Starvation and rapamycin differentially regulate host cell lysosome exocytosis and invasion by Trypanosoma cruzi metacyclic forms Rafael Miyazawa Martins, Renan Melatto Alves, Silene Macedo and Nobuko Yoshida* Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, R. Botucatu, 862, 6° andar, 04023-062 São Paulo, Brasil. Summary The molecular mechanisms of host cell invasion by T. cruzi metacyclic trypomastigotes (MT), the developmental forms that initiate infection in the mammalian host, are only partially understood. Here we aimed at further identifying the target cell components involved in signalling cascades leading to MT internalization, and demonstrate for the first time the participation of mammalian target of rapamycin (mTOR). Treatment of human epithe- lial HeLa cells with mTOR inhibitor rapamycin reduced lysosomal exocytosis and MT invasion. Downregulation of phosphatidylinositol 3-kinase and protein kinase C also impaired exocytosis and MT internalization. The recombinant protein based on gp82, the MT surface molecule that mediates cell adhesion/invasion, induced exocytosis in HeLa cells. Such an effect has not previously been attributed to any T. cruzi surface molecule. Rapa- mycin treatment diminished gp82 binding as well. Cell invasion assays under conditions that pro- moted lysosome exocytosis, such as 1 h incuba- tion in starvation medium PBS ++ , increased MT invasion, whereas pre-starvation of cells for 1–2 h had an opposite effect. In contrast to MT, invasion of tissue culture trypomastigotes (TCT) increased upon host cell pre-starvation or treatment with rapamycin, a novel finding that discloses quite dis- tinctive features of the two infective forms in a key process for infection. Introduction The virulence of many bacteria and protozoan parasites that are pathogenic to humans is critically dependent on their ability to invade host cells. By exploiting host cell components or even subverting cellular functions, many of these microorganisms accomplish their internalization. Re-arrangement of target cell actin cytoskeleton, for instance, constitutes a strategy used by diverse entero- pathogenic bacteria, such as Shigella, Salmonella. List- eria and Yersisnia. To induce host cell actin remodelling, Shigella and Salmonella use a specialized protein secre- tion system to inject into target cells virulence factors that trigger a variety of signalling pathways, whereas Listeria and Yersinia engage transmembrane cell adhesion pro- teins (Stebbins and Galán, 2001; Cossart and Sansonetti, 2004). Invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi requires the activation of sig- nalling cascades that lead to intracellular Ca 2+ mobiliza- tion (Docampo and Moreno, 1996; Burleigh and Andrews, 1998; Yoshida, 2006) and disruption of actin cytoskeleton (Rodriguez et al., 1995; Cortez et al., 2006). Trypanosoma cruzi causes Chagas’ disease, which was predominantly a neglected disease of poor, rural and forgotten populations in Latin America, but is now spread- ing to non-endemic countries, posing a new worldwide challenge (Coura and Viñas, 2010; Schmunis and Yadon, 2010). In endemic regions, oral transmission of T. cruzi has been responsible for outbreaks of acute cases of Chagas’ disease (Coura et al., 2002; Coura, 2006; Bastos et al., 2010; Noya et al., 2010), with the involvement of metacyclic forms from insect vectors. In natural infections, metacyclic trypomastigotes (MT) are the parasite forms that first contact the host cells. They are internalized in a membrane-bounded vacuole, then escape to the cyto- plasm and differentiate into the replicative amastigote forms, which later transform into trypomastigotes that are released in the circulation upon host cell rupture and disseminate to diverse organs and tissues. Studies using MT generated in liquid media and tissue culture-derived trypomastigotes (TCT) as counterparts of insect-borne and bloodstream parasites, respectively, have revealed some common features of the mammalian cell invasion (Burleigh and Andrews, 1998; Yoshida, 2006). However, Received 22 November, 2010; revised 24 February, 2011; accepted 25 February, 2011. *For correspondence. E-mail nyoshida@ unifesp.br; Tel. (+55) 11 5549 5159; Fax (+55) 11 55711095. Cellular Microbiology (2011) 13(7), 943–954 doi:10.1111/j.1462-5822.2011.01590.x First published online 24 Apr 2011 © 2011 Blackwell Publishing Ltd cellular microbiology