http://repositorio.unicamp.br/jspui/bitstream/REPOSIP/96385/1/2-s2.0-0027941410.pdf

Cytosolic-free Calcium Elevation In Trypanosoma Cruzi Is Required For Cell Invasion

An anion exchange column of DE-52 has been used to purify Eimeria sporozoites from a post- excystation mixture of oocysts, oocyst shells, sporocysts, sporocyst shells, and sporozoites. The mean recovery from several experiments was 94% and virtually all non-sporozoite material was removed. Infectivity studies in vitro with sporozoites showed that they were viable after purification and were at least as infectious as the unpurified sporozoites; furthermore, oocysts in the crude preparation could be recovered from the DE-52 cellulose by resuspending them in a 20% (w/v) sodium chloride solution.

Purification of Eimeria Sporozoites by DE-52 Anion Exchange Chromatography

A 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 proteasomes. Here, we show that the transformation in axenic medium of trypomastigotes of Trypanosoma cruzi into amastigote-like organisms, 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 of lactacystin in vivo. Our results document the essential role of proteasomes in the stage-specific transformation of a protozoan.

https://rupress.org/jem/article-pdf/184/5/1909/1396318/1909.pdf

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

Summary A 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.

Proteasome Activity Is Required for the Stage-specific Transformation of a Protozoan Parasite By Jorge Gonzfilez,* E Juarez Ramalho-Pinto,~ Ute Frevertfi

The survival of a eukaryotic protozoan as an obligate parasite in the interior of a eukaryotic host cell implies its adaptation to an environment with a very different ionic composition from that of its extracellular habitat. This is particularly important in the case of Ca2+, the intracellular concentration of which is 3 orders of magnitude lower than the extracellular value. Ca2+ entry across the plasma membrane is a widely recognized mechanism for Ca2+ signaling, needed for a number of intracellular processes, and obviously, it would be restricted in the case of intracellular parasites. Here we show that Trypanosoma cruzi amastigotes possess a higher Ca2+ content than the extracellular stages of the parasite. This correlates with the higher expression of a calcium pump, the gene for which was cloned and sequenced. The deduced protein product (Tca1) of this gene has a calculated molecular mass of 121,141 Da and exhibits 34 to 38% identity with vacuolar Ca2+-ATPases of Saccharomyces cerevisiae and Dictyostelium discoideum, respectively. The tca1 gene suppresses the Ca2+ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca2+ accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that Tca1 colocalizes with the vacuolar H+-ATPase to the plasma membrane and to intracellular vacuoles of T. cruzi. These vacuoles were shown to have the same size and distribution as the calcium-containing vacuoles identified by the potassium pyroantimoniate-osmium technique and as the electron-dense vacuoles observed in whole unfixed parasites by transmission electron microscopy and identified in a previous work (D. A. Scott, R. Docampo, J. A. Dvorak, S. Shi, and R. D. Leapman, J. Biol. Chem. 272:28020-28029, 1997) as being acidic and possessing a high calcium content (i.e., acidocalcisomes). Together, these results suggest that acidocalcisomes are distinct from other previously recognized organelles present in these parasites and underscore the ability of intracellular parasites to adapt to the hostile environment of their hosts.

Ca2+ content and expression of an acidocalcisomal calcium pump are elevated in intracellular forms of Trypanosoma cruzi.

Since the in vitro production of trypomastigote stages of Trypanosoma cruzi in cell culture is frequently limited by (1) host cell overgrowth and (2) by the unequal redistribution of parasites after cell division resulting in asynchronous release of trypomastigotes, a culture system was devised in which host cell mitosis was inhibited by irradiation prior to parasite infection. L-6 rat myoblast cells when exposed to 3000 rad. of gamma radiation lost their ability to divide but remained susceptible to infection with, and capable of supporting the intracellular growth of, T. cruzi. Using this approach it proved possible to have virtually 100% of cells infected and achieve much better synchronization of trypomastigote release than with conventional culture systems. Additionally, the total number of parasites provided approached 1 x 10(9) trypomastigotes/150 cm2 flask, a significant increase over other culture systems. Preliminary studies with Plasmodium fallax an Eimeria tenella indicate that irradiated host cells may be utilized to advantage for the cultivation of other intracellular protozoa.

D. M. Schmatz

Papers

Cultivation of Trypanosoma cruzi in irradiated muscle cells: improved synchronization and enhanced trypomastigote production