Migration through host cells activates Plasmodium sporozoites for infection

Plasmodium sporozoites, the infective stage of the malaria parasite transmitted by mosquitoes, migrate through several hepatocytes before infecting a final one. Migration through hepatocytes occurs by breaching their plasma membranes, and final infection takes place with the formation of a vacuole around the sporozoite. Once in the liver, sporozoites have already reached their target cells, making migration through hepatocytes prior to infection seem unnecessary. Here we show that this migration is required for infection of hepatocytes. Migration through host cells, but not passive contact with hepatocytes, induces the exocytosis of sporozoite apical organelles, a prerequisite for infection with formation of a vacuole. Sporozoite activation induced by migration through host cells is an essential step of Plasmodium life cycle.     

Expression of human CD81 differently affects host cell susceptibility to malaria sporozoites depending on the Plasmodium species

Plasmodium sporozoites can enter host cells by two distinct pathways, either through disruption of the plasma membrane followed by parasite transmigration through cells, or by formation of a parasitophorous vacuole (PV) where the parasite further differentiates into a replicative exo-erythrocytic form (EEF). We now provide evidence that following invasion without PV formation, transmigrating Plasmodium falciparum and Plasmodium yoelii sporozoites can partially develop into EEFs inside hepatocarcinoma cell nuclei. We also found that rodent P. yoelii sporozoites can infect both mouse and human hepatocytes, while human P. falciparum sporozoites infect human but not mouse hepatocytes. We have previously reported that the host tetraspanin CD81 is required for PV formation by P. falciparum and P. yoelii sporozoites. Here we show that expression of human CD81 in CD81-knockout mouse hepatocytes is sufficient to confer susceptibility to P. yoelii but not P. falciparum sporozoite infection, showing that the narrow P. falciparum host tropism does not rely on CD81 only. Also, expression of CD81 in a human hepatocarcinoma cell line is sufficient to promote the formation of a PV by P. yoelii but not P. falciparum sporozoites. These results highlight critical differences between P. yoelii and P. falciparum sporozoite infection, and suggest that in addition to CD81, other molecules are specifically required for PV formation during infection by the human malaria parasite.     

Inhibition of Plasmodium sporozoites infection by targeting the host cell

There is a great need of new drugs against malaria because of the increasing spread of parasite resistance against the most commonly used drugs in the field. We found that monensin, a common veterinary antibiotic, has a strong inhibitory effect in Plasmodium berghei and Plasmodium yoelii sporozoites hepatocyte infection in vitro. Infection of host cells by another apicomplexan parasite with a similar mechanism of host cell invasion, Toxoplasma tachyzoites, was also inhibited. Treatment of mice with monensin abrogates liver infection with P. berghei sporozoites in vivo. We also found that at low concentrations monensin inhibits the infection of Plasmodium sporozoites by rendering host cells resistant to infection, rather than having a direct effect on sporozoites. Monensin effect is targeted to the initial stages of parasite invasion of the host cell with little or no effect on development, suggesting that this antibiotic affects an essential host cell component that is required for Plasmodium sporozoite invasion.     

Migration through host cells by apicomplexan parasites.

In what appears to be an essential prelude to establish a successful infection in the mammalian host, Plasmodium sporozoites move rapidly through several host cells breaching the cell plasma membranes in the process. This mode of invasion precedes the 'traditional' mode in which the sporozoite enters by invagination of the host cell membrane and develops within a parasitophorous vacuole. Here we revisit the existing literature that supports the presence of similar invasive behaviors in other apicomplexan parasites.     

Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade host cells

Malaria infection is initiated when Anopheles mosquitoes inject Plasmodium sporozoites into the skin. Sporozoites subsequently reach the liver, invading and developing within hepatocytes. Sporozoites contact and traverse many cell types as they migrate from skin to liver; however, the mechanism by which they switch from a migratory mode to an invasive mode is unclear. Here, we show that sporozoites of the rodent malaria parasite Plasmodium berghei use the sulfation level of host heparan sulfate proteoglycans (HSPGs) to navigate within the mammalian host. Sporozoites migrate through cells expressing low-sulfated HSPGs, such as those in skin and endothelium, while highly sulfated HSPGs of hepatocytes activate sporozoites for invasion. A calcium-dependent protein kinase is critical for the switch to an invasive phenotype, a process accompanied by proteolytic cleavage of the sporozoite's major surface protein. These findings explain how sporozoites retain their infectivity for an organ that is far from their site of entry.     

