Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.
Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.
Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf; m.wolters@uke.de.
Manuel
M
Wolters
UKE Microscopy Imaging Facility, University Medical Center Hamburg-Eppendorf.
Bernd
B
Zobiak
Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf.
Theresa
T
Nauth
Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf.
Martin
M
Aepfelbacher
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D014178:Translocation, Genetic; D000069296:Type III Secretion Systems; D015008:Yersinia enterocolitica / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity*; D001618:beta-Lactamases / Q000032:analysis / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D000069296:Type III Secretion Systems; C063239:yopE protein, Yersinia; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1