Cysteine proteases of malaria parasites

A number of cysteine proteases of malaria parasites have been described, and many more putative cysteine proteases are suggested by analysis of the Plasmodium falciparum genome sequence. Studies with protease inhibitors have suggested roles for cysteine proteases in hemoglobin hydrolysis, erythrocyte rupture, and erythrocyte invasion by erythrocytic malaria parasites. The best characterised Plasmodium cysteine proteases are the falcipains, a family of papain-family (clan CA) enzymes. Falcipain-2 and falcipain-3 are hemoglobinases that appear to hydrolyse host erythrocyte hemoglobin in the parasite food vacuole. This function was recently confirmed for falcipain-2, with the demonstration that disruption of the falcipain-2 gene led to a transient block in hemoglobin hydrolysis. A role for falcipain-1 in erythrocyte invasion was recently suggested, but disruption of the falcipain-1 gene did not alter parasite development. Other papain-family proteases predicted by the genome sequence include dipeptidyl peptidases, a calpain homolog, and serine-repeat antigens. The serine-repeat antigens have cysteine protease motifs, but in some the active site Cys is replaced by a Ser. One of these proteins, SERA-5, was recently shown to have serine protease activity. As SERA-5 and some other serine-repeat antigens localise to the parasitophorous vacuole in mature parasites, they may play a role in erythrocyte rupture. The P. falciparum genome sequence also predicts more distantly related (clan CD and CE) cysteine proteases, but biochemical characterisation of these proteins has not been done. New drugs for malaria are greatly needed, and cysteine proteases may provide useful new drug targets. Cysteine protease inhibitors have demonstrated potent antimalarial effects, and the optimisation and testing of falcipain inhibitor antimalarials is underway.     

Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte

Escape from the host erythrocyte by the invasive stage of the malaria parasite Plasmodium falciparum is a fundamental step in the pathogenesis of malaria of which little is known. Upon merozoite invasion of the host cell, the parasite becomes enclosed within a parasitophorous vacuole, the compartment in which the parasite undergoes growth followed by asexual division to produce 16-32 daughter merozoites. These daughter cells are released upon parasitophorous vacuole and erythrocyte membrane rupture. To examine the process of merozoite release, we used P. falciparum lines expressing green fluorescent protein-chimeric proteins targeted to the compartments from which merozoites must exit: the parasitophorous vacuole and the host erythrocyte cytosol. This allowed visualization of merozoite release in live parasites. Herein we provide the first evidence in live, untreated cells that merozoite release involves a primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte plasma membrane. We have confirmed, with the use of immunoelectron microscopy, that parasitophorous vacuole membrane rupture occurs before erythrocyte plasma membrane rupture in untransfected wild-type parasites. We have also demonstrated selective inhibition of each step in this two-step process of exit using different protease inhibitors, implicating the involvement of distinct proteases in each of these steps. This will facilitate the identification of the parasite and host molecules involved in merozoite release.     

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

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.     

A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression

The process of merozoite release in Plasmodium falciparum involves rupture of the parasitophorous vacuole membrane and erythrocyte plasma membrane. Through the use of protease inhibitors that halt the merozoite release, a number of parasite proteases, especially serine, aspartic, and cysteine proteases, have been implicated in the schizont rupture. To understand the precise role of cysteine proteases in the merozoite release, in the present study, we treated P. falciparum cultures with siRNAs corresponding to falcipain-1, falcipain-2, and falcipain-3, the three papain-family proteases of the parasite. Treatment of malaria parasites with either of the falcipain siRNAs considerably reduced parasite growth. Morphological examination of the siRNA treated parasite cultures revealed that most of the parasites in falcipain-2 siRNA treated cultures were arrested at schizont stage. Analysis of a transgenic P. falciparum line expressing chimeric-GFP upon treatment with falcipain-2 siRNA revealed block in the rupture of erythrocyte membrane at the time of merozoite egression. These results suggest that falcipain-2 is an important parasitic protease that participates in hemoglobin degradation and in the merozoite release.     

