The Study Of The Function Of Legionella Pneumophila Effectors Using The Alternative Host Saccharomyces Cerevisiae

Legionella pneumophila as the pathogen of Legionnaires' disease is an ideal model for the studies on the relation between intracellular pathogen and its host. The key to the intracellular survival and replication of L. pneumophilla is the secretion of large amount of effector proteins delivered by its type IVB secretion system into the host cell, which interferes the endocytosis of the macrophage cell by hindering the fusion between lysosome and endosome and the lysis of the invader bacteria or repressing the host protein synthesis or apoptosis, resulting in the successful intracellular growth and proliferation. However, identification and characterization of these effectors has been difficult, often limited by the lack of detectable signal and functional redundancy. Saccharomyces cerevisiae as a single-cell organism sharing many conserved fundamental biochemical and cellular process among eukaryotes is widely used in the fields of molecular genetics and cellular biology due to the simple culture and fast growth, well-depicted genetic background and powerful genetic manipulating. Recent studies show that expression of L. pneumophilla effector in the budding yeast can cause detectable aberrance of the normal cell growth, providing an easy approach for the studies on the relation between L. pneumophilla effector and the yeast. The sub-cellular localization of effector protein is also a beneficial clue in deciphering its function. Meanwhile, multi-copy suppressor screening system has been proved to be a powerful tool for identification and characterization of protein-protein interaction in the budding yeast. In this study, several L. pneumophilla effectors Ceg20 (also known as lpg1137), LegK(also known as lpg1483), RalF (also known as lpg1950)and LegS2 (also known as lpg2176) are investigated. The results showed that expression of L. pneumophilla effector in the budding yeast cause growth defect in a more or less way, suggesting the difference in the mechanism of these effectors. The results also revealed LegS2 probably localize to yeast mitochondria and expression of LegS2 reduce yeast cell oxygen consumption, indicating LegS2 plays its role via interfering with yeast mitochondria. In addition, a preliminary multi-copy suppressor screening for the identification of the yeast target protein of the LegK1 was also performed.