Kinome-wide RNAi screen implicates at least 5 host hepatocyte kinases in Plasmodium sporozoite infection

Plasmodium sporozoites, the causative agent of malaria, are injected into their vertebrate host through the bite of an infected Anopheles mosquito, homing to the liver where they invade hepatocytes to proliferate and develop into merozoites that, upon reaching the bloodstream, give rise to the clinical phase of infection. To investigate how host cell signal transduction pathways affect hepatocyte infection, we used RNAi to systematically test the entire kinome and associated genes in human Huh7 hepatoma cells for their potential roles during infection by P. berghei sporozoites. The three-phase screen covered 727 genes, which were tested with a total of 2,307 individual siRNAs using an automated microscopy assay to quantify infection rates and qRT-PCR to assess silencing levels. Five protein kinases thereby emerged as top hits, all of which caused significant reductions in infection when silenced by RNAi. Follow-up validation experiments on one of these hits, PKCsigma (PKCzeta), confirmed the physiological relevance of our findings by reproducing the inhibitory effect on P. berghei infection in adult mice treated systemically with liposome-formulated PKCsigma-targeting siRNAs. Additional cell-based analyses using a pseudo-substrate inhibitor of PKCsigma added further RNAi-independent support, indicating a role for host PKCsigma on the invasion of hepatocytes by sporozoites. This study represents the first comprehensive, functional genomics-driven identification of novel host factors involved in Plasmodium sporozoite infection.     

Targeting Plasmodium host cells: survival within hepatocytes

Upon entering their host, Plasmodium sporozoites travel directly to the liver. Once there, they migrate through several hepatocytes before they infect a final one. During migration, sporozoites breach the plasma membrane of traversed hepatocytes, but to infect they must form a parasitophorous vacuole, in which the intra-hepatic form of the parasite grows and multiplies. During this period there is a remarkable parasite multiplication, but little is known about the requirements and strategies that are developed to be successful. Hepatocyte growth factor and its receptor on hepatocytes might enhance early Plasmodium development within these cells. We anticipate that this might be the basis for further studies on host-cell requirements for Plasmodium development.     

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.     

The elusive malaria sporozoite in the mammalian host

Malaria infection is initiated when Plasmodium sporozoites are injected into a host during the bite of an infected mosquito. In the mammal, the sporozoite must rapidly reach an intravacuolar niche within a hepatocyte, where it will generate the parasite stage that invades red blood cells and causes the symptoms of the disease. Herein, we describe our understanding of the way in which sporozoites travel from the site of the mosquito bite to the liver, arrest in the liver, cross the sinusoidal barrier and eventually gain access to hepatocytes. We also highlight some of the recent advances in our understanding of these processes at the molecular level.     

Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection

Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha), a gene containing regions with high homology to adenylyl cyclases. PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACalpha and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.     

Alternative invasion pathways for Plasmodium berghei sporozoites

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a natural malaria infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that infection by Plasmodium falciparum and Plasmodium yoelii sporozoites depends on CD81 and cholesterol-dependent tetraspanin-enriched microdomains (TEMs) on the hepatocyte surface. Here we have analyzed the role of CD81 and TEMs during infection by sporozoites from the rodent parasite Plasmodium berghei. We found that depending on the host cell type, P. berghei sporozoites can use several distinct pathways for invasion. Infection of human HepG2, HuH7 and HeLa cells by P. berghei does not depend on CD81 or host membrane cholesterol, whereas both CD81 and cholesterol are required for infection of mouse hepatoma Hepa1-6 cells. In primary mouse hepatocytes, both CD81-dependent and -independent mechanisms participate in P. berghei infection and the relative contribution of the different pathways varies, depending on mouse genetic background. The existence of distinct invasion pathways may explain why P. berghei sporozoites are capable of infecting a wide range of host cell types in vitro. It could also provide a means for human parasites to escape immune responses and face polymorphisms of host receptors. This may have implications for the development of an anti-malarial vaccine targeting sporozoites.     

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.     

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.     

CO-opting the host HO-1 pathway in tuberculosis and malaria

Infection with Mycobacterium tuberculosis and Plasmodium species results in upregulation of the host heme oxygenase-1 pathway. In tuberculosis infection, this leads to upregulation of the bacterial "dormancy regulon," whereas in malaria, it enhances the efficiency with which sporozoites develop into exoerythrocytic stages. Here we discuss these findings as well as some of the interesting questions they raise.     

Distinct malaria parasite sporozoites reveal transcriptional changes that cause differential tissue infection competence in the mosquito vector and mammalian host

The malaria parasite sporozoite transmission stage develops and differentiates within parasite oocysts on the Anopheles mosquito midgut. Successful inoculation of the parasite into a mammalian host is critically dependent on the sporozoite's ability to first infect the mosquito salivary glands. Remarkable changes in tissue infection competence are observed as the sporozoites transit from the midgut oocysts to the salivary glands. Our microarray analysis shows that compared to oocyst sporozoites, salivary gland sporozoites upregulate expression of at least 124 unique genes. Conversely, oocyst sporozoites show upregulation of at least 47 genes (upregulated in oocyst sporozoites [UOS genes]) before they infect the salivary glands. Targeted gene deletion of UOS3, encoding a putative transmembrane protein with a thrombospondin repeat that localizes to the sporozoite secretory organelles, rendered oocyst sporozoites unable to infect the mosquito salivary glands but maintained the parasites' liver infection competence. This phenotype demonstrates the significance of differential UOS expression. Thus, the UIS-UOS gene classification provides a framework to elucidate the infectivity and transmission success of Plasmodium sporozoites on a whole-genome scale. Genes identified herein might represent targets for vector-based transmission blocking strategies (UOS genes), as well as strategies that prevent mammalian host infection (UIS genes).     

