Liver stage | LISP2::mCherry expression in liver stages.
Fluorescent signals were not observed at 12 hpi. At 24 hpi, weak mCherry signals were observed in a small proportion of parasites in the marginal space surrounding the GFP signals, i.e. were localized in PVM (parasitophorous vacuole membrane) and seemed confined in secretory vesicles. At 36 hpi, signals were observed in the periphery of the PVM of all LS parasites, and delineating particles inside the parasite cytoplasm. At 48 hpi, the distribution of fluorescent signal in schizonts was similar to that at 36 hpi, but a proportion of the parasites had already developed into the cytomere stage, in which mature schizonts enter the process of lobulation in preparation for merogony. At 53 hpi, when most parasites were in the cytomere stage, red fluorescent particles inside PV had decreased significantly. Fluorescent signals were distributed evenly in the vacuolar space around the lobular structure of the cytomere stage. These results suggested that, following synthesis in the endoplasmic reticulum (ER), LISP2 was carried by secretory vesicles through the cytoplasm of parasites to the vacuolar space, where it was released (see also 'Additional information). |
Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal tagged version of LISP2 and GFP under the control of the constitutive promoter of the elongation factor 1 alpha (eef1a) gene of P. berghei.
Protein (function)
PBANKA_100300 encodes a 2172 amino acid protein with a large repeat region of approximately 1000 amino acids. This region is composed of several repeat motifs, particularly 12 copies of a 56-amino acid motif. Analyses of the amino acid sequence with various structure analysis tools indicates that it has an N-terminal signal sequence but no other motifs for membrane association such as transmembrane domains or a GPI-anchor motif, suggesting that the encoded product has the structure of a secreted protein. It contains a predicted Plasmodium 6-cysteine motif. Several studies analysing expression of this protein in P. berghei (RMgm-593, RMgm-799) provide evidence for specific expression in liver stages. The protein has been named LISP2 and sequestrin.
Analyses of a mutant lacking expression of LISP (RMgm-799) indicate that the protein is involved in the development of maturing liver stages. Evidence is presented that the absence of LISP2 impairs merozoite formation in liver schizonts.
Phenotype
Analyses of a mutant lacking expression of LISP (RMgm-799) indicate that the protein is involved in the development of maturing liver stages. Evidence is presented that the absence of LISP2 impairs merozoite formation in liver schizonts.
mCherry:LISP2 is expressed in maturing liver stages. Evidence is presented for export to the parasitophorous vacuole (membrane). Additional analyses using anti-LISP2 antibodies provide evidence for for export of LISP2 into to the cytoplasm and nucleus of host hepatocytes.
Additional information
To investigate whether both proteins are exported through the same pathway, LISP2::mCherry parasites were double stained for mCherry and LISP1. At 42 hpi, secretory vesicle-like particles were detected inside the parasites with anti-LISP1 antibodies. Vesicles stained with anti-mCherry antibodies overlapped with anti-LISP1 antibodies, indicating that these two proteins were exported by the same vesicles. Magnifying the view clearly showed that LISP2 is surrounded by LISP1, suggesting that the membrane, with which LISP1 was associated, bound the vesicles. The membrane stained with anti-LISP1 antibodies also surrounded vesicles on the PVM. At 53 hpi, the space formed between the meroblasts and PVM was filled with LISP2. Vesicles were mainly located on the PVM and surrounded by the membrane stained with anti-LISP1 antibodies, as at 42 hpi. At this stage, the PVM was stained more clearly with anti-LISP1 antibodies than that at 42 hpi, suggesting that LISP1 accumulated in the PVM.
Immunofluorescence analysis of LISP expression using anti-LISP2 antibodies at 48 hpi showed strong signals in the infected HepG2 cells and LS parasites, which was quite different from the observation using the LISP2::mCherry parasites. The complex shape of the host cells was clearly marked by antibody in infected, but not uninfected HepG2 cells, indicating that LISP2 was exported to the host cell and distributed throughout the cytoplasm. Importantly, LISP2 was also localized in the nucleus of all infected HepG2 cells, and the signals in the nucleus were stronger than those in the cytoplasm. This distribution suggested that LISP2 was actively transported from the cytoplasm to the nucleus of the host cell. At 55 hpi, while most parasites had developed into the cytomere stage, signals were observed in the host cell and in PV space surrounding the cytomere, as observed in LISP2::mCherry parasites, and secretory vesicles were scarcely observed in parasites. Therefore LISP2 appears to be produced in the schizont stage, immediately carried to the host cell through the PVM, distributed in the cytoplasm and transported to the nucleus.
Evidence is presented in this study for processing of LISP2 before export into the host hepatocyte which may explain the different locations of mCherry::LISP2 (as shown by fluorescence microscopy) and LISP2 (as show by immuno-fluorescence microscopy using LISP2-antibodies)
Other mutants
RMgm-593 - A mutant expressing GFP under the control of the promoter of PBANKA_100300 (named lisp2 or sequestrin)
RMgm-799 - A mutant lacking expression of PBANKA_100300 (named lisp2 or sequestrin) |