Name: transformation of probiotic yeast and their recovery from gastrointestinal immune tissues following oral gavage in mice
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Description: Watch this Scientific Journal Video about Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice at JoVE.com

The authors acknowledge funding through the Children&#39;s Center for Immunology and Vaccines and an NIH New Innovator Award (1DP2AI112242-01) awarded to Tracey J. Lamb. The authors also thank Natalya P. Degtyareva for the generous contribution of rad1 S. cerevisiae.

1. UV Mutagenesis to Generate Auxotrophic Yeast Strains
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	<li>Generate survival curves to determine needed doses of UV irradiation
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		<li>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 &#176;C O/N&#160;to saturation for at least 8 hr.</li>
		<li>Determine the cell concentration of O/N cultures using a spectropho...

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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&#39...

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 boulardii2, requires knowledge of laboratory techniques not traditionally combined within a given study, ranging from yeast and molecular biology to animal handling techniques and immunological methods. Thus although the individual procedures described herein are not in themselves novel laboratory protocols, the goal of this manuscript is to present a unified introduction to techniques needed for experimental testing of probiotic yeast as drug delivery vehicles to the murine gastrointestinal tract. Provided is a compilation of essential protocols for: 1) generation of auxotrophic mutant strains of yeast that can easily be genetically manipulated; 2) transformation of yeast cultures to express heterologous protein; 3) administration of recombinant yeast to the intestine via oral gavage; and 4) recovery of viable recombinant probiotic yeast from the murine intestine and assessment of their heterologous protein expression.
First, although numerous positive and negative selection methods exist for the manipulation of yeast species, negative selection such as through the use of auxotrophic markers increases both the efficiency and ease with which yeast can be transformed and selected. Positive selection of transformants using antibiotics, in contrast, significantly increases the cost of yeast manipulation. Furthermore, selection of yeast on antibiotic-containing solid media can allow for increased growth of untransformed background colonies relative to selection of auxotrophic yeast on synthetic drop out solid media (unpublished observations). Auxotrophic yeast is strains which lack enzymes critical for the synthesis of essential amino acids or uracil. Such yeast can grow only if supplemented with the missing metabolite or metabolic gene, thus enabling negative selection when yeast is plated onto synthetic drop out media that lacks the essential metabolite. Many commonly used Saccharomyces cerevisiae laboratory strains are in fact already auxotrophic mutants3. Industrial, clinical, and probiotic yeast strains, however, are typically prototrophic with the ability to synthesize all required nutrients. To enable more efficient genetic manipulation of such yeast, auxotrophic genes can be selectively targeted to generate strains that can be selected without antibiotics. Specific targeting of auxotrophic marker genes can be achieved through PCR-mediated gene disruption relying on homologous recombination or more recently through CRISPR/Cas9 targeting4-6. Alternatively, UV mutagenesis can quickly generate auxotrophic mutants even in yeast strains for which transformation with multiple plasmids is technically difficult7. While PCR targeting and CRISPR/Cas9 have been described extensively elsewhere, presented in part one of this manuscript is a detailed protocol describing a UV mutagenesis approach to create auxotrophic strains that will allow for negative selection rather than positive antibiotic selection of yeast transformants.
The next necessary step in the use of such auxotrophic strains for oral delivery of heterologous protein is yeast transformation with plasmid DNA. Since the first successful transformation of yeast spheroplasts reported for Saccharomyces cerevisiae in 19788, numerous modifications have been characterized to increase the efficiency and ease with which yeast species can be genetically modified. Use of electroporation for the successful transformation of DNA into S. cerevisiae was first described in 19859 and has since been improved via the addition of 1 M sorbitol incubation to osmotically support cells10. Electroporation efficiency has furthermore been shown to depend on the yeast species and strain, cell number and phase of growth, electroporation volume, field strength, and specific buffers11. Lithium acetate (LiOAc) transformation, originally described by Ito et al.12, is among the most commonly used transformation protocols as it requires no special equipment. Additional analyses showed that the efficiency of LiOAc yeast transformation greatly increases when cells are collected in mid-log phase of growth and are heat shocked in the presence of polyethylene glycol (PEG) and DNA at 42 &#176;C12. Incubation of whole intact yeast with PEG is essential for efficient transformation, possibly through improving attachment of DNA to the cell membrane as well as via other effects on the membrane13. Lithium itself also increases the permeability of intact cells14. Although most laboratory S. cerevisiae strains can easily be transformed using LiOAc transformation3, other yeast species may be more efficiently transformed using alternative protocols. Pichia pastoris, for example, is most efficiently transformed via electroporation rather than LiOAc transformation13. It is crucial, therefore, to test multiple methods of transformation and to optimize incubation periods and reagent concentrations when attempting to genetically modify an uncharacterized yeast strain. This manuscript thus describes both LiOAc transformation and electroporation as techniques for the transformation of auxotrophic mutant and wild type S. boulardii. Interested readers are directed to recent reviews for thorough descriptions of the evolution of yeast transformation, alternative protocols, and further discussions of possible mechanisms of action13,15. Transformation of yeast with plasmid encoding an easily detectable protein is furthermore essential for downstream testing in order to ensure proper expression and function of heterologous protein. Myriad different proteins may be selected depending on the ultimate purpose of the therapeutic study and the antibodies available for protein detection by immunoblotting, ELISA, and other techniques. Protocols for these techniques have been thoroughly described elsewhere16,17, and can be used to determine levels of heterologous protein production from transformed yeast by comparison to standard curves. For purposes of demonstration and to show successful production of a very commonly used protein in yeast biology, this manuscript presents transformation with plasmid encoding green fluorescent protein (GFP), which allows for subsequent detection using fluorescence microscopy.
Equally important to the production of probiotic organisms that express heterologous protein is the proper administration and detection of these microorganisms within gastrointestinal tissues, as described in parts three and four. Administration of recombinant yeast via oral gavage allows for delivery of controlled quantities of yeast directly into the stomach, from which C57BL/6 mice are naturally incapable of vomiting18. However, improper animal handling and gavage can lead to esophageal damage and perforation, gastric perforation, tracheal administration, and aspiration pneumonia19,20. Poor technique and inexperience can furthermore increase variability in murine immune responses and experimental results, which have been attributed to animal stress upon oral gavage21,22. Practice in the proper technique can thus not only attenuate animal discomfort, but can also increase precision of experimental results. This manuscript describes and demonstrates animal handling and oral gavage for the administration of controlled doses of recombinant yeast.
Finally, it is vital to confirm successful delivery of recombinant yeast by analyzing lymphoid tissues for the presence of yeast and heterologous protein. The gastrointestinal immune tissues which can most easily and predictably be examined for the presence of yeast are the Peyer&#39;s patches. Peyer&#39;s patches are secondary lymphoid organs along the small intestine that are key sites of mucosal immune response induction23. Antigens from the lumen are transferred transcellularly through microfold (M) cells in the epithelium and are released into the Peyer&#39;s patches, thus exposing enclosed antigen presenting cells to intestinal luminal contents. Although particle uptake across the intestinal epithelium can also be achieved by goblet cells, these cells have been shown to only take up particles less than 0.02 &#181;m in diameter24. Transepithelial dendrites extended from CD103+ dendritic cells (DC) also take up small particles from the intestinal lumen25; however, there are currently no reports demonstrating that CD103+ DCs take up particles larger than bacteria. Thus, intact probiotic yeast, of average size between 3-6 &#181;m in diameter, are most likely to be taken up by M cells and transferred to the Peyer&#39;s patches. Described here is a protocol for collection and screening of Peyer&#39;s patches for viable recombinant yeast, although this procedure can also be easily adapted for evaluating uptake of probiotic bacteria.
In summary, assessing recombinant probiotic yeast for the delivery of therapeutic proteins to the intestine requires proficiency in laboratory techniques spanning molecular biology to animal handling and immunology. Presented here are protocols for 1) the generation and screening of auxotrophic yeast strains which can be easily negatively selected without antibiotics, 2) alternative protocols to transform yeast and enable expression of heterologous protein, 3) demonstrations of proper animal handling techniques and oral gavage for intragastric delivery of recombinant yeast, and 4) protocols for Peyer&#39;s patch dissection and screening for viable recombinant yeast and functional heterologous protein. Combined, these protocols will allow for the generation and testing of a probiotic yeast strain capable of delivering heterologous therapeutic protein to the gastrointestinal tract.

