Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Summary

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Abstract

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Introduction

Probiotic microorganisms are an intriguing potential means of efficiently and economically delivering heterologous proteins to the gastrointestinal tract. These organisms are capable of surviving passage through the gastrointestinal tract yet do not colonize it¹, enabling controlled dosing and limiting exposure to the drug expressed. Furthermore, the ability to easily engineer these organisms to produce heterologous protein on a large scale renders them an economical alternative to synthetic delivery particles. However, development of such an approach, as recently demonstrated using an auxotrophic strain of the probiotic yeast Saccharomyces bou....

Protocol

1. UV Mutagenesis to Generate Auxotrophic Yeast Strains

1. Generate survival curves to determine needed doses of UV irradiation
   1. Prepare YPD (yeast extract peptone dextrose) media and other reagents listed in Table 1 according to standard procedures²⁶ and inoculate single colonies into 5-10 ml of YPD media. Incubate cultures on a roller drum at 30 °C O/N to saturation for at least 8 hr.
   2. Determine the cell concentration of O/N cultures using a spectrophotometer by diluting cells 1:10 in water in a plastic cuvette. Dilute cells to a concentration of 10⁷ cells/ml in 20 ml sterile distilled wa....

Results

Generation of a survival curve following UV irradiation requires plating of diluted yeast cells such that distinct colony forming units (CFU) are able to form. Each 500 µl sample collected as described above contains approximately 5 x 10⁶ cells; however, greater than 100 colonies per plate are difficult to accurately distinguish. Plating undiluted sample as well as serial 1:10 dilutions of irradiated cells thus ensures that CFU can be enumerated at each UV dose, as demonstrated.......

Discussion

Together, the protocols herein describe the essential steps necessary for the development and testing of auxotrophic probiotic yeast strains for delivery of heterologous therapeutic protein to the intestine. This manipulation and testing of recombinant probiotic yeast requires techniques and resources with which any individual laboratory may not currently be familiar. Thus, although numerous previous studies have described the above protocols for multiple yeast and mouse strains, these methods have not to the authors'.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SmartSpec 3000 Spectrophotometer	BioRad	170-2501	Example of spectrophotometer for determining cell concentration and OD600 of yeast cultures
New Brunswick Roller Drum	Eppendorf	M1053-4004	Example of roller drum for yeast culture incubation
UV Stratalinker 2400	Stratagene	400075-03	Example stratalinker
Stuart Colony Counter SC6PLUS	11983044	Fisher Scientific	Plate stand with magnification records colony count upon sensing pressure from pen
Scienceware Colony Counter	F378620002	Bel-Art Scienceware	Hand held colony counter pen
Replica plating device	Fisherbrand	09-718-1	Example of replica plating stand and pads
Velveteen squares	Fisherbrand	09-718-2
L shaped sterile cell spreaders	Fisherbrand	14665230
Deoxyribonucleic acid, single stranded from salmon testes	Sigma-Aldrich	D7656-1ML	Example carrier DNA for yeast LiOAc transformation
Gavage needles	Braintree Scientific	N-PK 002	For mice 15-20 g, the suggested needle is a 22 gauge (1.25 mm ball), 1 in long, straight reusable gavage needle. For mice weighing greater than 20 g, 20 gauge or larger straight or curved gavage needles may be used
1 ml sterile slip-tip disposable tuberculin syringe	Becton Dickinson	BD 309659
Blunt forceps such as Electron Microscopy Sciences 7" (178 mm) serrated tip, broad grip forceps	Electron Microscopy Sciences	77937-28	Example of blunt forceps needed for dissection
Straight and curved dissection scissors	Electron Microscopy Sciences	72966-02 and 72966-03	Examples of scissors needed for dissection
IMDM	Life technologies	12440053