Migration of Plasmodium sporozoites through cells before infection.

Intracellular bacteria and parasites typically invade host cells through the formation of an internalization vacuole around the invading pathogen. Plasmodium sporozoites, the infective stage of the malaria parasite transmitted by mosquitoes, have an alternative mechanism to enter cells. We observed breaching of the plasma membrane of the host cell followed by rapid repair. This mode of entry did not result in the formation of a vacuole around the sporozoite, and was followed by exit of the parasite from the host cell. Sporozoites traversed the cytosol of several cells before invading a hepatocyte by formation of a parasitophorous vacuole, in which they developed into the next infective stage. Sporozoite migration through several cells in the mammalian host appears to be essential for the completion of the life cycle.

Identification of electrophoretically separated proteases from midgut and hemolymph of adult Anopheles stephensi mosquitoes.

Digestion of blood within the mosquito midgut is mediated primarily by a series of proteases, and several previous studies have described protease activity within homogenates of the midgut of the malaria vector Anopheles stephensi. We have expanded on these previous data by resolving protease isoforms from the midgut as well as the hemolymph of adult An. stephensi mosquitoes via gel electrophoresis and zymography. Using this procedure, we have been able to identify multiple isozymes of trypsin, chymotrypsin, and aminopeptidase. We were able to detect an increase in the intensity of some of these protease bands plus the appearance of new bands 24 hr after mosquitoes had taken a blood meal. Furthermore, we detected 2 endogenous trypsin isozymes within the hemolymph. There was no upregulation of these hemolymph isozymes after a blood meal, thus suggesting that they may not be involved in digestion of the blood meal by the mosquito.

Delayed migration of Plasmodium sporozoites from the mosquito bite site to the blood.

Studies were done on delivery of Plasmodium yoelii sporozoites by Anopheles stephensi into the skin of BALB/C mice. When infected mosquitoes fed on a portion of the ear, 81% of these positive control mice developed parasitemia. When the fed-upon site was excised immediately or 5 min postfeeding, a highly significant, smaller percentage of these experimental mice developed parasitemia. When the delay in removal of mosquito-bitten tissue was extended to 15 min, no significant difference was found between this group and positive control mice. These findings show that mosquito-injected sporozoites tend to remain at the bite site for at least 5 min after the mosquito bite. By approximately 15 min, the first wave of migrating sporozoites has left the bite site and moved into the general circulation. These findings have implications concerning possible host obstruction of sporozoite migration in skin by either anti-sporozoite antibodies or by a cutaneous hypersensitivity reaction to mosquito bite.

Electrophoretic separation and identification of phenoloxidases in hemolymph and midgut of adult Anopheles stephensi mosquitoes.

Anopheles mosquitoes frequently respond to invading malaria parasites with a rejection mechanism consisting of phenoloxidase-mediated melanization of ookinetes in the mosquito midgut epithelium. The relative roles of hemolymph vs. midgut phenoloxidase in this rejection mechanism is unclear. We have separated and identified phenoloxidase isozymes from midgut and hemolymph of Anopheles stephensi by native gel electrophoresis followed by zymography. The isozymes from the 2 sites had distinctively different electrophoretic characteristics. Hemolymph possessed 2 phenoloxidase-positive bands, both of which were bifunctional molecules that oxidized monophenol as well as o-diphenol substrates. Midgut extract possessed 3 bands that migrated more rapidly than those of the hemolymph. None of these midgut bands had detectable monophenoloxidase activity; they possessed, however, a broad spectrum of diphenoloxidase activity in their ability to oxidize both o- and p-diphenol substrates, as well as the laccase substrate syringaldazine. The 2 most rapidly migrating midgut PO bands could be distinguished from the more slowly migrating band through their insensitivity to inhibition by the chelating agent tropolone. A question to be resolved in the future relates to the relative roles of hemolymph vs. midgut phenoloxidase in mosquito defense against invasive parasites.

