Falstatin, a cysteine protease inhibitor of Plasmodium falciparum, facilitates erythrocyte invasion

Abstract

Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum cysteine proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum cysteine proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.

Figure 1. Alignment of Falstatin with Chagasin and Putative Cysteine Protease Inhibitors of Other Plasmodial Species

Sequences were aligned using the CLUSTALW program. Amino acid identities with falstatin are in blue, and amino acids of falstatin are numbered. The predicted N-terminal signal sequence is underlined.

Figure 2. Expression and Purification of Falstatin

Falstatin was expressed in E. coli and purified by Ni-NTA affinity chromatography and ion exchange (IEX) chromatography. Protein was resolved by SDS-PAGE and stained with Coomassie blue. The positions of molecular weight markers (kDa) are indicated.

Figure 3. Activity of Falstatin against Different Classes of Proteases

Equal amounts (4 μg) of proteases (FP2, falcipain-2; FP3, falcipain-3; trypsin; α-chymo, α-chymotrypsin; pepsin; renin; collagenase; MM-2, matrix-metalloprotease-2) were mixed with 350 μl of appropriate buffers containing falstatin (1.5 μg) for 15 min, FITC-casein (20 μg) was added, and hydrolysis of the substrate with and without falstatin was compared for each protease. Error bars represent the standard deviations of results from two different assays, each performed in duplicate.

Figure 4. Mechanism of Interaction of Falstatin with Falcipain-2

Activity (arbitrary fluorescent units (FU) per minute) against Z-Leu-Arg-AMC is shown for indicated mixtures of falcipain-2 (FP2) and inactivated falcipain-2 (FP2^E-64) with falstatin. Reaction components were incubated for 15 min before the addition of substrate and measurement of fluorescence over time. Error bars represent the standard deviations of results from two different assays, each performed in duplicate.

Figure 5. Stage-Specific Expression of Falstatin

Extracts from highly synchronized parasites were collected every 8 h, separated by SDS-PAGE, and evaluated by immunoblotting with anti-falstatin antibodies. Each sample of early-ring, late-ring, early-trophozoite, late-trophozite, early-schizont, or late-schizont extracts corresponded to 1.3 × 10⁷ parasitized cells. The positions of molecular weight markers (kDa) are indicated. ER, early-ring; LR, late-ring; ET, early-trophozite; LT, late-trophozite; ES, early-schizont; LS, late-schizont.

Figure 6. Immunolocalization of Falstatin

(A) Immunofluorescence microscopy. Erythrocytes infected with synchronized 3D7 or W2 parasites were collected every 8 h, stained with DAPI and anti-falstatin antibodies and FITC-second antibody, and then evaluated by immunofluorescence microscopy. (B) Immunoelectron microscopy. Late-schizont stage parasites were incubated with anti-falstatin antibodies and gold-conjugated second antibody and then evaluated by electron microscopy. Labels show individual merozoites (M) and erythrocyte cytosol (EC).

Figure 7. Release of Falstatin with Schizont Lysis

Synchronized late-schizont-infected erythrocytes were cultured in Albumax-free medium. At the indicated time points culture media were collected and concentrated, falstatin was immunoprecipitated with anti-falstatin antiserum and anti-rat IgG beads, the beads were washed, bound proteins were solubilized in sample buffer, and falstatin was resolved by SDS-PAGE and identified by immunoblotting. Controls with pre-immune serum did not immunoprecipitate any detectable proteins. The positions of molecular weight markers are indicated (kDa). Schizont and ring parasitemias at the indicated time points are shown below the gel.

Figure 8. Inhibitor Competition

The indicated amounts of falstatin and anti-falstatin antibody were incubated with lysates from asynchronous parasite cultures before addition of [¹²⁵I] DCG04, electrophoresis, and analysis by autoradiography. Results with increasing concentrations of falstatin (A), increasing concentrations of antibody (B), and increasing falstatin in the presence of antibody (C) are shown. Labels above the gels represent concentrations of falstatin and antibody (μg/ml). Proteins are labeled based on known migration patterns that were previously confirmed by mass spectrometry. FP, falcipain; DPAP1, dipeptidyl aminopeptidase1[28].

Figure 9. Inhibition of Falstatin Function by Anti-Falstatin Antibodies

Hydrolysis of the peptide substrate Z-Leu-Arg-AMC by falcipain-2 (FP2; 19.8 nM), falcipain-3 (FP3; 27.1 nM), or trophozoite extract (TE; corresponding to 5.5 × 10⁶ parasites per reaction) was evaluated in the absence or presence of falstatin (31 nM) and the indicated quantities of anti-falstatin antibodies in 350 μl of 100 mM sodium acetate, 8 mM DTT (pH 6.0). Reaction components were incubated for 15 min before addition of substrate, and activity was measured as arbitrary fluorescence units over time (FU/min). Error bars represent the standard deviations of results from two different assays, each performed in duplicate.

Figure 10. Effect of Anti-Falstatin Antibodies on Cultured Parasites

Schizont-infected erythrocytes were combined with fresh erythrocytes in culture medium with 0.5% DMSO or PBS, pre-immune serum, or the indicated concentration of E-64d (in DMSO) or antibody (in PBS). Smears were then made and stained with Giemsa, and percentages of schizonts after 12 h (A), rings after 20 h (B), and total parasites (C) were counted. In a separate experiment (D), purified schizonts were incubated for 20 h with PBS, control pre-immune serum (50 μg/ml), rat antiserum against P. falciparum farnesyl pyrophosphate synthetase (control Ab; 50 μg/ml), or the indicated concentrations of antibodies (Ab; μg/ml) in PBS with or without preincubation for 10 min with 2 μg falstatin (F). Errors bars indicate standard deviations from means of two different assays, each done in triplicate. PI, pre-immune serum.