The peritrophic membrane as a barrier: its penetration by Plasmodium gallinaceum and the effect of a monoclonal antibody to ookinetes

We studied the point at which a monoclonal antibody (mAb C5) to a surface protein (Pgs25) on Plasmodium gallinaceum ookinetes blocked the infection of Aedes aegypti mosquitoes. The antibody did not block the development of zygotes to ookinetes in vitro. Development of ookinetes to oocysts in the mosquito was blocked to the same extent whether zygotes grew to ookinetes in the presence of mAb C5 or the antibody was added after the ookinetes had reached full development. When ookinetes developed in vitro in the presence of mAb C5, antibody remained on the surface of the parasite for the next 50 hr and did not block attachment to the peritrophic membrane. When ookinetes were fed to mosquitoes, two subpopulations of mosquitoes were observed (high numbers of oocysts per midgut and low numbers of oocysts per midgut). mAb C5 reduced the number of oocysts per midgut in the subpopulation that had low numbers of oocysts. The subpopulation that had high numbers of oocysts was unaffected by antibody, indicating that the antibody did not block invasion of the midgut epithelium. When mAb C5 was fed with gametocytes, the parasites invaded the epithelium at the same time (between 30 and 35 hr after the blood meal) as in controls, although at a markedly reduced rate. The ultrastructural observations were consistent with a block of parasites within the peritrophic membrane and not with a block at the epithelium, as parasites were not seen to accumulate within the space between the peritrophic membrane and the epithelium. The mechanism by which mAb C5 to Pgs25 of P. gallinaceum blocks the penetration of the peritrophic membrane remains undefined. We present evidence that the parasite modifies the peritrophic membrane during penetration, an observation first made for Babesia microti during penetration of the peritrophic membrane in Ixodes ticks. Ookinetes in the absence of antibodies appeared to disrupt the layers of the peritrophic membrane, suggesting an enzymatic mechanism for penetration.     

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.     

SOAP,a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development

An essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite-producing oocysts on the mosquito midgut wall. For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion. After invasion ookinete-to-oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components. Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP). The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus. It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine-rich domains. Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds. Moreover, SOAP interacts strongly with mosquito laminin in yeast-two-hybrid assays. Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts. These results identify SOAP as a key molecule for ookinete-to-oocyst differentiation in mosquitoes.     

CTRP is essential for mosquito infection by malaria ookinetes

The malaria parasite suffers severe population losses as it passes through its mosquito vector. Contributing factors are the essential but highly constrained developmental transitions that the parasite undergoes in the mosquito midgut, combined with the invasion of the midgut epithelium by the malaria ookinete (recently described as a principal elicitor of the innate immune response in the Plasmodium-infected insect). Little is known about the molecular organization of these midgut-stage parasites and their critical interactions with the blood meal and the mosquito vector. Elucidation of these molecules and interactions will open up new avenues for chemotherapeutic and immunological attack of parasite development. Here, using the rodent malaria parasite Plasmodium berghei, we identify and characterize the first microneme protein of the ookinete: circumsporozoite- and TRAP-related protein (CTRP). We show that transgenic parasites in which the CTRP gene is disrupted form ookinetes that have reduced motility, fail to invade the midgut epithelium, do not trigger the mosquito immune response, and do not develop further into oocysts. Thus, CTRP is the first molecule shown to be essential for ookinete infectivity and, consequently, mosquito transmission of malaria.     

Plasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites

Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites.     

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.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called "regenerative" cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

The establishment of Plasmodium berghei in mosquitoes of a refractory and a susceptible line of Anopheles atroparvus

The events between the ingestion of Plasmodium berghei-infected mouse blood and the establishment of the ookinetes in the epithelium of the midgut in refractory (R) and susceptible (S) Anopheles atroparvus are described. Simultaneously fed, fully engorged female mosquitoes were randomly assigned to dissection at 22, 28, 32, 48 h and 10 days (controls) after the infective feed (post-infection: p.i.). Serial transverse sections of 6 micron were cut. Every 10th section was studied. The maturation of ookinetes was monitored at 16, 19 and 22 h p.i. The infections in R and S mosquitoes developed similarly with regard to the maturation of ookinetes and the number of mature ookinetes in the lumen of the midgut. The semiquantitative evaluation of the envelopment of the food bolus by the peritrophic layer showed that this layer cannot function as a physical barrier against migrating ookinetes. In the midgut epithelium the number of ookinetes decreased significantly with time in both R and S mosquitoes, but a similar number of penetrations was recorded for both types of mosquito. In S mosquitoes maximal 1% of the ookinetes present in the midgut formed an oocyst. In both R and S mosquitoes a substantial loss of parasites was found, first in the lumen of the midgut and second after penetration of the midgut epithelium by the mature ookinetes. Relatively few parasites develop into oocysts in S, but hardly any do so in R individuals. The factors in control of refractoriness are likely to operate on early oocyst development.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called “regenerative” cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