Anopheles stephensi salivary glands bear receptors for region I of the circumsporozoite protein of Plasmodium falciparum.

In the mosquito, Plasmodium sporozoites rupture from oocysts found on the midgut wall, circulate in the hemolymph and invade salivary glands where they wait to be injected into a vertebrate host during a bloodmeal. The mechanisms by which sporozoites specifically attach to and invade salivary glands are not known but evidence suggests that it is a receptor-mediated process. Here we show that the major surface protein of sporozoites, the circumsporozoite protein (CS), binds preferentially to salivary glands when compared to other organs exposed to the circulating hemolymph. In addition, we show that a peptide encompassing region I, a highly conserved sequence found in all rodent and primate Plasmodium CS proteins, inhibits binding of CS to mosquito salivary glands.

Inhibition of mosquito salivary gland apyrase activity by antibodies produced in mice immunized by bites of Anopheles stephensi mosquitoes.

Mice (BALB/c) were immunized to mosquito saliva by repeated bites of Anopheles stephensi mosquitoes. Studies were conducted on the ability of these mice to develop antibodies against the apyrase component of the saliva. By means of immunoprecipitation procedures and Western blot analysis, we demonstrated the presence of antiapyrase antibodies to the mosquito saliva. Furthermore, these antibodies were able to inhibit apyrase activity. Serum titers of 1:20 were able to inhibit approximately 90% of salivary gland apyrase activity, while titers of 1:160 retained the ability to inhibit more than 50% of apyrase activity. Parallel inhibition assays with immunoglobulin G (IgG) from immunized versus nonimmunized mice showed that the inhibitory activity of serum from immunized mice could be accounted for by its IgG component. Mosquito salivary gland apyrase has previously been shown to facilitate mosquito feeding by inhibiting aggregation of platelets at the mosquito bite site. However, our studies have shown that mosquitoes feeding on immunized mice had no deficiency in probing these mice for a blood meal, even in the face of high titers of anti-apyrase antibodies.

Plasmodium sporozoites possess both positively and negatively charged sites accessible on their surface.

Studies were conducted on the distribution of anionic and cationic sites on the surface of intact Plasmodium berghei and P. yoelii sporozoites. Anionic and cationic ferritins were used as probes for electron microscope studies and fluorescein conjugates of the same charged ferritins were used for correlative studies by fluorescence light microscopy. We found that the surfaces of mature sporozoites from mosquito salivary glands possess both negatively and positively charged sites that are accessible to charged ferritin molecules. Sporozoites obtained from oocysts expressed negatively charged sites but were inconsistent in their expression of positively charged sites. The charged nature of the ferritin probes was confirmed by their binding to cellulose particles or cellular components with known negative or positive charges. The electrostatic nature of the binding of ferritin to sporozoites was established by showing that binding could be blocked by competition with high concentrations of NaCl. The most likely source of these surface charges is circumsporozoite (CS) protein, the major protein covering the surface of sporozoites. Circumsporozoite proteins contain a cluster of positively charged amino acids, which may play a role as components of a surface ligand capable of binding to a liver receptor.

Induction of hepatic inflammatory response by Plasmodium berghei sporozoites protects BALB/c mice against challenge with Plasmodium yoelii sporozoites.

BALB/c mice are about 2,000 times less susceptible to sporozoites of Plasmodium berghei than to Plasmodium yoelii. Associated with this is the innate cellular response mounted after injection with P. berghei. Host inflammatory cells do not normally attack P. yoelii during their development as exoerythrocytic forms (EEFs) in the liver. We used P. berghei sporozoites to induce host inflammation that might act against developing P. yoelii EEFs. Mice injected with P. berghei sporozoites followed 1 hr later with P. yoelii had a 58% reduction in P. yoelii EEFs. To establish whether this was due to events that occurred before vs. after invasion of hepatocytes by P. yoelii sporozoites, mice received P. yoelii sporozoites that were allowed to invade for 1 hr before subsequent injection with P. berghei; these mice showed minimal reduction in P. yoelii EEFs. Thus, most of the deleterious effects of P. berghei sporozoites appear to have been directed against P. yoelii sporozoites prior to their invasion of hepatocytes. Plasmodium yoelii that had already invaded were relatively unaffected. Further timing experiments showed that this effect was induced only by viable P. berghei sporozoites, which may thus induce rapid changes in sinusoid physiology leading to host resistance against P. yoelii sporozoites.

