A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection†

Abstract

SummaryPlasmodium sporozoites are injected into the mammalian host during mosquito blood feeding and carried by the blood stream to the liver, where they infect hepatocytes and develop into erythrocyte-invasive forms. To reach the hepatocytes, sporozoites must cross the liver sinusoidal cell layer, which separates the hepatocytes from the circulatory system. Little is known about the molecular mechanisms by which sporozoites breach this cellular barrier. Here we report that a protein with a membrane attack complex/ perforin (MACPF)-related domain is involved in this step. This molecule is specifically expressed in liverinfective sporozoites and localized in micronemes, organelles engaged in host cell invasion. Gene disruption experiments revealed that this protein is essential for the membrane-wounding activity of the sporozoite and is involved in its traversal of the sinusoidal cell layer prior to hepatocyte-infection. Disruptants failed to leave the circulation, and most of them were eliminated from the blood by liver perfusion. Our results suggest that rupture of the host plasma membrane by the pore-forming activity of this molecule is essential for cell passage of the sporozoite. This report is the first to demonstrate an important role of a MACPF-related protein in host cell invasion by a pathogenic microorganism.

A long and winding road: The Plasmodium sporozoite's journey in the mammalian host

“…In addition, op/op mice which have reduced numbers of Kupffer cells have somewhat lower parasite burdens when inoculated with sporozoites by mosquito bite [49]. However, treating mice with CL, which as stated earlier eliminates Kupffer cells and creates large gaps in the sinusoidal barrier [49], leads to enhanced liver infection with wild type sporozoites and restores the infectivity of SPECT 1, SPECT 2 and CelTOS knockout sporozoites [25,27,28] indicating that Kupffer cell passage is not obligatory for hepatocyte infection. In the normal hepatic sinusoid, however, the majority of sporozoites probably gain access to the hepatic parenchyma by crossing Kupffer cells.…”

“…Sporozoites in which one of the SPECT proteins or CelTOS was deleted using gene transfection technology show similar phenotypes: In vitro they lack the ability to traverse cells but when placed directly on hepatocytes they invade and develop normally. In vivo these mutants lack infectivity for the mammalian host when injected intravenously, a phenotype that can be reversed by pretreating mice with liposomeencapsulated dichloromethylene diphosponate (CL) which depletes the liver of Kupffer cells and leaves gaps in the sinusoidal barrier thus allowing sporozoites direct access to hepatocytes [25,27,28]. It is likely that these proteins are also required for sporozoite exit from the dermis and in the case of the SPECT 1 knockout this has been recently directly demonstrated by intravital microscopy [Amino and Menard, unpublished data].…”

“…Although we are still far from a molecular understanding of this process, four proteins involved in cell traversal recently have been identified. Three of these, SPECT 1 (sporozoite microneme protein essential for cell traversal) and SPECT 2 (also called perforin-like protein 1; PLP1) and CelTOS (cell traversal protein for ookinetes and sporozoites) were identified using EST databases [25][26][27][28] while the fourth, a phospholipase (PL) was found using an RNA subtraction screen that identified genes up-regulated in salivary gland sporozoites [29,30]. Expression of SPECT 1, SPECT 2 and PL are specific to salivary gland sporozoites whereas CelTOS is expressed by both ookinetes and salivary gland sporozoites.…”

Cellular and molecular interactions between the apicomplexan parasitesPlasmodiumandTheileriaand their host cells

“…In exiting the midgut, the ookinete traverses a number of midgut epithelial cells before reaching the basal lamina and rounding up to become an oocyst (Zieler & Dvorak, 2000). The process of cell transmigration is similar to that displayed by sporozoites in traversing hepatocytes, again requiring the action of perforin-like proteins (Ecker et al, 2007;Ishino et al, 2005;Kadota et al, 2004). In both processes, a prerequisite of parasite transmigration is secretion of the parasite protein CelTOS into the cytoplasm of the traversed cell (Kariu et al, 2006).…”

“…Activation of sporozoites is believed to occur by their contact with the intracellular environment of the hepatocyte and at least in part appears to be due to their exposure to intracellular concentrations of potassium (Kumar et al, 2007). To gain entry into a hepatocyte for traversal, sporozoites puncture the cell membrane using a perforin-like protein, spect2, or pplp1 (Ishino et al, 2005;Kadota et al, 2004). Sporozoite activation by cell traversal does not, however, appear to be essential, as genetically modified spect2-negative parasites, which were unable to traverse hepatocytes, can successfully infect hepatocytes in by , and the activation and suppression of host cell death are indicated by → and respectively.…”