Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal cmyc-tagged version of the 'rhoptry' protein and expresses GFP under control of the constitutive eef1a promoter.
Protein (function)
See below more details about the different rhoptry proteins
Phenotype
See below for detailed information of absence/presence of expression in merozoites and sporozoites
Additional information
In this study the following rhoptry proteins of P. falciparum were analysed for expression in P. berghei merozoites and sporozoites. The following localisation was detected in sporozoites:
ron2, PBANKA_1315700; rhoptry
ron4, PBANKA_0932000; rhoptry
ron5, PBANKA_0713100; rhoptry
ron6, PBANKA_0311700; apical end
ralp1, PBANKA_0619700; rhoptry
asp/ron1, PBANKA_1003600; rhoptry
rap1, PBANKA_1032100; rhoptry
ron3, PBANKA_1464900; rhoptry
rama, PBANKA_0804500; rhoptry
rhoph1a, PBANKA_1400600; not detected
rhoph2, PBANKA_0830200; not detected
rhoph3, PBANKA_0416000; not detected
Transgenic parasite lines were generated by single-crossover homologous recombination to express each rhoptry protein fused with a C-terminal c-Myc tag. Ten transgenic parasite lines were successfully isolated. Specific antibodies against recombinant protein were prepared for ASP/RON1 and RAMA, since they are predicted to have C-terminal glycosylphosphatidylinositol (GPI) anchored domains and therefore the modification of their C-terminal structure might disrupt their function. Specific antibodies against the middle region of RON3 were also prepared, because it was demonstrated that a 40 kDa fragment of C-terminal RON3 is cleaved during schizont maturation in P. falciparum
Protein lysates of 1.5 × 105 schizonts and oocyst-derived sporozoites of each transgenic parasite line expressing c-Myc tagged rhoptry protein or WT-GFP were analyzed by western blotting using anti-c-Myc antibodies or specific antibodies against ASP/RON1, RAMA, and RON3. In schizonts all examined c-Myc tagged rhoptry proteins, except for RON3, were detected at the expected size of full-length, demonstrating that c-Myc fused rhoptry proteins are successfully expressed. In addition, the processed forms of RON4 and RALP1 were detected at ∼60 and 40 kDa. In the case of RON3, anti-c-Myc antibodies detected ∼40 kDa fragment as reported in P.falciparum, while anti-RON3 antibodies recognized two bands, near 250 kDa, corresponding to the full-length and processed RON3. It was confirmed that aroughly 40kDa fragment of the C-terminal region in RON3 is cleaved in P.berghei mature schizonts as well as in P. falciparum. Antibodies against ASP/RON1 and RAMA recognized corresponding proteins at the size of expected full- and processed-proteins, confirming the specificity of these antibodies. RhopH1A and RhopH3 proteins were not detected in sporozoites, while RhopH2 was detected as a far weaker band compared to that in schizonts, which is in good agreement with the transcriptional data. In addition, RON2 production in sporozoites was significantly less than in schizonts.
To determine the precise localization of rhoptry proteins, immuno-electron microscopy (IEM) was performed using schizont stage merozoites and oocyst sporozoites. In P. berghei merozoites, RON2, RON4, RON5, RALP1, and ASP/RON1, which are categorized as rhoptry neck proteins in Pf merozoites, were confirmed to localize to the rhoptry neck region. In addition, RAP1, RON3, RhopH1A, RhopH2, and RhopH3 are observed in the rhoptry bulb region, as reported for Pf merozoites. RAMA is observed on the rhoptry membrane at the bulb region. RON6 could not be detected by anti-c-Myc antibodies, possibly because its protein amount in merozoites is not sufficient to be observed by IEM.
In sporozoites formed inside oocysts, it was confirmed that three components for the RhopH complex do not accumulate in rhoptries, as expected from the observation of far less amounts of transcripts and proteins in sporozoites compared to merozoites. Other than the RhopH complex, all proteins examined are localized to rhoptries in sporozoites as well as in merozoites. However, most proteins are distributed throughout rhoptries in sporozoites, despite their sub-localization in merozoites, suggesting that sub-compartmentation in rhoptries might be different between merozoites and sporozoites. This is consistent with the observation that the sub-localization of RON11 in rhoptries differs between merozoites and sporozoites. Only ASP/RON1 tends to accumulate in the thinner part in rhoptries near the tip of sporozoites.
RON2, RON4, RON5, RALP1, RAP1, and RAMA were detected in rhoptries of sporozoites residing in salivary glands, indicating that rhoptry proteins reside in rhoptries after sporozoite invasion of salivary glands, presumably available for subsequent invasion of hepatocytes in mammalian hosts. ASP/RON1 and RON3 could not be detected by anti-ASP/RON1 or anti-c-Myc antibodies, which might due to less target or c-Myc tagged protein amounts in salivary gland sporozoites.
Other mutants
Tokunaga N, Ishino T
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