Electron microscopic analysis of circumsporozoite protein trail formation by gliding malaria sporozoites.

Immunoelectron microscopic techniques were utilized to characterize the morphology of circumsporozoite protein-containing trails deposited on various substrates by gliding Plasmodium berghei and Plasmodium falciparum sporozoites. The basic components of the trails are beadlike particles, 25 to 90 nm in diameter, which are devoid of unit membrane and have an electron-lucent center. Trails were captured on formvar-covered grids coated with anticircumsporozoite protein monoclonal antibodies and compared with trails produced on uncoated formvar; the results suggest that material containing circumsporozoite protein forms the matrix within which the particles are embedded. The trails exhibit morphological features similar to those displayed by circumsporozoite precipitation reactions; of note is the demonstration of sheaths of circumsporozoite protein-containing material that emanate from sporozoites prior to their gliding. The sheaths narrow into accumulations of electron-dense material, which eventually taper to form typical trails. The structural manifestation of sheaths and other morphological details of the formed trails enables us to correlate sporozoite behavior during trail formation with the motile actions of gliding sporozoites observed by light microscopy.

Specific inflammatory cell infiltration of hepatic schizonts in BALB/c mice immunized with attenuated Plasmodium yoelii sporozoites.

We compared immunization of BALB/c mice with radiation-attenuated versus killed sporozoites of the rodent malaria parasite, Plasmodium yoelii. We employed a suboptimal schedule of only two immunizations, in expectation that some parasites might break through the resultant low level immunity and that it might thus be possible to study the response of the host against these 'breakthrough' schizonts. As a measure of protective immunity, we used histological means to determine the percentages of challenge sporozoites prevented from completing development into hepatic schizonts within the liver. Immunization with attenuated sporozoites led to almost complete protection, whereas immunization with similar dosages of killed sporozoites led to approximately a 75% protection. Fluorescent antibody titers against sporozoites were similar in both sets of immunized animals. However, serum from mice immunized with attenuated sporozoites had a protective effect upon passive transfer into immunologically naive mice subsequently challenged with normal sporozoites; serum from mice immunized with killed sporozoites had no such effect. When mice suboptimally immunized with attenuated sporozoites were challenged, we observed breakthrough schizonts being infiltrated with inflammatory cells, primarily mononuclear cells, and neutrophils; partial depletion of CD4+ or CD8+ cells within these mice prior to challenge prevented the infiltration of breakthrough schizonts. Thus, cellular infiltration of schizonts was apparently secondary to earlier action by lymphocytes. This infiltration was also not observed in mice immunized with killed sporozoites. The more effective protective immunity induced by attenuated sporozoites could be due to their ability to release antigen into the cytoplasm of hepatocytes that they invade or their ability to continue differentiating, thereby presenting new antigens that are not seen after immunization with killed sporozoites.

Early hepatic stages of Plasmodium berghei: release of circumsporozoite protein and host cellular inflammatory response.

