Distribution of cytoskeletal structures and organelles of the host cell during evolution of the intracellular parasitism by Trypanosoma cruzi.

The distribution of microtubules, microfilaments, mitochondria, Golgi complex and endosomes/lysosomes was analyzed in Vero cells allowed to interact for different periods of time with the pathogenic protozoan Trypanosoma cruzi and observed by confocal laser scanning microscopy. Microtubules were revealed using a mouse monoclonal anti-alpha-tubulin antibody. Actin filaments were revealed using phalloidin-rhodamine. To identify mitochondria, endosomes/lysosomes and the Golgi complex the cells were labelled with Rhodamine 123, Lucifer yellow and C6-NBD-ceramide, respectively. During cell invasion actin filaments concentrate at the site of parasite penetration in some, but not in all cells, probably depending upon the mechanism used by the trypomastigote form to penetrate into the host cells. Following internalization the trypomastigote form gradually changes into the amastigote form, disruption of the parasitophorous vacuole membrane takes place and the amastigote form enters in direct contact with host cell structures and organelles, and starts to divide. The presence of the parasite in the cytoplasm of the host cell did not induce significant changes in the distribution of actin filaments, microtubules, the Golgi complex, mitochondria and endosomes/lysosomes during the first 48 h of infection. Amastigote forms were seen close to the microtubules. After 72 h of interaction, the number of microtubules and microfilaments around the parasites was reduced and lysosomes and mitochondria were seen in between the parasites.

The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction

In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein phosphorylation, revealed by immunocytochemistry, and sites of actin assembly, revealed by the use of labeled phaloidin. The results obtained show that proteins localized in the interaction sites are phosphorylated. The process of formation of the parasitophorous vacuole was monitored by labeling the host cell surface with fluorescent probes for lipids (PKH26), proteins (DTAF) and sialic acid (FITC-thiosemicarbazide) before interaction with the parasites. Evidence was obtained indicating transfer of components of the host cell surface to the parasite surface in the beginning of the interaction process. We also analyzed the distribution of cytoskeletal structures (microtubules and microfilaments visualized with specific antibodies), mitochondria (visualized with rhodamine 123), the Golgi complex (visualized with C6-NBD-ceramide) and the endoplasmic reticulum (visualized with anti-reticulin antibodies and DIOC6) during the evolution of intracellular parasitism. The results obtained show that some, but not all, structures change their position during evolution of the intracellular parasitism.

The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction.

In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein phosphorylation, revealed by immunocytochemistry, and sites of actin assembly, revealed by the use of labeled phaloidin. The results obtained show that proteins localized in the interaction sites are phosphorylated. The process of formation of the parasitophorous vacuole was monitored by labeling the host cell surface with fluorescent probes for lipids (PKH26), proteins (DTAF) and sialic acid (FITC-thiosemicarbazide) before interaction with the parasites. Evidence was obtained indicating transfer of components of the host cell surface to the parasite surface in the beginning of the interaction process. We also analyzed the distribution of cytoskeletal structures (microtubules and microfilaments visualized with specific antibodies), mitochondria (visualized with rhodamine 123), the Golgi complex (visualized with C6-NBD-ceramide) and the endoplasmic reticulum (visualized with anti-reticulin antibodies and DIOC6) during the evolution of intracellular parasitism. The results obtained show that some, but not all, structures change their position during evolution of the intracellular parasitism.