PbCap380, a novel oocyst capsule protein, is essential for malaria parasite survival in the mosquito

An essential requisite for transmission of Plasmodium, the causative agent of malaria, is the successful completion of a complex developmental cycle in its mosquito vector. Of hundreds of ookinetes that form in the mosquito midgut, only few transform into oocysts, a loss attributed to the action of the mosquito immune system. However, once oocysts form, they appear to be resistant to mosquito defences. During oocyst development, a thick capsule forms around the parasite and appears to function as a protective cover. Little information is available about the composition of this capsule. Here we report on the identification and partial characterization of the first Plasmodium oocyst capsule protein (PbCap380). Genetic analysis indicates that the gene is essential and that PbCap380(-) mutant parasites form oocysts in normal numbers but are gradually eliminated. As a result, mosquitoes infected with PbCap380(-) parasites do not transmit malaria. Targeting of the oocyst capsule may provide a new strategy for malaria control.     

The role of Plasmodium berghei ookinete proteins in binding to basal lamina components and transformation into oocysts.

The ookinete is a motile form of the malaria parasite that travels from the midgut lumen of the mosquito, invades the epithelial cells and settles beneath the basal lamina. The events surrounding cessation of ookinete motility and its transformation into an oocyst are poorly understood, but interaction between components of the basal lamina and the parasite surface has been implicated. Here we report that interactions occur between basal lamina constituents and ookinete proteins and that these interactions inhibit motility and are likely to be involved in transformation to an oocyst. Plasmodium berghei ookinetes bound weakly to microtitre plate wells coated with fibronectin and much more strongly to wells coated with laminin and collagen IV. A 1:1 mixture of collagen and laminin significantly enhanced binding. Binding increased with time of incubation up to 10 h and different components showed different binding profiles with time. Two parasite molecules were shown to act as ligands for basal lamina components. Western blots demonstrated that the surface molecule Pbs21 bound strongly to laminin but not to collagen IV whereas a 215 kDa molecule (possibly PbCTRP) bound to both laminin and collagen IV. Furthermore up to 90% inhibition of binding of ookinetes to collagen IV/laminin combination occurred if parasites were pre-incubated with anti-Pbs21 monoclonal antibody 13.1. Some transformation of ookinetes to oocysts occurred in wells coated with laminin or laminin/collagen IV combinations but collagen IV alone did not trigger transformation. No binding or transformation occurred in uncoated wells. Our data support the suggestion that ookinete proteins Pbs21 and a 215 kDa protein may have multiple roles including interactions with midgut basal lamina components that cause binding, inhibit motility and trigger transformation.     

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.     

Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion

Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and species-specific transmembrane adhesins, which interact with an intracellular motor complex shared across parasite stages and species. How gliding is regulated by extracellular factors and intracellular signalling mechanisms is largely unknown, but current evidence suggests an important role for cytosolic calcium as a second messenger. Studying a Plasmodium berghei gene deletion mutant, we here provide evidence that a calcium-dependent protein kinase, CDPK3, has an important function in regulating motility of the ookinete in the mosquito midgut. We show that a cdpk3- parasite clone produces morphologically normal ookinetes, which fail to engage the midgut epithelium, due to a marked reduction in their ability to glide productively, resulting in marked reduction in malaria transmission to the mosquito. The mutant was successfully complemented with an episomally maintained cdpk3 gene, restoring mosquito transmission to wild-type level. cdpk3- ookinetes maintain their full genetic differentiation potential when microinjected into the mosquito haemocoel and cdpk3- sporozoites produced in this way are motile and infectious, suggesting an ookinete-limited essential function for CDPK3.     