After injection of Plasmodium berghei sporozoites into Norway-Brown rats, we were able to localize these sporozoites and the early hepatic trophozoites developing from them in histological sections of the liver stained with a sensitive immunogold-silver procedure. Sporozoites invading hepatocytes released substantial quantities of circumsporozoite protein into the hepatocyte cytoplasm. This intrahepatic cytoplasmic distribution reached a maximal level at about 4 h post-sporozoite injection. As the hepatic parasites continued to differentiate, circumsporozoite protein became undetectable within the cytoplasm of the hepatocytes and became localized around the periphery of each parasite. There was generalized cellular inflammation within the liver of the host, which first became evident at around 4 h post-sporozoite injection and progressed to the formation of well-defined granulomas by 24 h. Such histopathological changes were not seen in rats injected with killed sporozoites, indicating that the cellular inflammation was induced by viable, infective sporozoites. We did not observe cellular infiltration specifically associated with any of the developing hepatic stages that we observed, even up to 28 h post-sporozoite inoculation. These results indicate that viable sporozoites induced rapid and generalized hepatic inflammation in host rats. However, sporozoites that successfully invaded hepatocytes and then proceeded to develop further did not appear to be the target of inflammatory cells until a period beginning at around 40 h post-sporozoite inoculation.

Role of host cellular response in differential susceptibility of nonimmunized BALB/c mice to Plasmodium berghei and Plasmodium yoelii sporozoites.

We found BALB/c mice to be on the order of 2,000 times more susceptible to Plasmodium yoelii than Plasmodium berghei sporozoites, as measured by the ability of these sporozoites to differentiate into microscopically detectable hepatic schizonts in the livers of immunologically naive mice. One of the factors that determine the relative insusceptibility of mice to P. berghei sporozoites is the innate cellular inflammatory response that the mice mount after injection with sporozoites. The cellular inflammatory response against P. berghei is initiated soon after sporozoite injection; by 24 h, substantial histopathological changes have developed within the liver. There is considerably less of a cellular inflammatory response against P. yoelii; significant histopathological changes within the liver are not observed until well after hepatic schizonts have begun to rupture at around 44 h postinjection of sporozoites. These differences in the cellular inflammatory response against two different, closely related species of sporozoites are of considerable interest. The data strongly suggest that the BALB/c-P. berghei sporozoite system is a relatively poor biological model for sporozoite immunization studies.

Eosinophil-rich, granulomatous inflammatory response to Plasmodium berghei hepatic schizonts in nonimmunized rats is age-related.

Inflammatory responses to Plasmodium hepatic schizonts within the livers of non-immunized animals have long been assumed to be initiated only after the parasites have matured and begun to burst. However, recent reports of inflammatory responses around hepatic schizonts suggested a re-examination of this issue. We injected Norway-Brown rats of various ages intravenously with Plasmodium berghei sporozoites and studied subsequent liver histopathology. We found that the ability of these rats to mount an inflammatory response is age-dependent. Young (4 weeks) rats had weak inflammatory responses against hepatic schizonts, whereas older (8-10 weeks) rats mounted a strong response. Older rats had many granulomatous reactions within the liver; eosinophils represented a pioneer component of the cellular infiltrate. There was a reduction in the numbers of surviving hepatic schizonts in the older rats, suggesting that these granulomatous and eosinophilic reactions had effectively destroyed some of the hepatic schizonts. We found clear evidence of inflammatory cells (eosinophils) infiltrating hepatic schizonts as early as 40 hours post-injection with sporozoites, a time well before any hepatic schizonts could have burst within the liver. This presents histological evidence that inflammatory cells can recognize and infiltrate intact hepatocytes containing schizonts in immunologically naive animals.

Malaria sporozoites release circumsporozoite protein from their apical end and translocate it along their surface.

Plasmodium sporozoites, the causative agents of malaria, release circumsporozoite (CS) protein into medium when under conditions simulating those that the parasites encounter in the bloodstream of the vertebrate host. CS protein of the rodent parasite, Plasmodium berghei, is released as the lower molecular weight form, Pb44. This release is substratum- and antibody-independent. Previous studies show that CS protein is released at the trailing, posterior end of motile sporozoites. Video and electron microscopic studies now demonstrate that CS protein is released at the apical end of cytochalasin b-immobilized sporozoites. We propose that CS protein released from the apical end, the leading end of gliding sporozoites, adheres to the sporozoite surface and is translocated posteriorly by a cytochalasin-sensitive and apparently actin-mediated surface motor, which drives gliding motility. This model explains the mechanism of both the circumsporozoite precipitation (CSP) reaction and formation of the CS protein trail by gliding sporozoites.

