetes remained on the surface of the microvilli. The disruptant ookinetes showed severe defect in motility, resulting in failure of parasite invasion of the midgut epithelium. When the disruptant ookinetes were cultured in vitro, they transformed into oocysts and sporozoites. These results demonstrate that PbGCbeta is essential for ookinete motility when passing through the midgut cells, but not for further development of the parasites.