Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Summary

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Abstract

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Introduction

The first report of procedures similar to those presented here characterized the Legionella pneumophila type IV effector SidD, a deAMPylase that modifies Rab1¹. Comparable techniques were used for the characterization of several L. pneumophila effectors^1,2,3. The assay was adapted to characterize a Coxiella burnetii type IV effector protein⁴, and recently the utility of this technique was expanded for the characterization of Chlamydia trachomatis inclusion membrane proteins^5<....

Protocol

1. Preparation of media and reagents

NOTE: Plates should be prepared before the day of the assay and are good for 1 month. Media and reagents can be made at any point and are good for 1 month.

1. Prepare 1 L of the glucose solution (10% w/v) by dissolving 100 g of D-(+)-glucose in 800 mL of distilled water in a 1, 000 mL beaker. Adjust the volume to 1 L with distilled water. Filter through a 0.2 µm sterile filter into a sterile 1 L media storage bottle.
2. Prepare 1 L of the galactose solution (10% w/v) by dissolving 100 g of D-(+)- galactose in 800 mL of distilled water in a 1 L beaker. Adjust the volume to 1 mL using ....

Results

Before the actual yeast suppressor screen can be performed, the effector protein of interest must be tested for toxicity in yeast. This is accomplished by expressing the protein of interest in yeast under the control of a galactose-inducible promoter. Growth on glucose (noninducing conditions) should first be compared to ensure toxicity is specifically due to the expression of the protein of interest and is not a general defect. As shown in Figure 3, toxicity manifests as smaller colonies an.......

Discussion

This protocol outlines step-by-step procedures for identifying host biological pathways targeted by bacterial effector proteins using a modified yeast toxicity and suppressor screen. The yeast strain used, S. cerevisiae W303, is auxotrophic for both uracil and leucine. Uracil auxotrophy of the strain is used to select yeast carrying the protein of interest on the pYesNTA-Kan vector while leucine auxotrophy is used to select for the yeast genomic library vector pYep13. The yeast genomic library plasmids carry 3&#.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Fisher Scientific	BP2641500
Galactose	MilliporeSigma	G0750-1KG
GeneJet Gel extraction kit	ThermoFisher Scientific	K0691
GeneJet PCR purification kit	ThermoFisher Scientific	K0701
GeneJet plasmid miniprep kit	Thermo	K0503
Glucose	MilliporeSigma	G8270-1KG
Herring Sperm DNA	Promega	D1811
KpnI-HF	New England Biolabs	R3142S
Lithium acetate dihydrate	MilliporeSigma	L6883-250G
Peptone	Fisher Scientific
Phusion High-Fidelity DNA Polymerase	New England Biolabs	M0530
Poly(ethylene glycol) 3350	MilliporeSigma	1546547-1G
pYep13	ATCC	37323
T4 DNA ligase	New England Biolabs	M0202S
Tryptophan	MilliporeSigma	470031-1G
XhoI-HF	New England Biolabs	R0146S
Yeast extract	Fisher Scientific	BP1422-500
Yeast miniprep kit	Zymo	D2001
Yeast nitrogen base without amino acids	MilliporeSigma	Y0626-250G
Yeast Synthetic Drop-out Medium Supplements	MilliporeSigma	Y1501-20G	without uracil
Yeast Synthetic Drop-out Medium Supplements	MilliporeSigma	Y1771-20G	without uracil, leucine, tryptophan