Blocking effect of a biotinylated protease inhibitor on the egress of Plasmodium falciparum merozoites from infected red blood cells

The malaria parasite Plasmodium falciparum invades human red blood cells. Before infecting new erythrocytes, the merozoites have to exit their host cell to get into the blood plasma. Knowledge about the mechanism of egress is scarce, but it is thought that proteases are basically involved in this step. We have introduced a biotinylated dibenzyl aziridine-2,3-dicarboxylate (bADA) as an irreversible cysteine protease inhibitor to study the mechanism of merozoite release and to identify the proteases involved. The compound acts on parasite proteins in the digestive vacuole and in the host cell cytosol, as judged by fluorescence microscopy. The inhibitor blocks rupture of the host cell membrane, leading to clustered merozoite structures, as evidenced by immunoelectron microscopy. Interestingly, bADA did not prevent rupture of the parasitophorous vacuole membrane (PVM) that surrounds the parasite during the period of intraerythrocytic maturation. The compound appears to be a valuable template for the development of inhibitors specific for individual plasmodial proteases, which would be useful tools to dissect the molecular mechanisms underlying the process of merozoite release and consequently to develop potent antimalarial drugs.     

Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum

Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.     

Malaria proteases mediate inside-out egress of gametocytes from red blood cells following parasite transmission to the mosquito

Malaria parasites reside in human erythrocytes within a parasitophorous vacuole. The parasites are transmitted from the human to the mosquito by the uptake of intraerythrocytic gametocytes during a blood meal, which in the midgut become activated by external stimuli and subsequently egress from the enveloping erythrocyte. Gametocyte egress is a crucial step for the parasite to prepare for fertilization, but the molecular mechanisms of egress are not well understood. Via electron microscopy, we show that Plasmodium falciparum gametocytes exit the erythrocyte by an inside-out type of egress. The parasitophorous vacuole membrane (PVM) ruptures at multiple sites within less than a minute following activation, a process that requires a temperature drop and parasite contact with xanthurenic acid. PVM rupture can also be triggered by the ionophore nigericin and is sensitive to the cysteine protease inhibitor E-64d. Following PVM rupture the subpellicular membrane begins to disintegrate. This membrane is specific to malaria gametocytes, and disintegration is impaired by the aspartic protease inhibitor EPNP and the cysteine/serine protease inhibitor TLCK. Approximately 15 min post activation, the erythrocyte membrane ruptures at a single breaking point, which can be inhibited by inhibitors TLCK and TPCK. In all cases inhibitor treatment results in interrupted gametogenesis.     

Plasmodium food vacuole plasmepsins are activated by falcipains

Intraerythrocytic malaria parasites use host hemoglobin as a major nutrient source. Aspartic proteases (plasmepsins) and cysteine proteases (falcipains) function in the early steps of the hemoglobin degradation pathway. There is extensive functional redundancy within and between these protease families. Plasmepsins are synthesized as integral membrane proenzymes that are activated by cleavage from the membrane. This cleavage is mediated by a maturase activity whose identity has been elusive. We have used a combination of cell biology, chemical biology, and enzymology approaches to analyze this processing event. These studies reveal that plasmepsin processing occurs primarily via the falcipains; however, if falcipain activity is blocked, autoprocessing can take place, serving as an alternate activation system. These results establish a further level of redundancy between the protease families involved in Plasmodium hemoglobin degradation.     

Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte.     

Cysteine protease inhibitors as chemotherapy for parasitic infections.

Analysis of the evolution, localization and biologic function of papain family cysteine proteases in metazoan and protozoan parasites has provided important and often surprising insights into the biochemistry and cellular function of this diverse enzyme family. Furthermore, the relative lack of redundancy of cysteine proteases in parasites compared to their mammalian hosts makes them attractive targets for the development of new antiparasitic chemotherapy. The treatment of experimental models of parasitic diseases with cysteine protease inhibitors has provided an important 'proof of concept' for the use of cysteine protease inhibitors in vivo. Evidence has now accumulated that cysteine protease inhibitors can selectively arrest replication of a microbial pathogen without untoward toxicity to the host. Furthermore, this can be achieved with reasonable dosing schedules and oral administration of the drug. Initial studies have confirmed the efficacy of cysteine protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. Work on Trypanosoma brucei, the agent of African trypanosomiasis, is preliminary but also promising. Target validation studies have shown that biotinylated or radiolabeled irreversible inhibitors specifically bind to the cysteine protease targets thought to represent the major activity within the parasite. In the case of T. cruzi, the effect of inhibitors appears to be predominantly in blocking protease processing. Transfection studies using variant constructs have supported this model. Finally, the generation of null mutants for the multiple protease genes in Leishmania mexicana has provided the first genetic support for the key role of this enzyme family in parasite virulence. Safety studies in rodents and analysis of uptake of inhibitors by parasites and host cells suggest that the selectivity of inhibitors for the parasite targets may reside in the lack of redundancy of parasite proteases, the higher concentration of host proteases in intracellular compartments, and differential uptake of inhibitors by parasites. Attempts to elicit resistance to cysteine protease inhibitors in parasite cultures suggest that mechanisms of induced resistance are independent of resistance to the traditional antiparasitic agents. This suggests that cysteine protease inhibitors may provide an alternative to traditional therapy in drug-resistant organisms.     

