Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes

Previous studies have shown that mosquitoes inject Plasmodium sporozoites into avascular portions of the skin of their rodent host rather than directly into the blood circulation. Then, over time, these sporozoites move into the circulation, from where they reach the liver to initiate a malaria infection. By use of intravital microscopy of the skin, we present direct morphological evidence of mosquito probing that introduces sporozoites into avascular tissue, of the migration of these sporozoites through the dermis and into blood vessels, and of the role of anti-sporozoite antibodies in blocking sporozoite invasion of these dermal blood vessels.     

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.     

Rhoptry neck protein 11 has crucial roles during malaria parasite sporozoite invasion of salivary glands and hepatocytes

The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells – mosquito salivary glands and mammalian hepatocytes – via involvement in sporozoite motility.     

Early transcriptional responses of HepG2-A16 liver cells to infection by Plasmodium falciparum sporozoites

Invasion of hepatocytes by Plasmodium sporozoites deposited by Anopheles mosquitoes, and their subsequent transformation into infective merozoites is an obligatory step in the initiation of malaria. Interactions between the sporozoites and hepatocytes lead to a distinct, complex and coordinated cellular and systemic host response. Little is known about host liver cell response to sporozoite invasion, or whether it is primarily adaptive for the parasite, for the host, or for both. Our present study used gene expression profiling of human HepG2-A16 liver cells infected with Plasmodium falciparum sporozoites to understand the host early cellular events and factors influencing parasite infectivity and sporozoite development. Our results show that as early as 30 min following wild-type, non-irradiated sporozoite exposure, the expressions of at least 742 genes was selectively altered. These genes regulate diverse biological functions, such as immune processes, cell adhesion and communications, metabolism pathways, cell cycle regulation, and signal transduction. These functions reflect cellular events consistent with initial host cell defense responses, as well as alterations in host cells to sustain sporozoites growth and survival. Irradiated sporozoites gave very similar gene expression pattern changes, but direct comparative analysis between liver gene expression profiles caused by irradiated and non-irradiated sporozoites identified 29 genes, including glypican-3, that were specifically up-regulated only in irradiated sporozoites. Elucidating the role of this subset of genes may help identify the molecular basis for the irradiated sporozoites inability to develop intrahepatically, and their usefulness as an immunogen for developing protective immunity against pre-erythrocytic stage malaria.     

A Cysteine Protease Inhibitor of Plasmodium berghei Is Essential for Exo-erythrocytic Development

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.     

Plasmodium sporozoites trickle out of the injection site

Plasmodium sporozoites make a remarkable journey from the skin, where they are deposited by an infected Anopheline mosquito, to the liver, where they invade hepatocytes and develop into exoerythrocytic stages. Although much work has been done to elucidate the molecular mechanisms by which sporozoites invade hepatocytes, little is known about the interactions between host and parasite before the sporozoite enters the blood circulation. It has always been assumed that sporozoites rapidly exit the injection site, making their interactions with the host at this site, brief and difficult to study. Using quantitative PCR, we determined the kinetics with which sporozoites leave the injection site and arrive in the liver and found that the majority of infective sporozoites remain in the skin for hours. We then performed sub-inoculation experiments which confirmed these findings and showed that the pattern of sporozoite exit from the injection site resembles a slow trickle. Last, we found that drainage of approximately 20% of the sporozoite inoculum to the lymphatics is associated with a significant enlargement of the draining lymph node, a response not observed after intravenous inoculation. These findings indicate that there is ample time for host and parasite to interact at the inoculation site and are of relevance to the pre-erythrocytic stage malaria vaccine effort.     

The ETRAMP Family Member SEP2 Is Expressed throughout Plasmodium berghei Life Cycle and Is Released during Sporozoite Gliding Motility

The early transcribed membrane proteins ETRAMPs belong to a family of small, transmembrane molecules unique to Plasmodium parasite, which share a signal peptide followed by a short lysine-rich stretch, a transmembrane domain and a variable, highly charged C-terminal region. ETRAMPs are usually expressed in a stage-specific manner. In the blood stages they localize to the parasitophorous vacuole membrane and, in described cases, to vesicle-like structures exported to the host erythrocyte cytosol. Two family members of the rodent parasite Plasmodium berghei, uis3 and uis4, localize to secretory organelles of sporozoites and to the parasitophorous membrane vacuole of the liver stages. By the use of specific antibodies and the generation of transgenic lines, we showed that the P. berghei ETRAMP family member SEP2 is abundantly expressed in gametocytes as well as in mosquito and liver stages. In intracellular parasite stages, SEP2 is routed to the parasitophorous vacuole membrane while, in invasive ookinete and sporozoite stages, it localizes to the parasite surface. To date SEP2 is the only ETRAMP protein detected throughout the parasite life cycle. Furthermore, SEP2 is also released during gliding motility of salivary gland sporozoites. A limited number of proteins are known to be involved in this key function and the best characterized, the CSP and TRAP, are both promising transmission-blocking candidates. Our results suggest that ETRAMP members may be viewed as new potential candidates for malaria control.     

Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface. Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus, is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.     

Molecular mechanism of host specificity in Plasmodium falciparum infection: role of circumsporozoite protein

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.     

Invasion of mammalian host cells by Plasmodium sporozoites

Malaria is transmitted through the bite of an infected mosquito, which introduces Plasmodium sporozoites into the mammalian host. Sporozoites rapidly reach the liver of the host where they are sequestered, a process probably mediated by circumsporozoite (CS) protein. Once in the liver, sporozoites migrate through several hepatocytes by breaching their plasma membranes before infecting a final hepatocyte with formation of a vacuole around the sporozoite, where development occurs into blood stage parasites. We propose that migration through several host cells activates sporozoites for ultimate productive invasion. This migration triggers sporozoite exocytosis, which is necessary for hepatocyte invasion, probably because it provides molecules, such as thrombospondin-related anonymous protein (TRAP), likely required for sporozoite invasion with the formation of a vacuole. How sporozoites migrate from the skin to the liver and invade hepatocytes remains unclear. Understanding this initial stage of malaria is crucial for the development of new approaches against the disease.     

Conserved Protective Mechanisms in Radiation and Genetically Attenuated uis3(-) and uis4(-) Plasmodium Sporozoites

Immunization with radiation attenuated Plasmodium sporozoites (RAS) elicits sterile protective immunity against sporozoite challenge in murine models and in humans. Similarly to RAS, the genetically attenuated sporozoites (GAPs) named uis3(-), uis4(-) and P36p(-) have arrested growth during the liver stage development, and generate a powerful protective immune response in mice. We compared the protective mechanisms in P. yoelii RAS, uis3(-) and uis4(-) in BALB/c mice. In RAS and GAPs, sterile immunity is only achieved after one or more booster injections. There were no differences in the immune responses to the circumsporozoite protein (CSP) generated by RAS and GAPs. To evaluate the role of non-CSP T-cell antigens we immunized antibody deficient, CSP-transgenic BALB/c mice, that are T cell tolerant to CSP, with P. yoelii RAS or with uis3(-) or uis4(-) GAPs, and challenged them with wild type sporozoites. In every instance the parasite liver stage burden was approximately 3 logs higher in antibody deficient CSP transgenic mice as compared to antibody deficient mice alone. We conclude that CSP is a powerful protective antigen in both RAS and GAPs viz., uis3(-) and uis4(-) and that the protective mechanisms are similar independently of the method of sporozoite attenuation.     

Sneaking in through the back entrance: the biology of malaria liver stages

Malaria infection is caused by sporozoites, the life cycle stage of Plasmodium that is transmitted by female anopheline mosquitoes. The inoculated sporozoites migrate in the skin, enter a capillary and use the bloodstream for the long haul to the liver. Here, the parasites invade hepatocytes and differentiate to thousands of merozoites that specifically infect red blood cells. Hepatocytes, however, are not directly accessible to sporozoites entering the liver sinusoid. The liver phase of the malaria life cycle can occur only if the parasites first cross the layer of sinusoidal cells that line the liver capillaries. Experimental observations show that sporozoite entry into the liver parenchyma involves a complex cascade of events, from binding to extracellular matrix proteoglycans via passage through Kupffer cells and transmigration through several hepatocytes, until the final host cell is found. By choosing the liver as their initial site of replication, Plasmodium sporozoites can exploit the tolerogenic properties of this unique immune organ to evade the host's immune response.     

The journey of the malaria sporozoite through its hosts: two parasite proteins lead the way

Malaria is transmitted to a mammalian host when the sporozoite stage of the Plasmodium parasite is injected by a mosquito vector. Sporozoites are unique in being able to interact with both hosts. Formed and released in the mosquito midgut, sporozoites bind to the salivary glands and invade their secretory cells. Once injected into the mammalian host, they home to the liver and invade hepatocytes. Recent work has shown that two sporozoite surface proteins, CS and TRAP, act in both hosts, perform multiple functions, and are each essential for the parasite at more than one step of its life cycle.     

