A Yeast Genetic Screen to Identify Host Cell Pathways Targeted by Legionella pneumophila.
pneumophila is a Gram-negative, facultative, intracellular bacterium and causative agent
of Legionnaires' disease, which presents as an atypical pneumonia and flu-like symptoms,
such as cough, fever and chills. Disease is caused by inhalation or aspiration of
aerosolized bacterium from a fresh water source which is then taken up by alveolar
macrophages. Inside its host ce... read morell, L. pneumophila resides and replicates in a
membrane-bound vacuole that avoids fusion with late endosomes and lysosomes. This
compartment is called the Legionella-containing vacuole (LCV). The pathogenicity of L.
pneumophila is partially attributed to its ability to evade the host immune system.
Central to L. pneumophila pathogenesis is the Icm/Dot type IVb secretion system. Through
this secretion system, L. pneumophila injects proteins into the host cell. Over 270
different bacterial proteins have been identified thus far. The roles of individual
Icm/Dot translocated substrates (IDTSs) in promoting replication vacuole formation have
been difficult to elucidate due to the fact that individual deletions of most substrates
of the L. pneumophila genome have no consequence on intracellular growth of the
bacterium. This lack of defect is thought to be due to functional redundancy, such that
multiple translocated substrates target a single pathway in the host cell. In order to
identify host cell pathways controlled by L. pneumophila that have not been previously
identified using traditional strategies of analyzing membrane trafficking and kinase
networks during intracellular growth, we performed a genetic screen in the model
organism Sacchromyces cerevisiae. In this study, we screened L. pneumophila IDTS yeast
expression library and identified LegA7 as a protein that reduced the ability of yeast
to adapt to conditions of high osmolarity. LegA7 does this by acting on host cell MAPK
pathway signaling. In yeast, this interaction is likely an indirect interaction since
LegA7 did not activate the high osmolarity glycerol pathway MAPK Hog1. LegA7 contains a
catalytic triad including residues C61, H166 and E179. All three residues are required
for LegA7 function. LegA7 also contains an ankyrin repeat domain important for its
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Genetics.
Advisors: Ralph Isberg, and Naomi Rosenberg.
Committee: Catherine Freudenreich, Andrew Wright, and Cammie Lesser.
Keyword: Genetics.read less