Espressione eterologa della proteina effettrice LtpN di Legionella pneumophila in Saccharomyces cerevisiae

Legionella species are gram-negative ubiquitous bacteria responsible of two types of disease: Legionnaires' disease and Pontiac fever. Legionella pneumophila, serogroups 1, 4 and 6, is the major responsible of Legionnaires' disease. Symptoms of this type of pneumonia are especially high fever, non-productive cough, dyspnea but also gastrointestinal ones, as diarrhea. Legionella pneumophila is found in freshwater reservoirs and infection happens upon inhalation of infected aerosols. Legionella pneumophila replicates in amoebas and human alveolar macrophages in a self-made compartment called Legionella-containing vacuole (LCV). To survive, Legionella pneumophila make use of the Dot/Icm type IV secretion system through which roughly 300 effector proteins are translocated into host cells, many of which are involved in the formation of LCV. The infective process, due to the action of effector proteins, results in cellular alterations that can consist on modulation of membrane trafficking, signal transduction and cytoskeleton dynamics. Saccharomyces cerevisiae has been used in this work as a model to study one of these effector proteins, the putative protease LtpN. Heterologous expression of LtpN in yeast led to toxicity in yeast, allowing the observation of phenotypic effects. In this work the wild type LtpN protein has been analyzed in parallel to other two versions of the same protein: LtpNC61A, that is an catalytically inactive version bearing a C61A point mutation in its catalytic residue, and LtpNSh, which lacks a putative N-terminal transmembrane domain. Through the overexpression of LtpN in S. cerevisiae it has been possible to demonstrate that it causes depolarization of the actin cytoskeleton, alteration of endocytic pathways, mitochondrial condensation and an interesting alteration of ER structure, all effects dependent on its catalytic activity.