Trypanosoma cruzi: interaction with vertebrate cells in vitro. 3. Selection for biological characteristics following intracellular passage

Two strains of Trypanosoma cruzi, House 510 and Limbo Tree Platform, were studied to determine whether maintenance in vertebrate cell culture would alter their biological characteristics. After having been maintained for several years in alternate mouse-insect passage using NIH general purpose male white mice and Rhodnius prolixus, respectively, the parasites were transferred into primary bovine embryo skeletal muscle cell culture and their intracellular doubling time and generation number quantified.A significant change appeared in the doubling time of each strain. Initially, the doubling times of the House 510 and Limbo Tree Platform strains were not significantly different. They were 8.6 ± 0.8 hr and 7.5 ± 0.5 hr, respectively. After a varying period in cell culture, these values changed to 11.4 ± 1.6 and 11.5 ± 1.3 hr, respectively. Other observations, unrelated to length of time in cell culture, included the appearance of many broad, trypomastigote forms with a decreased ability to reach host cells and a varying generation number ranging from 7 to 9.     

Trypanosoma cruzi: interaction with vertebrate cells in vitro. 2. Quantitative analysis of the penetration phase

A technique is described for quantifying the in vitro penetration of vertebrate cells by trypomastigotes of Trypanosoma cruzi. It was found that the parasites are distributed among host cells in a manner described by the negative binomial distribution. The rate at which trypomastigotes penetrate bovine embryonic skeletal muscle cells (BESM) decreased exponentially in time in this system. The rate of the exponential decrease was dependent upon the concentration of parasites, being faster for more concentrated suspensions of trypomastigotes. A significantly lower penetration rate of canine kidney and HeLa cells was found when compared to bovine embryonic skeletal muscle cells. Within a single population of BESM cells, the smaller cells were penetrated more rapidly than the larger ones per unit cell area.     

Trypanosoma cruzi: in vitro interactions between cultured amastigotes and human skin-muscle cells.

Established cultures of human skin-muscle cells were used for determining the parasite—host cell relationship of Trypanosoma cruzi amastigotes (12–16 passages) cultured in a cell-free medium (F-69) at 37 C. The medium used for this experiment was tissue culture fluid M-199 enriched with 10% fetal bovine serum and relatively high concentrations of ATP, ADP and AMP. Amastigotes entered skin-muscle cells incubated at 32 or 35 C, multiplied and completed their intracellular life cycle in about 7 days. At 35 C, 23.6% of cells became infected in 7 days and at 32 C, 43.6% were infected in 5 days. The higher infection rate of cultured cells at 32 C was probably due to more frequent and prolonged cell-parasite contact, as amastigotes multiplied in the tissue culture medium and remained viable for a longer period at the lower temperature. As a control, epimastigotes were used to infect skinmuscle cells. Epimastigotes transformed into metacyclic trypomastigotes before entering host cells, multiplied, and completed the intracellular life cycle. We conclude that the amastigotes cultured in F-69 at 37 C are biologically similar to intracellular amastigotes from the vertebrate host, in that both can multiply and complete the life cycle intracellulary.     

Trypanosoma cruzi: fusogenic ability of membranes from cultured epimastigotes in interaction with human syncytiotrophoblast

Trypanosoma cruzi epimastigotes cultured in vitro were disrupted by successive freezing and thawing and subsequent sonication. The total homogenate was fractionated by differential centrifugation to obtain an enriched plasma membrane fraction. The proteins of subcellular parasite fractions were labeled with 131I and their binding to membrane fractions from human placenta syncytiotrophoblast was studied. Syncytiotrophoblast fractions enriched in plasma showed higher specific activity for binding an enriched T. cruzi plasma membrane fraction compared with other fractions of syncytiotrophoblast. The properties of this interaction were studied with digestive enzymes (trypsin and phospholipase A2). The results showed that both proteins and lipids could be involved in this interaction. The Ca2+ requirements for the membrane-membrane interaction are different for the two membranes studied. Also the enriched plasma membrane T. cruzi fraction had a higher capacity to induce fusion processes than the other subcellular fractions. The above results indicate that a preferential syncytiotrophoblast-T. cruzi interaction may occur between the two cell surfaces as compared to intracellular membranes and that the parasite surface is able to induce an instability process leading to membrane fusion. These results may have implications in regard to the mechanism of entry of the parasite into cells.     

Trypanosoma cruzi: Interaction with Vertebrate Cells. DNA Synthesis and Growth of Intracellular Amastigotes and their Relationship to Host Cell DNA Synthesis and Growth

SYNOPSIS DNA synthesis of intracellular Trypanosoma cruzi amastigotes, following the infection of bovine embryo skeletal muscle (BESM) cells, was studied by autoradiography. After penetration, there was a prereplicative lag period (∼12 h) followed by a synchronous round of DNA synthesis which was found to be independent of parasite number/BESM cell and the host cell DNA synthesis cycle. Parasite reproduction occurred, for the first time, at ∼ 21 h postinfection. It was concluded that T. cruzi trypomastigotes are in the G₁/G, phase of their cell division cycle and that after penetration parasite reproduction occurs independent of events controlling host cell DNA synthesis and growth. The early synchronous growth of intracellular amastigotes should facilitate further studies on the biochemical events controlling trypomastigote-to-amastigote transformation and amastigote reproduction. A further application is envisaged for studies on the mode of action of drugs with trypanocidal activity.