Stage-specific antimalarial activity of cysteine protease inhibitors

Cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are promising targets for antimalarial chemotherapy. We evaluated cultured parasites for the stage-specific expression of cysteine proteases and sensitivity to cysteine protease inhibitors. Protease activity and inhibitor sensitivity varied markedly over time. Cysteine protease activity was greatest in early trophozoites, while sensitivity to cysteine protease inhibitors was greatest in mature trophozoites. Our results indicate the importance of considering the stage-specific effects of antimalarials and are consistent with the conclusion that the principal antimalarial activity of cysteine protease inhibitors is due to a block in hemoglobin hydrolysis.     

Membrane transformation during malaria parasite release from human red blood cells

Three opposing pathways are proposed for the release of malaria parasites from infected erythrocytes: coordinated rupture of the two membranes surrounding mature parasites; fusion of erythrocyte and parasitophorus vacuolar membranes (PVM); and liberation of parasites enclosed within the vacuole from the erythrocyte followed by PVM disintegration. Rupture by cell swelling should yield erythrocyte ghosts; membrane fusion is inhibited by inner-leaflet amphiphiles of positive intrinsic curvature, which contrariwise promote membrane rupture; and without protease inhibitors, parasites would leave erythrocytes packed within the vacuole. Therefore, we visualized erythrocytes releasing P. falciparum using fluorescent microscopy of differentially labeled membranes. Release did not yield erythrocyte ghosts, positive-curvature amphiphiles did not inhibit release but promoted it, and release of packed merozoites was shown to be an artifact. Instead, two sequential morphological stages preceded a convulsive rupture of membranes and rapid radial discharge of separated merozoites, leaving segregated internal membrane fragments and plasma membrane vesicles or blebs at the sites of parasite egress. These results, together with the modulation of release by osmotic stress, suggest a pathway of parasite release that features a biochemically altered erythrocyte membrane that folds after pressure-driven rupture of membranes.     

Biophysics of malarial parasite exit from infected erythrocytes

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.     

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.     

Leishmania tropica: cysteine proteases are essential for growth and pathogenicity

Leishmania parasites are responsible for a diverse collection of diseases of humans and other animals. Cysteine proteases are putative virulence factors of leishmania parasites. There are differences in the susceptibility of specific stages in different Leishmania species to cysteine protease inhibitors. Here, we establish a key role of cysteine proteases in growth, viability, and pathogenicity of Leishmania tropica by using a specific cysteine protease inhibitor (N-Pip-F-hF-VS Phenyl). Reduction or arrest of promastigote growth occurred at inhibitor concentration of 5 and 100 microM, respectively. This shows an essential role for cysteine proteases in viability and growth of L. tropica promastigotes. It confirms that the promastigote stage of L. tropica more closely resembles that of Leishmania major than that of Leishmania mexicana, which is refractory to this inhibitor. Pathogenicity of L. tropica amastigotes in mice, as assessed by footpad swelling, was also reduced by treatment with the cysteine protease inhibitor. This suggests that cysteine proteases are essential for pathogenicity of L. tropica amastigote in mammalian host, similar to both L. major and L. mexicana.     

