No abstract

Plasmodium sporozoite invasion of liver cells has been an extremely elusive event to study. In the prevailing model, sporozoites enter the liver by passing through Kupffer cells, but this model was based solely on incidental observations in fixed specimens and on biochemical and physiological data. To obtain direct information on the dynamics of sporozoite infection of the liver, we infected live mice with red or green fluorescent Plasmodium berghei sporozoites and monitored their behavior using intravital microscopy. Digital recordings show that sporozoites entering a liver lobule abruptly adhere to the sinusoidal cell layer, suggesting a high-affinity interaction. They glide along the sinusoid, with or against the bloodstream, to a Kupffer cell, and, by slowly pushing through a constriction, traverse across the space of Disse. Once inside the liver parenchyma, sporozoites move rapidly for many minutes, traversing several hepatocytes, until ultimately settling within a final one. Migration damage to hepatocytes was confirmed in liver sections, revealing clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes, and by elevated serum alanine aminotransferase activity. In summary, malaria sporozoites bind tightly to the sinusoidal cell layer, cross Kupffer cells, and leave behind a trail of dead hepatocytes when migrating to their final destination in the liver.

SummaryRhomboid-like proteases cleave membrane-anchored proteins within their transmembrane domains. In apicomplexan parasites substrates include molecules that function in parasite motility and host cell invasion. While two Plasmodium rhomboids, ROM1 and ROM4, have been examined, the roles of the remaining six rhomboids during the malaria parasite's life cycle are unknown. We present systematic gene deletion analyses of all eight Plasmodium rhomboid-like proteins as a means to discover stage-specific phenotypes and potential functions in the rodent malaria model, P. berghei. Four rhomboids (ROM4, 6, 7 and 8) are refractory to gene deletion, suggesting an essential role during asexual blood stage development. In contrast ROM1, 3, 9 and 10 were dispensable for blood stage development and exhibited no, subtle or severe defects in mosquito or liver development. Parasites lacking ROM9 and ROM10 showed no major phenotypic defects. Parasites lacking ROM1 presented a delay in blood stage patency following liver infection, but in contrast to a previous study blood stage parasites had similar growth and virulence characteristics as wild type parasites. Parasites lacking ROM3 in mosquitoes readily established oocysts but failed to produce sporozoites. ROM3 is the first apicomplexan rhomboid identified to play a vital role in sporogony.

Accumulation of infectious Plasmodium sporozoites in Anopheles spp. salivary glands marks the final step of the complex development of the malaria parasite in the insect vector. Sporozoites are formed inside midgutassociated oocysts and actively egress into the mosquito hemocoel. Traversal of the salivary gland acinar cells correlates with the sporozoite's capacity to perform continuous gliding motility. Here, we characterized the cellular role of the Plasmodium berghei sporozoite invasion-associated protein 1 (SIAP-1). Intriguingly, SIAP-1 orthologs are found exclusively in apicomplexan hemoprotozoa, parasites that are transmitted by arthropod vectors, e.g., Plasmodium, Babesia, and Theileria species. By fluorescent tagging with mCherry, we show that SIAP-1 is expressed in oocyst-derived and salivary gland-associated sporozoites, where it accumulates at the apical tip. Targeted disruption of SIAP-1 does not affect sporozoite formation but causes a partial defect in sporozoite egress from oocysts and abolishes sporozoite colonization of mosquito salivary glands. Parasites with the siap-1(؊) mutation are blocked in their capacity to perform continuous gliding motility. We propose that arthropod-transmitted apicomplexan parasites specifically express secretory factors, such as SIAP-1, that mediate efficient oocyst exit and migration to the salivary glands.Protozoan parasites of the phylum Apicomplexa have adopted an obligate intracellular life-style in a wide range of animal hosts. Apicomplexan parasites share many characteristics, including a set of apical specialized secretory organelles, active substrate-dependent locomotion, and compartmentalization of biosynthetic pathways in apicoplasts. Transmission to the vertebrate hosts occurs via tailor-made motile parasite stages, termed sporozoites, which are formed inside oocysts, the only extracellular replication phase during the complex apicomplexan life cycles. However, apicomplexan parasites differ fundamentally in their transmission modes. Coccidian parasites, such as Toxoplasma gondii, the causative agent of toxoplasmosis, and cryptosporidia, which can cause life-threatening diarrhea, form oocysts that are taken up orally via contaminated food or water, respectively. After accidental ingestion, sporozoites are liberated from the oocysts and breach or directly invade the intestinal endothelium to commence an infection. In marked contrast, sporozoites of apicomplexan hemoprotozoa are inoculated intradermally by arthropod vectors, e.g., Anopheles mosquitoes or ticks. Sporozoites then actively leave the inoculation site and enter the blood circulation (3). In the case of Plasmodium species, the causative agents of malaria, sporozoites are formed in midgut-associated oocysts (16), actively exit the oocyst (1, 23), and penetrate the distal portion of the lateral salivary gland lobes where they eventually accumulate, rendering the infected mosquito infectious to the vertebrate host (4).Colonization of the salivary glands by Plasmodium parasites is driven by a number ...