Immune response to pre-erythrocytic stages of malaria parasites

Immunization with radiation-attenuated Plasmodium spp. sporozoites induces sterile protective immunity against parasite challenge. This immunity is targeted primarily against the intrahepatic parasite and appears to be sustained long term even in the absence of sporozoite exposure. It is mediated by multifactorial mechanisms, including T cells directed against parasite antigens expressed in the liver stage of the parasite life cycle and antibodies directed against sporozoite surface proteins. In rodent models, CD8+ T cells have been implicated as the principal effector cells, and IFN-gamma as a critical effector molecule. IL-4 secreting CD4+ T cells are required for induction of the CD8+ T cell responses, and Th1 CD4+ T cells provide help for optimal CD8+ T cell effector activity. Components of the innate immune system, including gamma-delta T cells, natural killer cells and natural killer T cells, also play a role. The precise nature of pre-erythrocytic stage immunity in humans, including the contribution of these immune responses to the age-dependent immunity naturally acquired by residents of malaria endemic areas, is still poorly defined. The importance of immune effector targets at the pre-erythrocytic stage of the parasite life cycle is highlighted by the fact that infection-blocking immunity in humans rarely, if ever, occurs under natural conditions. Herein, we review our current understanding of the molecular and cellular aspects of pre-erythrocytic stage immunity.     

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.     

The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics.     

Variants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Colombian isolates

Phenotypic diversity has been described in the central repeated region of the circumsporozoite protein (CSP) from Plasmodium vivax. Two sequences VK210 (common) and VK247 (variant) have been found widely distributed in P. vivax isolates from several malaria endemic areas around the world. A third protein variant called P. vivax-like showing a sequence similar to the simian parasite P. simioovale has also been described. Here, using an immunofluorescent test and specific monoclonal antibodies, we assessed the presence of two of these protein variants (VK210 and VK247) in laboratory produced sporozoite. Both sequences were found in parasite isolates coming from different geographic regions of Colombia. Interestingly, sporozoites carrying the VK247 sequence were more frequently produced in Anopheles albimanus than sporozoites with the VK210 sequence. This difference in sporozoites production was statistically significant (p <0.05, Kruskal-Wallis); not correlation was found with parameters as the total number of parasites or gametocytes in blood from human donors used to feed mosquitoes. Previous studies in the same region have shown a higher prevalence of anti-VK210 antibodies which in theory may suggest their role in blocking the development of sporozoites carrying the CSP VK210 sequence.     

A rodent malaria, Plasmodium berghei, is experimentally transmitted to mice by merely probing of infective mosquito, Anopheles stephensi

We found that infection of a rodent malaria, Plasmodium berghei, occurred when the sporozoites were injected into the skin, the muscle, the peritoneal cavity and the tail end. Mice, which were injected with sporozoites in the tail end and had the site cut 5 min later, did not develop malaria. We also found that mice developed malaria when malaria infective mosquitoes, Anopheles stephensi, were forced not to take blood but only to probe into the skin. Moreover, the mice probed by the infective mosquitoes were protected from malaria infection if the site was treated with Kyu (heat treatment) after the mosquitoes had probed. These findings indicate that malaria infection occurs not only by blood feeding of the infective mosquito but also by probing of the mosquito. Sporozoites injected into the skin remain at the injected site for at least 5 min, then migrate to the blood vessels and invade into the blood stream. At present, the mechanism is not clear, although we propose here the existence of the skin stage of malaria parasites before the liver stage and the blood stage.     

Environmental constraints guide migration of malaria parasites during transmission

Migrating cells are guided in complex environments mainly by chemotaxis or structural cues presented by the surrounding tissue. During transmission of malaria, parasite motility in the skin is important for Plasmodium sporozoites to reach the blood circulation. Here we show that sporozoite migration varies in different skin environments the parasite encounters at the arbitrary sites of the mosquito bite. In order to systematically examine how sporozoite migration depends on the structure of the environment, we studied it in micro-fabricated obstacle arrays. The trajectories observed in vivo and in vitro closely resemble each other suggesting that structural constraints can be sufficient to guide Plasmodium sporozoites in complex environments. Sporozoite speed in different environments is optimized for migration and correlates with persistence length and dispersal. However, this correlation breaks down in mutant sporozoites that show adhesion impairment due to the lack of TRAP-like protein (TLP) on their surfaces. This may explain their delay in infecting the host. The flexibility of sporozoite adaption to different environments and a favorable speed for optimal dispersal ensures efficient host switching during malaria transmission.