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. 2004 Mar 30;101(13):4384-9.
doi: 10.1073/pnas.0307720101. Epub 2004 Mar 15.

Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum

Puran S Sijwali  1 , Philip J Rosenthal
Affiliations

Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum (VSports app下载)

Puran S Sijwali et al. Proc Natl Acad Sci U S A. .

Abstract (V体育2025版)

Erythrocytic malaria parasites degrade hemoglobin in an acidic food vacuole to acquire free amino acids and maintain parasite homeostasis. Hemoglobin hydrolysis appears to be a cooperative process requiring cysteine proteases (falcipains) and aspartic proteases (plasmepsins), but the specific roles of different enzymes in this process are unknown. We previously showed that falcipain-2 is a major trophozoite food vacuole cysteine protease. To characterize the specific role of falcipain-2, we disrupted the falcipain-2 gene and assessed the effect of this alteration. Falcipain-2-knockout trophozoites had markedly diminished cysteine protease activity and swollen, dark staining food vacuoles, consistent with a block in hemoglobin hydrolysis, as caused by cysteine protease inhibitors VSports手机版. However, more mature stages of knockout parasites were indistinguishable from wild-type parasites and developed normally. The knockout parasites had decreased and delayed expression of falcipain-2, which appeared to be directed by increased transcription of a second copy of the gene (falcipain-2'). Expression of other falcipains and plasmepsins was similar in wild-type and knockout parasites. Compared with wild-type, knockout parasites were about 3 times more sensitive to the cysteine protease inhibitors E-64 and leupeptin, and over 50-fold more sensitive to the aspartic protease inhibitor pepstatin. Our results assign a specific function for falcipain-2, the hydrolysis of hemoglobin in trophozoites. In addition, they highlight the cooperative action of cysteine and aspartic proteases in hemoglobin degradation by malaria parasites. .

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"V体育官网" Figures

Fig. 1.
Fig. 1.
Schematic of falcipain-2 gene disruption. The WT falcipain-2 locus, transfection plasmid (pDC-PFP2/GFPA), and integration locus after a single crossover are shown. Thick lines represent regulatory regions, thin dotted lines indicate the plasmid backbone, and thick dotted lines represent the chromosomal DNA. Bent arrows indicate orientations, horizontal arrows represent the locations of primers, and vertical arrows indicate the locations of restriction endonuclease sites. The sizes of restriction fragments are shown in kilobases in parentheses.
Fig. 2.
Fig. 2.
Verification of falcipain-2 gene disruption. (A) Ethidium bromide-stained agarose gel showing PCR products from WT (lanes 2 and 5) and FP2KO (lanes 3 and 6) gDNAs and pDC-PFP2/GFPA plasmid DNA (lanes 4 and 7) when primers specific for WT falcipain-2 (1F/2R) and the integration locus (1F/3R) are used. The sizes of DNA markers (lane 1) are indicated (kb). (B) Southern blot of PacI (P) and PacI–FokI (P-F) gDNA digests of WT and FP2KO parasites probed with the falcipain-2 proregion. Fragments of 11.5 kb (P) and 1.6 and 3.9 kb (P-F) for FP2KO gDNA and of 4 kb (P) and 1.9 kb (P-F) for WT gDNA indicate integration of a single copy of pDC-PFP2/GFPA into the proregion of falcipain-2 by single-crossover homologous recombination. The weaker signals of 15 kb (P) and 4 kb (P-F) correspond to falcipain-2′.(C) Immunoblots of early (24–28 h) trophozoite lysates (TL) and vacuolar fractions (V) of WT and FP2KO parasites (2 × 107 per lane) probed with antibodies to falcipain-2 (FP2) and falcipain-3 (FP3). The positions of molecular mass standards are indicated (kDa).
Fig. 3.
Fig. 3.
Effect of falcipain-2 gene disruption on parasite development. Synchronized WT, FP2KO, and E-64-treated (10 μM, beginning at 0 h) (WT-E64) parasites were collected at different stages, stained with Giemsa stain, and evaluated by light microscopy (A). Extracts were assessed for the hydrolysis of Z-Leu-Arg-AMC [4.8 × 106 parasites per reaction, measured as fluorescent intensity units (FIU)/min, B] and native hemoglobin (1.2 × 107 parasites and 2 μg of hemoglobin per reaction for2hat37°C) visualized by Coomassie staining after SDS/PAGE (C), each in 100 mM sodium acetate, pH 5.5, with 10 mM DTT (B) or 2.5 mM reduced glutathione (C). Error bars represent the standard deviations of means from three replicate assays.
Fig. 4.
Fig. 4.
Evaluation of trophozoite hemoglobin hydrolysis. Early (24-h) trophozoites of FP2KO, WT, and E-64-treated (10 μM, beginning at 0 h) (WT-E64) parasites were evaluated by Giemsa staining (A) and electron microscopy (B). Erythrocytes infected with equal numbers of these trophozoites were lysed with saponin, washed to remove erythrocyte hemoglobin, and solubilized in reducing sample buffer, and proteins were resolved by SDS/PAGE and stained with Coomassie blue (C). Hb represents a hemoglobin control.
Fig. 5.
Fig. 5.
Expression of proteases in FP2KO parasites. Synchronized FP2KO and WT parasites were collected at the indicated time points, and immunoblots (2 × 107 parasites per lane) were probed with monospecific antibodies to falcipain-1 (FP1), falcipain-2 (FP2), falcipain-3 (FP3), plasmepsin I (PM I), plasmepsin II (PM II), histoaspartic protease (HAP), and plasmepsin IV (PM IV).
Fig. 6.
Fig. 6.
Quantitation of falcipain transcript levels. cDNA from synchronized FP2KO and WT parasites collected at the indicated time points was evaluated for transcript levels of an 18S rRNA control, falcipain-2 (FP2), falcipain-2′ (FP2′), falcipain-3 (FP3), and falcipain-1 (FP1) by real-time PCR, and normalized genome equivalents (transformed by 104) were determined. Error bars represent standard deviations of means from three replicate experiments.
Fig. 7.
Fig. 7.
PCR analysis of transcription loci. The ethidium bromide-stained agarose gel shows amplification from gDNA and pooled multistage cDNA of WT and FP2KO parasites by using primers specific for full-length WT falcipain-2 (WFP2), recombined falcipain-2 (RFP2), falcipain-3 (FP3), and falcipain-2′ (FP2′). The positions of DNA size markers (M) are indicated (kb).
Fig. 8.
Fig. 8.
Multiplication rates and inhibitor sensitivity. (A) WT and FP2KO parasites were cultured in complete medium for three life cycles, and parasitemias were assessed at the end of each cycle. (B) Parasites were incubated with various concentrations of chloroquine (Cq), artemisinin (Art), E-64, leupeptin (Leup), and pepstatin (Pep) for one full life cycle, beginning at the ring stage, parasitemias were then determined, and IC50 values were calculated. Error bars represent the standard deviations of means from three (two for Cq and Art) independent experiments, each performed in duplicate. IC50 values for WT and FP2KO parasites are shown (μM, except nM for Cq and Art).
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References (VSports在线直播)

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