Host cell traversal is important for progression of the malaria parasite through the dermis to the liver

The malaria sporozoite, the parasite stage transmitted by the mosquito, is delivered into the dermis and differentiates in the liver. Motile sporozoites can invade host cells by disrupting their plasma membrane and migrating through them (termed cell traversal), or by forming a parasite-cell junction and settling inside an intracellular vacuole (termed cell infection). Traversal of liver cells, observed for sporozoites in vivo, is thought to activate the sporozoite for infection of a final hepatocyte. Here, using Plasmodium berghei, we show that cell traversal is important in the host dermis for preventing sporozoite destruction by phagocytes and arrest by nonphagocytic cells. We also show that cell infection is a pathway that is masked, rather than activated, by cell traversal. We propose that the cell traversal activity of the sporozoite must be turned on for progression to the liver parenchyma, where it must be switched off for infection of a final hepatocyte.     

A surface phospholipase is involved in the migration of plasmodium sporozoites through cells

Plasmodium sporozoites, injected by mosquitoes into the skin of the host, traverse cells during their migration to hepatocytes where they continue their life cycle. The mechanisms used by the parasite to rupture the plasma membrane of the host cells are not known. Here we report the presence of a phospholipase on the surface of Plasmodium berghei sporozoites (P. berghei phospholipase; Pb PL) and demonstrate that it is involved in the establishment of a malaria infection in vivo. Pb PL is highly conserved among the Plasmodium species. The protein is about 750 amino acids, with a predicted signal sequence and a carboxyl terminus that is 32% identical to the vertebrate lecithin:cholesterol acyltransferase, a secreted phospholipase. Pb PL contains a motif characteristic of lipases and a catalytic triad of a serine, aspartate, and histidine that is found in several phospholipases. We have verified its lipase and membrane lytic activity in vitro, using recombinant baculovirus-expressed protein. To study its role in vivo, we have disrupted the P. berghei PL open reading frame and generated mutants in its active site. During an infection through mosquito bite, the infectivity of the knock-out parasites in the liver is decreased by approximately 90%. The prepatent period of the resulting blood infection is 1 day longer as compared with wild type. Further, the mutant sporozoites are impaired in their ability to cross epithelial cell layers. Thus, the Pb PL functions as a lipase to damage cell membranes and facilitates sporozoite passage through cells during their migration from the skin to the bloodstream.     

HGF/MET signalling protects Plasmodium-infected host cells from apoptosis

Plasmodium, the causative agent of malaria, migrates through several hepatocytes before initiating a malaria infection. We have previously shown that this process induces the secretion of hepatocyte growth factor (HGF) by traversed cells, which renders neighbour hepatocytes susceptible to infection. The signalling initiated by HGF through its receptor MET has multifunctional effects on various cell types. Our results reveal a major role for apoptosis protection of host cells by HGF/MET signalling on the host susceptibility to infection. Inhibition of HGF/MET signalling induces a specific increase in apoptosis of infected cells leading to a great reduction on infection. Since HGF/MET signalling is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways, we determined the impact of these pathways on Plasmodium sporozoite infection. Although inhibition of either of these pathways leads to a reduction in infection, inhibition of PI3-kinase/Akt pathway caused a stronger effect, which correlated with a higher level of apoptosis in infected host cells. Altogether, the results show that the HGF/MET signalling requirement for infection is mediated by its anti-apoptotic signal effects. These results demonstrate for the first time that active inhibition of apoptosis in host cell during infection by Plasmodium is required for a successful infection.     

Heme oxygenase-1 is an anti-inflammatory host factor that promotes murine plasmodium liver infection

The clinically silent Plasmodium liver stage is an obligatory step in the establishment of malaria infection and disease. We report here that expression of heme oxygenase-1 (HO-1, encoded by Hmox1) is upregulated in the liver following infection by Plasmodium berghei and Plasmodium yoelii sporozoites. HO-1 overexpression in the liver leads to a proportional increase in parasite liver load, and treatment of mice with carbon monoxide and with biliverdin, each an enzymatic product of HO-1, also increases parasite liver load. Conversely, mice lacking Hmox1 completely resolve the infection. In the absence of HO-1, the levels of inflammatory cytokines involved in the control of liver infection are increased. These findings suggest that, while stimulating inflammation, the liver stage of Plasmodium also induces HO-1 expression, which modulates the host inflammatory response, protecting the infected hepatocytes and promoting the liver stage of infection.