Biochemical characterization of Plasmodium falciparum hemozoin.

Hemozoin, the pigment granule which develops within the blood stage food vacuole of the malaria parasite Plasmodium falciparum, was biochemically characterized. Hemozoin was found to be composed of 65% protein, 16% ferriprotoporphyrin-IX (hematin), 6% carbohydrate, and trace amounts of lipid and nucleic acids. The overwhelming majority of the protein component is a mixture of native and denatured human globin non-covalently associated with the metalloporphyrin. Immunoelectron microscopy, employing anti-human hemoglobin as a probe, identified in situ association of hemoglobin with hemozoin. Hemozoin produced within diabetic blood had a higher proportion of carbohydrate, suggesting that the carbohydrate component comes from non-enzymatic glycosylation of hemoglobin.

Plasmodium sporozoite interactions with macrophages in vitro: a videomicroscopic analysis.

Studies of in vitro interactions between Plasmodium berghei sporozoites and peritoneal macrophages from mice and rats were performed. A videomicroscopic analysis was made of interactions observed by phase-contrast microscopy. Our results showed a diversity of dynamic interactions between sporozoites and macrophages that included no interaction, surface interaction without sporozoite interiorization, active sporozoite penetration, active penetration with subsequent sporozoite escape, macrophage destruction, and the formation of "tethers" or web-like structures by sporozoites that had actively invaded macrophages. Sporozoites are thus clearly capable of actively invading host macrophages and are not restricted to being phagocytosed for interiorization. The formation of "tethers" by the moving sporozoite might function in vivo by anchoring the sporozoite to the cells lining the lumen of the liver sinusoid. Active sporozoite motility appears to be a functional phenomenon involved in sporozoite invasion of host liver cells.

Plasmodium sporozoite-host cell interactions during sporozoite invasion.

In vitro and in vivo studies were performed to clarify the nature of some interactions between Plasmodium berghei sporozoites and rodent host cells. Videomicroscopic observations were made on in vitro interactions between sporozoites and cultured host cells (rodent peritoneal macrophages, W138 human lung fibroblasts, and HepG2 human hepatoma cells). The results showed a diversity of dynamic interactions and sporozoite activities, including active sporozoite penetration, often followed by sporozoite escape, killing of invaded macrophages, phagocytosis of sporozoites by macrophages, and elaboration of a weblike structure by the sporozoite, following its escape from cells. Other studies were performed to examine host cell inflammatory responses to challenge sporozoites in the livers of rats that had been previously immunized with radiation-attenuated sporozoites. The results showed a variety of focal inflammatory infiltrates in the livers of the challenged animals. The relevance of these observations to the problems of sporozoite invasion into natïve and immunized animals is discussed.

Malaria sporozoites leave behind trails of circumsporozoite protein during gliding motility.

As Plasmodium sporozoites undergo gliding motility in vitro, they leave behind trails of circumsporozoite (CS) protein that correspond to their patterns of movement. This light microscopic observation was made using Plasmodium berghei sporozoites, a monoclonal antibody (MAb H4) directed against the immunodominant repetitive epitope of the CS protein of P. berghei, and an immunogold-silver staining (IGSS) technique. Sporozoites pretreated with agents that inhibit sporozoite motility and invasiveness did not produce trails. Sporozoites that glided on microscope slides coated with MAb H4 left behind considerably longer CS protein trails than those on uncoated slides, and the staining of these trails was more intense. The fact that the CS protein is an exoantigen continuously released as trails by motile sporozoites, together with our previous finding that anti-CS protein antibodies inhibit sporozoite motility, strongly suggests that the CS protein plays a role in gliding motility. The sensitive IGSS technique used in this study may be a useful tool in the study of the translocation of surface proteins during gliding of other apicomplexans, other protists, and bacteria.