The autophagosome route
More recently, a third invasion route employed by T. cruzi has been described, which involves elements of the autophagy pathway.11 Autophagy is a conserved catalytic pathway in mammalian cells, involved in the turnover of old or surplus organelles and macromolecules by their engulfment into phagosomes and subsequent delivery to endosomes and/or lysosomes for their degradation and reuse.51 The products of several autophagy related genes form part of the core molecular autophagy machinery, and are essential for the formation of phagosomes.51 Among them, microtubule-associated protein 1A/1B-light chain 3 (LC3), a soluble protein distributed ubiquitously in mammalian cells, constitutes one of the best characterized markers used to monitor autophagy and autophagy-related processes.52
Using CHO cells overexpressing a GFP-tagged version of the autophagic marker LC3, Romano et al.11 evidenced colocalization of GFP-LC3 with T. cruzi parasito- phorous vacuoles by confocal microscopy, indicating interaction of the parasite with the autophagic compartments during host cell invasion. Abundant phagosomes displaying LC3 on their membrane were recruited to the parasite entry site at early time points (1-h postinfection), but no LC3 was detectable in association with the parasite at later time points, when the parasite is free on the cytoplasm and no longer contained in the parasitophorous vacuole (48—72 h).
Induction of autophagy by cell starvation or pharmacological means (rapamycin) increases host cell invasion by the parasite, along with LC3 colocalization with parasitophorous vacuoles.11 Subsequent studies have shown that the parasitophorous vacuoles for different strains of T. cruzi (Brazil, K98, and CL Brenner) associate with LC3 in various other cell types, including rat myoblasts, mouse cardiomyo- cytes, and epithelial cells, suggesting that the usage of this invasion route is not limited to a particular parasite strain/host cell combination, and that it constitutes a widespread phenomenon.53 Additionally, Vanrell et al.54 showed that blocking the synthesis of polyamines (ubiquitous low molecular weight polycations involved in nucleic acid/protein and protein/protein interactions) through the inhibition of the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine results
in suppression of autophagy in mammalian cells, thus preventing cell invasion by