Vesicular ATPase-overexpressing cells determine the distribution of malaria parasite oocysts on the midguts of mosquitoes

In Plasmodium-infected mosquitoes, oocysts are preferentially located at the posterior half of the posterior midgut. Because mosquitoes rest vertically after feeding, the effect of gravity on the ingested blood has been proposed as the cause of such a biased distribution. In this paper, we examined the oocyst distribution on the midguts of mosquitoes that were continuously rotated to nullify the effect of gravity and found that the typical pattern of oocyst distribution did not change. Invasion of the midgut epithelium by ookinetes was similarly found to be biased toward the posterior part of the posterior midgut. We examined whether the distribution of oocysts depends on the distribution of vesicular ATPase (V-ATPase)-overexpressing cells that Plasmodium ookinetes preferentially use to cross the midgut epithelium. An antiserum raised against recombinant Aedes aegypti V-ATPase B subunit indicated that the majority of V-ATPase-overexpressing cells in Ae. aegypti and Anopheles gambiae are localized at the posterior part of the posterior midgut. We propose that the typical distribution of oocysts on the mosquito midgut is attributable to the presence and the spatial distribution of the V-ATPase-overexpressing cells in the midgut epithelium.     

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.     

Differential infectivity of Plasmodium for mosquitoes

The four human malarias - Plasmodium falciparum, P. vivax, P. ovale and P. malariaecan - canonly be transmitted by mosquitoes of the genus Anopheles, although not all species (nor all strains) of these mosquitoes are equally susceptible. Moreover, there are many other plasmodial parasites of other mammals and birds, that can infect other genera of mosquito. What determines this level of vector-parasite specificity? Malarial gametocytes, ingested by a feeding mosquito, must transform to gametes, fuse to form zygotes, and then, as ookinetes, migrate to the mosquito's midgut epithelium to develop as oocysts that release sporozoites to infect the mosquito's salivary glands. During this process, the blood- fed mosquito is developing its peritrophic membrane lining the gut. In this article, the Guthors examine these parallel processes in three sets of mosquito-parasite models, suggesting that parasite-vector specificity may depend on a balance between speed of parasite development versus speed of formation of the peritrophic membrane which can act as a barrier to ookinete migration and establishment in the midgut epithelium.     

Ookinete destruction within the mosquito midgut lumen explains Anopheles albimanus refractoriness to Plasmodium falciparum (3D7A) oocyst infection

Previous studies have shown that the central American mosquito vector, Anopheles albimanus, is generally refractory to oocyst infection with allopatric isolates of the human malaria parasite Plasmodium falciparum. However, the reasons for the refractoriness of A. albimanus to infection with such isolates of P. falciparum are unknown. In the current study, we investigated the infectivity of the P. falciparum clone 3D7A to laboratory-reared A. albimanus and another natural vector of human malaria, Anopheles stephensi. Plasmodium falciparum gametocytes grown in vitro were simultaneously fed to both mosquito species and the progress of malaria infection compared. In 22 independent paired experimental feeds, no mature oocysts were observed on the midguts of A. albimanus 10days after bloodfeeding. In contrast, high levels of oocyst infection were found on the midguts of simultaneously fed A. stephensi. Direct immunofluorescence microscopy and light microscopical examination of Giemsa-stained histological sections were used to identify when the P. falciparum clone 3D7A failed to establish mature oocyst infections in A. albimanus. Similar densities of macrogametes/zygotes, and immature retort-form and mature ookinetes were found within the bloodmeals of both mosquito species. However, in A. albimanus, ookinetes were seldom associated with the peritrophic matrix, and were neither observed in the ectoperitrophic space nor the midgut epithelium. In contrast, ookinetes were frequently observed in these midgut compartments in A. stephensi. Additionally, young oocysts were observed on the midguts of A. stephensi but not A. albimanus 2days after bloodfeeding. Vital staining of the immature retort-form and mature ookinetes found within the luminal bloodmeal, demonstrated that a significantly greater proportion of these malaria parasite stages were non-viable in A. albimanus compared with A. stephensi. Overall, our observations indicate that ookinetes of the P. falciparum clone 3D7A are destroyed within the bloodmeal of A. albimanus and that the midgut lumen, rather than the midgut epithelium, is the site of mosquito refractoriness in this particular malaria parasite-mosquito vector combination.