Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Summary

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Abstract

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.

Introduction

Type III secretion systems are specialized protein-export machines utilized by different genera of gram-negative bacteria to directly deliver bacterially encoded effector proteins into eukaryotic target cells. While the secretion machinery itself is highly conserved, specialized sets of effector proteins have evolved among the different bacterial species to manipulate cellular signaling pathways and facilitate specific bacterial virulence strategies¹. In case of Yersinia, up to seven effector proteins, so called Yops (Yersinia outer proteins), are translocated upon host cell contact and act together to subvert immune cell responses such as....

Protocol

1. Peak Emission and Cross-talk Determination (See Also Figure 2)

1. In order to determine the emission peaks and amount of cross-talk between the donor (coumarin derivate V450) and acceptor (fluorescein) dyes, perform spectral scans of cells labeled with both individual fluorophores at 405 nm excitation.
2. Determine a 10 nm bandwidth within the peak of each dye that will be used for the donor and acceptor channels (here 455-465 nm and 525-535 nm). Plot the normalized intensity over the wavelength and calculate the average percentage of cross-talk of the donor dye in the acceptor channel and vice versa (Figure 2).

Results

To demonstrate the capability of the described method to quantitatively analyze effector translocation into target cells, two Yersinia strains with different translocation kinetics were studied: Y. enterocolitica wild type strain WA-314 (serogroup O8, harboring the virulence plasmid pYVO8²²) and its derivative WA-314ΔYopE (WA-314 harboring pYVO8ΔYopE²³). Earlier work showed that Y. enterocolitica mutants lacking functional YopE exhibit a significantly higher Yop translocat.......

Discussion

We here successfully applied a TEM-1 beta-lactamase reporter based assay for quantitative analysis of effector translocation by Y. enterocolitica. Many different variations of this sensitive, specific and relatively straightforward technique have been described in the literature. In this study laser scanning microscopy was conducted for most sensitive and precise detection of fluorescence signals. Specifically the correction for cross-talk between the donor and acceptor dyes and the individually adjustable detec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LB-Agar	Roth	X969.2
LB-Medium	Roth	X968.2
Dulbecco’s Phosphate Buffered Saline	Sigma-Aldrich	D8662-500ML
96-well plate (black, clear bottom)	Greiner Bio One	655087
DMEM, high glucose, GlutaMAX Supplement, pyruvate	Gibco/Life Technologies	31966-047
Probenecid	Sigma-Aldrich	P8761-25G
LiveBLAzer FRET-B/G Loading Kit with CCF4-AM	Life Technologies	K1095
Lectin from Triticum vulgaris (wheat) FITC conjugate	Sigma-Aldrich	L4895	Used for peak emission and cross-talk determination
V450 Rat anti-Mouse CD8a	BD Bioscience	560469	Used for peak emission and cross-talk determination
Immersol W immersion oil	Zeiss	444969-0000-000	Refractive index = 1.3339 @ 23 °C
TCS SP5 II confocal laser scanning microscope	Leica microsystems		2x GaAsP-Hybrid detectors, 4 channel spectrometer, acusto optical beam spliter, motorized XY stage, adjustable pinhole, objective 20X HC PL APO CS IMM/CORR, 405 nm diode laser 50 mW
Imaris 7.6 software	Bitplane		Plugins included ImarisXT and MeasurementPro
MatLab compiler runtime	MathWorks
Prism 5	GraphPad software