Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli.
Bacteria use type III secretion systems (TTSS) to translocate effector proteins into host cells. Better understanding of the TTSS and its effectors' functions will require assays to measure their activities in vivo and in real time. We designed a real-time, high-throughput translocation assay that utilizes fusions of effector genes to the beta-lactamase reporter gene, positioned under the effector's native promoter and chromosomal location. Using this assay, we simultaneously and quantitatively analyzed the translocation kinetics of six core enteropathogenic E. coli effectors, EspF, EspG, EspH, EspZ, Map, and Tir. A distinct order in the efficiencies of effector translocation was observed. Translocation efficiency was determined by multiple factors, including the intrabacterial effector concentration, effector-chaperone interactions, the efficiency of bacterial attachment to the host cells, and possibly also by a translocation autoinhibition mechanism. The described real-time translocation assay could be easily adapted for varied applications in the study of bacterial pathogenesis.
Mills
Erez
E
Department of Molecular Genetics and Biotechnology, The Hebrew University Faculty of Medicine, Jerusalem 91904, Israel.
Baruch
Kobi
K
Charpentier
Xavier
X
Kobi
Simi
S
Rosenshine
Ilan
I
D001422:Bacterial Adhesion; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D004926:Escherichia coli / Q000472:pathogenicity / Q000502:physiology*; D004927:Escherichia coli Infections / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism; D021381:Protein Transport; D014774:Virulence; D001618:beta-Lactamases / Q000235:genetics
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D001618:beta-Lactamases