Abstract
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 β-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.
| Original language | English |
|---|---|
| Pages (from-to) | 104-113 |
| Number of pages | 10 |
| Journal | Cell Host and Microbe |
| Volume | 3 |
| Issue number | 2 |
| DOIs | |
| State | Published - 14 Feb 2008 |
Bibliographical note
Funding Information:We thank Dr. G. Schreiber for providing BlaM and BLIP; G. Frankel and B. Kenny for antibodies; and G. Boger, C. Nadler-Yona, M. Simovitch, G. Yerushalmi, and N.Q. Balaban for useful discussion. This work was supported by grants from the Israel Science Foundation founded by The Israel Academy of Science and Humanities, the Israel-United States Binational Foundation, the Center of Study of Emerging Disease, the EraNet-PathoGenomic program, and the Abisch-Frenkel Foundation. E.M. is a Golda Meir Fellow.
Keywords
- MICROBIO