Development of a novel functional high-throughput screening system for pathogen effectors in the yeast Saccharomyces cerevisiae.
Our understanding of the molecular mechanisms of bacterial pathogenesis has been improved especially by the discovery of host cell contact-dependent secretion systems such as the type-III secretion system (T3SS) found in numerous pathogens. Although the identification of pathogen effectors translocated into host cells through T3SS is essential to the understanding of pathogenesis, their general sequence uniqueness confound attempts to identify such proteins by sequence homology. Here we report the development of a functional high-throughput screening system for pathogen effectors in yeast that consists of a Gateway(TM)-compatible Tet-Off inducible expression vector and a yeast strain expressing a reporter, facilitating identification of the effectors affecting host vesicular trafficking pathways. We evaluated this system and optimized the screening condition using several known pathogen effectors. We found this system useful in functional characterization of pathogen effector and it can be adapted to functional high-throughput screening as well.
Tabuchi
Mitsuaki
M
Department of Molecular Genetics, Kawasaki Medical School, Kirashiki, Okayama, Japan.
Kawai
Yasuhiro
Y
Nishie-Fujita
Makiko
M
Akada
Rinji
R
Izumi
Terumi
T
Yanatori
Izumi
I
Miyashita
Naoyuki
N
Ouchi
Kazunobu
K
Kishi
Fumio
F
D001419:Bacteria / Q000235:genetics / Q000472:pathogenicity*; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004318:Doxycycline / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*
D003470:Culture Media; D004318:Doxycycline