Plasmodium protease ROM1 is important for proper formation of the parasitophorous vacuole

Apicomplexans are obligate intracellular parasites that invade host cells by an active process leading to the formation of a non-fusogenic parasitophorous vacuole (PV) where the parasite replicates within the host cell. The rhomboid family of proteases cleaves substrates within their transmembrane domains and has been implicated in the invasion process. Although its exact function is unknown, Plasmodium ROM1 is hypothesized to play a role during invasion based on its microneme localization and its ability to cleave essential invasion adhesins. Using the rodent malaria model, Plasmodium yoelii, we carried out detailed quantitative analysis of pyrom1 deficient parasites during the Plasmodium lifecycle. Pyrom1(-) parasites are attenuated during erythrocytic and hepatic stages but progress normally through the mosquito vector with normal counts of oocyst and salivary gland sporozoites. Pyrom1 steady state mRNA levels are upregulated 20-fold in salivary gland sporozoites compared to blood stages. We show that pyrom1(-) sporozoites are capable of gliding motility and traversing host cells normally. Wildtype and pyrom1(-) sporozoites do not differ in the rate of entry into Hepa1-6 hepatocytes. Within the first twelve hours of hepatic development, however, only 50% pyrom1(-) parasites have developed into exoerythrocytic forms. Immunofluorescence microscopy using the PVM marker UIS4 and transmission electron microscopy reveal that the PV of a significant fraction of pyrom1(-) parasites are morphologically aberrant shortly after invasion. We propose a novel function for PyROM1 as a protease that promotes proper PV modification to allow parasite development and replication in a suitable environment within the mammalian host.     

Expression,Characterization, and Cellular Localization of Knowpains,Papain-Like Cysteine Proteases of the Plasmodium knowlesi Malaria Parasite

Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries against knowpains for developing broadly effective compounds active against multiple human malaria parasites.     

Enhanced detection of Plasmodium vivax liver stages by cytocentrifugation

Malaria parasites circulating in the blood or developing in the mosquito host can easily be seen and studied. We know much less about malaria parasites in the liver not only because of their location, but also because there are so few of them. Hepatic parasites are most often grown within hepatoma cells in culture, stained and visualized on slides under direct microscopy. In this report, Chitraporn Karnasuta and George Watt investigate the potential of cytocentrifugation as a tool for improving the detection of liver-stage malaria parasites.     

Cryptostatin, a chagasin-family cysteine protease inhibitor of Cryptosporidium parvum

Cysteine proteases of pathogenic protozoan parasites play pivotal roles in the life cycle of parasites, but strict regulation of their activities is also essential for maintenance of parasite physiology and interaction with hosts. In this study, we identified and characterized cryptostatin, a novel inhibitor of cysteine protease (ICP) of Cryptosporidium parvum. Cryptostatin showed low sequence identity to other chagasin-family ICPs, but 3 motifs (NPTTG, GXGG, and RPW/F motifs), which are evolutionarily conserved in chagasin-family ICPs, were found in the sequence. The overall structure of cryptostatin consisted of 8 β-strands that progressed in parallel and closely resembled the immunoglobulin fold. Recombinant cryptostatin inhibited various cysteine proteases, including papain, human cathepsin B, human cathepsin L, and cryptopain-1, with K i's in the picomolar range. Cryptostatin was active over a wide pH range and was highly stable under physiological conditions. The protein was thermostable and retained its inhibitory activity even after incubation at 95°C. Cryptostatin formed tight complexes with cysteine proteases, so the complexes remained intact in the presence of sodium dodecyl sulfate and β-mercaptoethanol, but they were disassembled by boiling. An immunogold electron microscopy analysis demonstrated diffused localization of cryptostatin within oocystes and meronts, but not within trophozoites, which suggests a possible role for cryptostatin in host cell invasion by C. parvum.     

Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC-restricted antigen processing

While interference with the class I MHC pathway by pathogen-encoded gene products, especially those of viruses, has been well documented, few examples of specific interference with the MHC class II pathway have been reported. Potential targets for such interference are the proteases that remove the invariant chain chaperone and generate antigenic peptides. Indeed, recent studies indicate that immature dendritic cells express cystatin C to modulate cysteine protease activity and the expression of class II MHC molecules [1]. Here, we show that Bm-CPI-2, a recently discovered cystatin homolog produced by the filarial nematode parasite Brugia malayi (W. F. Gregory et al., submitted), inhibits multiple cysteine protease activities found in the endosomes/lysosomes of human B lymphocyte lines. CPI-2 blocked the hydrolysis of synthetic substrates favored by two different families of lysosomal cysteine proteases and blocked the in vitro processing of the tetanus toxin antigen by purified lysosome fractions. Moreover, CPI-2 substantially inhibited the presentation of selected T cell epitopes from tetanus toxin by living antigen-presenting cells. Our studies provide the first example of a product from a eukaryotic parasite that can directly interfere with antigen presentation, which, in turn, may suggest how filarial parasites might inactivate the host immune response to a helminth invader.