A Parasite Cysteine Protease Is Key to Host Protein Degradation and Iron Acquisition

Abstract

Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.Proteases are ubiquitous enzymes that function in virtually all biological phenomena. Two of the major groups of proteases, Clan CA (papain-like) cysteine proteases and Clan SA (trypsin-like) serine proteases, underwent an evolutionary inversion whereby the more abundant cysteine proteases of primitive eukaryotes gave way to serine proteases with the evolution of arthropods (1) (merops.sanger.ac.uk/). Therefore, an analysis of the role of cysteine proteases in protozoa can provide insights into differential use and molecular evolution of this protease class.Trypanosoma brucei is a protozoan parasite and the causative agent of human African trypanosomiasis, a fatal disease that is transmitted by the bite of the tsetse fly. Only four drugs are available to treat human African trypanosomiasis: two for the first stage of the disease when parasites proliferate in the blood (pentamidine and suramin) and two for the second stage when parasites have established infection in the cerebrospinal fluid (melarsoprol and eflornithine). These drugs cause serious side effects and are expensive to manufacture and administer (2). There is an obvious and urgent need to develop new chemotherapies to treat human African trypanosomiasis.Two Clan CA cysteine proteases have been identified in T. brucei: rhodesain, which is cathepsin L-like and most abundant (3), and tbcatB, a cathepsin B-like enzyme (4). Treatment of parasites in culture with the nonspecific cysteine protease inhibitor, benzyloxycarbonyl-phenylalanyl-alanyl diazomethane (Z-Phe-Ala-CHN 2 ) 2 is lethal to cultured parasites at 10 M (5). Parasites treated with this inhibitor exhibit altered cell morphology, are unable to undergo cytokinesis, ...

Iron metabolism in trypanosomatids, and its crucial role in infection

“…Unlike the mammalian transferrin receptor, which remains tightly bound to its cargo throughout, ESAG6/7 loses affinity for apotransferrin at low pH. The apotransferrin is then degraded by the cathepsin B-like protein, TbcatB in the lysosome (Maier and Steverding, 1996 ;O'Brien et al 2008), while the receptor is recycled back to the flagellar pocket via TbRab11 positive vesicles (Steverding et al 1995 ;Jeffries et al 2001). The degraded transferrin fragments are recycled to the surface and exocytosed, also by a Rab11-dependent mechanism (Pal et al 2003 ;Hall et al 2005).…”

The Oligopeptidase B of Leishmania Regulates Parasite Enolase and Immune Evasion

“…Residual activity is the percentage of OPB activity remaining as compared with preincubation with solvent-only controls. (20,33). Complete libraries for each of the P1-P4 positions were used, containing 160,000 tetrapeptide substrates (Fig.…”

Non-canonical Maturation of Two Papain-family Proteases in Toxoplasma gondii

“…A fourth cathepsin, falcipain 1, resides in intracellular vesicles and is dispensable for the intraerythrocytic stages (11) but plays an important yet undefined role in parasite development mosquitoes (12). African trypanosome cathepsin B is important for parasite survival probably due to its role in degrading endocytosed host transferrin for iron acquisition (13).…”