Proteasome activity is required for the stage-specific transformation of a protozoan parasite.

Abstract

SummaryA prominent feature of the life cycle of intracellular parasites is the profound morphological changes they undergo during development in the vertebrate and invertebrate hosts. In eukaryotic cells, most cytoplasmic proteins are degraded in proteasornes. Here, we show that the transformation in axenic medium of trypomastigotes of Trypanosoma cruzi into amastigote-like orgamsms, and the intracellular development of the parasite from amastigotes into trypomastigotes, are prevented by lactacystin, or by a peptide aldehyde that inhibits proteasome function. Clasto-lactacystin, an inactive analogue of lactacystin, and cell-permeant peptide aldehyde inhibitors of T. cruzi cysteine proteinases have no effect. We have also identified the 20S proteasomes from T. cruzi as a target oflactacystin in vivo. Our results document the essential role of proteasomes in the stage-specific transformation of a protozoan. Infection by Trypanosoma cruzi, the causative agent of Chagas' disease, is initiated by metacyclic trypomastigotes present in the feces of triatomine bugs. The trypomastigotes invade host cells and enter the cytoplasm, where they transform into amastigotes. The amastigotes replicate and, a few days later, transform back into trypomastigotes, rupture the host cells, and invade the bloodstream (1). Thus, on two occasions during its intracellular stage, T. cruzi undergoes shape and volume changes, restructures its flagellum and kinetoplast, and synthesizes new sets of surface molecules. These striking modifications are precisely timed, take place in an orderly fashion, and must involve selective degradation of cytoplasmic proteins.In eukaryotic cells, most proteins in the cytoplasm and nucleus are degraded not in lysosomes, hut within proteasomes, after they are marked for destruction by covalent attachment of ubiquitin (Ub) l molecules (2-5). In addition 1Abbreviatzons used in this paper: BSA, bovine serum albumin; CAPS, (3-[Cyclohexylarmno]-l-Propanesulfonlc acid), Ch-L, Chymotrypsln-hke; EDTA, ethylene dl-amino tetra acetic acid; E-64, trans-epoxysuccmyl-L-leucylamldo-3-methyl-butane ethyl ester; FCS, fetal calf serum; F1TC, fluoresceln lsothiocyanate; MES, (2-[N-morphohno]-ethanesulfonlc aod); MG-132, carboxybenzoxyl-leucinyl-leucinyl-leuclnal-H; PGPH, peptidylglutamyl peptlde hydrolase; T-L, Trypsin-like; Ub, ub~qultin.to their role in nonlysosomal protein turnover, proteasomes are involved in specific cellular functions, including the following: the programmed inactivation of mitotic cychns, transcription factors, and transcriptional regulators; the elimination of mutated or damaged proteins; and antigen presentation. The function of the proteasomes is also tightly regulated, and their structure may vary to match function (6-7).The experiments described below were designed to document the participation ofproteasomes in the developmental pathways of protozoan parasites. T. cruzi has an advantage as an experimental model because its trypomastigote form can be induced to change rapidly into amastigotes in axeni...

The autophagic pathway is a key component in the lysosomal dependent entry ofTrypanosoma cruziinto the host cell

“…8 Therefore, we next asked about the by incubating TCT in acidic pH, therefore D-MEM stabilized (buffered) at pH = 5 was used as a positive control medium to induce differentiation. 38 Furthermore, D-MEM pH = 7 was used as control for normal nutritional conditions. As shown in the Figure 4B, starvation or rapamycin significantly induced the in vitro differentiation of TCT to amastigotes.…”

“…In these experiments the differentiation to amastigotes was started earlier at 1 h in starvation medium (PBS) or in the presence of rapamycin and seems to be more effective than the pH = 5 that is the typical condition described in the literature to induce this differentiation. 38 Moreover, as we have mentioned above, rapamycin is a specific Tor kinase inhibitor; the presence of this kinase in trypanosomatids was predicted in the genome data base. 49,50 Here, for the first time, we demonstrated the presence of the Tor kinase in T. cruzi at a functional level by using a compound that specifically inhibits its enzymatic action.…”

Autophagy Is Involved in Nutritional Stress Response and Differentiation in Trypanosoma cruzi

“…Transitions between the hosts as well as changes in the replicative environment are accompanied by extensive metabolic and morphological changes of the parasite. Proteasome and cruzipain were suggested to be involved in these differentiation processes (16,17); however, the molecular mechanism has not been elucidated. Because differentiation in higher organisms is known to involve autophagy, it was reasonable to hypothesize that autophagy could be involved in T. cruzi differentiation.…”

Proteasome‐dependent cyst formation and stage‐specific ubiquitin mRNA accumulation in Entamoeba invadens

“…In eukaryotes proteasomes consist of a hollow cylindrical 20S core aggregation of two subunit rings of proteolytically active b-subunits sandwiched between two rings of a-subunits, which in turn interact directly with additional components involved in recognizing which proteins are ubiquitinated and destined to be degraded [14,15]. The composition of the latter specificity-determining components, as well as the core b-subunits, can change in at least one cell type in response to stimulation by g-interferon, which ultimately triggers novel gene transcription [16].Proteasome function has recently been found to play a role in the life cycle progression of Trypanosoma cruzi parasites [17] and in the cell cycle progression of T. brucei parasites [18]. Cyst formation by E. invadens parasites has also been found to be cell cycle dependent [19].…”