Long-term live imaging reveals cytosolic immune responses of host hepatocytes againstPlasmodiuminfection and parasite escape mechanisms

Abstract

Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition.

IFN-γ protects hepatocytes againstPlasmodium vivaxinfection via LAP-like degradation of sporozoites

“…In human hepatocytes stimulated with IFN-γ, Boonhok et al (1) demonstrate that a LAP-like process occurs when LC3 is deposited onto the PVM of sporozoites, followed by lysosomal fusion and degradation. This differs from a previously described autophagic pathway, with similarities to selective autophagy, described for P. berghei and P. yoelii sporozoites (5,6). In this pathway, ubiquitin and p62 localize with the PVM in unstimulated hepatocytes, whereas PI3K and Beclin 1 do not.…”

“…On the other hand, autophagy can also be hijacked to deliver nutrients to support the growth of invading pathogens. In malaria, both autophagic clearance and autophagy-mediated growth have recently been shown to play an important role in the development of rodent Plasmodium sporozoites in the liver (5,6). In the basal state, both Plasmodium berghei and Plasmodium yoelii parasites hijack the hepatocyte cell autophagy pathway to gain nutrients to support their rapid growth (5,6).…”

LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malariaPlasmodium vivaxliver stage

“…Note that in this study, we investigated an autophagy/autophagyrelated process induced by IFN-γ, which is different from the recently described basal autophagy process that was shown to be hijacked by mouse malarial parasites to facilitate their development in murine models (31,32). This is similar to the effect seen with T. gondii, in which basal autophagy is subverted by the parasite for its own growth (33), whereas the parasite can be eliminated by induced autophagy/autophagy-related process in response to IFN-γ (12,(34)(35)(36)(37).…”

Plasmodium berghei EXP-1 interacts with host Apolipoprotein H during Plasmodium liver-stage development

“…A total of 50,000 and 25,000 Huh7 cells grown on coverslips or eight-well Lab-Tek chamber slides (Nunc), respectively, were seeded 1 d before infection with 10,000 Pb sporozoites and fixed at the indicated time points (10,15,18,21,24, and 48 hpi) after sporozoite addition. Cells grown on coverslips were fixed with 4% (vol/vol) paraformaldehyde for 15 min, washed three times with PBS, permeabilized in 0.2% (vol/vol) saponin or Triton in PBS for 20 min, blocked for 1 h with 0.1% (vol/vol) Triton in 1% (wt/vol) BSA in PBS, and incubated with the appropriate primary antibodies in blocking buffer for 1 h at room temperature or overnight at 4°C.…”

“…However, little is known about intrahepatic Plasmodium stages with regard to interactions between the parasite and the host hepatocyte and their potential for nutrient uptake and/or exchange. Small molecules (up to 800 Da) can cross the PVM freely via specialized transport channels (14), whereas larger molecules might reach the parasite via association and possibly fusion of late endosomes, lysosomes, or amphisomes with the PVM (15)(16)(17)(18)(19).…”