Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Summary

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Abstract

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

Protocol

I. Designing a Yeast Expression System for Type III Effectors

Calibrating a yeast system appropriate for expression of the type III effector(s) of interest is an important task and may require some trial and error. Factors of major relevance that should be considered and optimized when designing such a system are: 1) the promoter driving expression of the effector(s), 2) the copy number of the effector gene, 3) the epitope tag used to verify protein expression, and 4) the yeast strain.

1) Promoter

Because bacterial type III effectors may be toxic to yeast cells, an inducible promoter sh....

Discussion

In this presentation, we illustrated how to use the budding yeast Saccharomyces cerevisiae as a heterologous system for the expression of type III bacterial effector proteins and how to identify effector-induced growth inhibition phenotypes. Importantly, these phenotypes can be utilized in genetic screens to identify suppressors of the negative impact of effectors on yeast growth. Suppressors may represent either direct targets of the effector studied or proteins that participate in cellular processes affected b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Yeast extract	Difco Laboratories	212750
Peptone	Difco Laboratories	211677
D-glucose	Sigma-Aldrich	G5767
Agar	Difco Laboratories	214010
Sodium hydroxide (NaOH)	Sigma-Aldrich	S8045
Yeast nitrogen base w/o amino acids	Difco Laboratories	291940
Yeast synthetic drop-out medium supplement	Sigma-Aldrich	Y2001
D-galactose	Sigma-Aldrich	G0750	>99%; <0.1% glucose
D-raffinose	Sigma-Aldrich	R0250	>98%
L-leucine	Sigma-Aldrich	L8000
Uracil	Sigma-Aldrich	U0750
L-tryptophan	Sigma-Aldrich	T0254
L-histidine	Sigma-Aldrich	H6034
DNA, single stranded, from salmon testes	Sigma-Aldrich	D7656
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D5879	Desiccate
Hydrochloric acid (HCl)	Sigma-Aldrich	H1758
Polyethylene glycol (PEG) 3350	Sigma-Aldrich	P4338
Lithium acetate (LiAc)	Sigma-Aldrich	L4958
Tris (base)	JT Baker	4109-02
Ethylenediamine-tetraacetic acid (EDTA)	Sigma-Aldrich	E5134
β-mercapt–thanol	Sigma-Aldrich	M6250
Glycerol	Sigma-Aldrich	G5516
Bromophenol blue	Sigma-Aldrich	B6131
Dodecyl sulfate sodium salt (SDS)	Merck & Co., Inc.	8.22050.1000
Centrifuge tubes (15 ml)	Corning	430052	Sterile
Spectrophotometer cuvette (10x4x45 mm)	Sarstedt Ltd	67.742
Inoculation loop	Sigma-Aldrich	Z643009	Sterile
Parafilm	Sigma-Aldrich	P7543
pH indicator strip, pH 6.5-10.0	Merck & Co., Inc.	1.09543.0001