LISP1 is important for the egress of Plasmodium berghei parasites from liver cells

Abstract

Most Apicomplexa are obligatory intracellular parasites that multiply inside a so-called parasitophorous vacuole (PV) formed upon parasite entry into the host cell. Plasmodium, the agent of malaria and the Apicomplexa most deadly to humans, multiplies in both hepatocytes and erythrocytes in the mammalian host. Although much has been learned on how Apicomplexa parasites invade host cells inside a PV, little is known of how they rupture the PV membrane and egress host cells. Here, we characterize a Plasmodium protein, called LISP1 (liver-specific protein 1), which is specifically involved in parasite egress from hepatocytes. LISP1 is expressed late during parasite development inside hepatocytes and locates at the PV membrane. Intracellular parasites deficient in LISP1 develop into hepatic merozoites, which display normal infectivity to erythrocytes. However, LISP1-deficient liver-stage parasites do not rupture the membrane of the PV and remain trapped inside hepatocytes. LISP1 is the first Plasmodium protein shown by gene targeting to be involved in the lysis of the PV membrane.

Fig. 1

Pblisp1 is specifically expressed in late liver stages and localizes to the PVM. A. Histogram representation of real-time RT-PCR analysis of lisp1 relative gene expression in P. berghei sporozoites (SPZ), HepG2 cells 5, 17, 40 and 50 h post infection and mixed blood stages (BS). The value was normalized to the expression of hsp70 mRNA in each sample. Error bars are standard deviation. B. Immunofluorescence analysis of frozen sections of rat liver 48 h post infection (LS) and purified BS schizonts. Samples were incubated with an anti-LISP1 antibody followed by FITC-conjugated secondary antibody and nuclei were stained with DAPI (scale bar 5 μm). C. Micrograph of confocal section of LS. HepG2 cells were fixed at 24, 36, 48 and 65 h post infection with WT ANKA sporozoites, incubated with an anti-LISP1 antibody followed by Alexa 488-conjugated secondary antibody and nuclei stained with DAPI (scale bars 20 μm). D. Micrograph of confocal section of LS. HepG2 cells were fixed 48 h post infection with WT ANKA sporozoites, incubated with anti-LISP1 and anti-EXP1 antibodies followed by Alexa 488-conjugated secondary antibodies and nuclei stained with DAPI (scale bar 20 μm).

Fig. 2

Targeted gene disruption of lisp1. A. Schematic representation of Lisp1I gene disruption. B. Schema of Lisp1Δ gene disruption. Shaded boxes indicate the regions of homology used for double-cross-over recombination; black boxes indicate the selectable marker; Met and the asterisk (*) indicate the initiation and stop codons respectively. C. Absence of LISP1 protein in Lisp1Δ parasites. Microscopy image of HepG2 cells 48 h post infection with WTGreen or Lisp1ΔGreen. LS were labelled with anti-LISP1 antibody and Alexa 647-conjugated secondary antibody (red); nuclei stained with DAPI (blue). D. Western blot analysis of extracts of HepG2 cells 48 h post infection with ANKA (WT) or Lisp1ΔGreen sporozoites. Arrowheads indicate the bands of LISP1 and human GAPDH. E. Lisp1I sporozoites have a decreased infectivity in the mammalian host. A total of 30 000 wild-type or Lisp1I sporozoites, from two independent Lisp1I clones, were injected intravenously into rats and blood parasitaemias were examined by Geimsa staining. The error bars show the standard errors from three rats.

Fig. 3

Lisp1-defective parasites develop normally, within a PVM, into late-stage LS. A. Histogram representation of the number of LS in vivo. A total of 300 000 wild-type or Lisp1I sporozoites were injected intravenously into rats and 48 h later the number of WT (white box) and Lisp1I (black box) LS were counted in the liver sections. Bars indicate the standard deviation from three rats. B. Confocal micrograph of PVM in Lisp1-defective parasites. HepG2 cells 36 and 48 h post infection with WT (Wild type) or Lisp1ΔGreen sporozoites. LS parasites (green) labelled with an anti-UIS4 antibody (red) and nuclei stained with DAPI (blue).

Fig. 4

Lisp1 mutant LS parasites are defective in the release of merozoites. Graph showing the ratio of Green : Red hepatic merozoites (hMZ) collected at 65 h from adherent HepG2 cells (left) or the supernatant (right) co-infected with Lisp1ΔGreen and WTRed or WTGreen and WTRed sporozoites and the ratios found in blood stages (iRBC) after injection of these merozoites into mice. The results obtained from the supernatant and adherent cells are indicated. The average and standard deviation from four mice are shown.

Fig. 5

Lisp1 mutant merozoites remain within a PVM. A. Histogram of the percentage of 58 h WTGreen and Lisp1ΔGreen unsegmented LS with no merozoites (US, lilac), and segmented LS containing merozoites (S) with (yellow) or without (orange) a visible PVM. B–F. Representative examples of hepatic schizonts 58 h (B–D) and 61 h (E and F) post infection. (B) WTGreen with PVM; (C) WTGreen without PVM; (D) Lisp1Δ LS with PVM; (E and F) Lisp1Δ LS at 61 h containing many merozoites within a membrane boundary; scale bars are 2 μm. Insets show mature merozoites; scale bar is 500 nm and arrow heads indicate membrane.

Fig. 6

Localization of SERA1 is modified in Lisp1 mutant LS parasites. HepG2 cells were infected with wild type (WT, top) or Lisp1ΔGreen sporozoites (Lisp1Δ, bottom) and 48 h later the LS were stained an anti-SERA1 antibody (red) and nuclei stained with DAPI (blue).