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			<td height="63" style="height:63px;width:216px;">SmartSpec 3000 Spectrophotometer</td>
			<td style="width:107px;">BioRad</td>
			<td style="width:107px;">170-2501</td>
			<td style="width:275px;">Example of spectrophotometer for determining cell concentration and OD600 of yeast cultures</td>
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			<td height="42" style="height:42px;width:216px;">New Brunswick Roller Drum</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:107px;">M1053-4004</td>
			<td style="width:275px;">Example of roller drum for yeast culture incubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV Stratalinker 2400</td>
			<td style="width:107px;">Stratagene</td>
			<td style="width:107px;">400075-03</td>
			<td style="width:275px;">Example stratalinker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stuart Colony Counter SC6PLUS</td>
			<td style="width:107px;">11983044</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:275px;">Plate stand with magnification records colony count upon sensing pressure from pen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scienceware Colony Counter&#160;</td>
			<td style="width:107px;">F378620002</td>
			<td style="width:107px;">Bel-Art Scienceware</td>
			<td style="width:275px;">Hand held colony counter pen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Replica plating device</td>
			<td style="width:107px;">Fisherbrand</td>
			<td>09-718-1</td>
			<td rowspan="2" style="width:275px;">Example of replica plating stand and pads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Velveteen squares</td>
			<td style="width:107px;">Fisherbrand</td>
			<td>09-718-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;L shaped sterile cell spreaders&#160;</td>
			<td style="width:107px;">Fisherbrand</td>
			<td style="width:107px;">14665230</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Deoxyribonucleic acid, single stranded from salmon testes</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:107px;">D7656-1ML</td>
			<td style="width:275px;">Example carrier DNA for yeast LiOAc transformation</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Gavage needles</td>
			<td style="width:107px;">Braintree Scientific</td>
			<td style="width:107px;">N-PK&#160;002</td>
			<td style="width:275px;">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</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1mL sterile slip-tip disposable tuberculin syringe&#160;</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:107px;">BD 309659</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Blunt forceps such as Electron Microscopy Sciences 7&#34; (178 mm) serrated tip, broad grip forceps</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">77937-28</td>
			<td style="width:275px;">Example of blunt forceps needed for dissection&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Straight and curved dissection scissors&#160;</td>
			<td style="width:107px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:107px;">72966-02 and 72966-03</td>
			<td style="width:275px;">Examples of scissors needed for dissection&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IMDM</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:107px;">12440053</td>
			<td style="width:275px;"></td>
		</tr>
	</tbody>
</table>

transformation of probiotic yeast and their recovery from gastrointestinal immune tissues following oral gavage in mice

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.

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 &#181;l 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 demonst...

Watch this Scientific Journal Video about Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice at JoVE.com

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

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.

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

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