<p>To enable our engineered bacteria to specifically function at the small intestinal lesions in IBD patients, we designed the colonization system. This system consists of two main components: one is the tetrathionate sensor TtrSR, and the other is the adhesion protein Als3. TtrSR is a two-component system from the marine bacterium Shewanella halifaxensis HAW-EB4, which can detect extracellular tetrathionate chemical signals and promote the expression of downstream genes in the signaling pathway. Als3 is a cell surface protein from Candida albicans, which acts as an adhesin, mediating adhesion to epithelial cells, endothelial cells, and extracellular matrix proteins. We chose Saccharomyces cerevisiae as the chassis organism for engineering the bacteria. By expressing the TtrSR system and Als3 protein in Saccharomyces cerevisiae, we will achieve specific colonization at the small intestinal lesions in IBD patients.</p>
<p>Our experimental design is divided into two parts: verify the functionality of the TtrSR system in Saccharomyces cerevisiae and test the adhesion ability of Saccharomyces cerevisiae after expressing Als3. We independently validated each component of the colonization system in the engineered Saccharomyces cerevisiae and ultimately plan to integrate them together.</p>
<p>To enable our engineered bacteria to specifically function at the small intestinal lesions in IBD patients, we designed the colonization system. This system consists of two main components: one is the tetrathionate sensor TtrSR, and the other is the adhesion protein Als3. TtrSR is a two-component system from the marine bacterium Shewanella halifaxensis HAW-EB4, which can detect extracellular tetrathionate chemical signals and promote the expression of downstream genes in the signaling pathway. Als3 is a cell surface protein from Candida albicans, which acts as an adhesin, mediating adhesion to epithelial cells, endothelial cells, and extracellular matrix proteins. We chose <i>Saccharomyces cerevisiae</i> as the chassis organism for engineering the bacteria. By expressing the TtrSR system and Als3 protein in <i>Saccharomyces cerevisiae</i>, we will achieve specific colonization at the small intestinal lesions in IBD patients.</p>
<p>Our experimental design is divided into two parts: verify the functionality of the TtrSR system in <i>Saccharomyces cerevisiae</i> and test the adhesion ability of <i>Saccharomyces cerevisiae</i> after expressing Als3. We independently validated each component of the colonization system in the engineered <i>Saccharomyces cerevisiae</i> and ultimately plan to integrate them together.</p>
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<p>To identify the lesion site, we need a molecule that specifically characterizes IBD. Current research suggests that thiosulfate and tetrathionate can serve as indicators of intestinal inflammation <sup>1</sup>, and the levels of thiosulfate or tetrathionate are directly proportional to the severity of intestinal inflammation. A previous study constructed a tetrathionate sensor in E. coli <sup>2</sup>. This system is the TtrSR two-component system (TCS) from the marine bacterium Shewanella halifaxensis HAW-EB4. The TCS includes TtrS, a membrane-bound sensor histidine kinase (SK), which can phosphorylate the cytoplasmic response regulator (RR) TtrR in the presence of tetrathionate. Phosphorylated TtrR activates the expression of downstream genes through the ttrB promoter (PttrB).</p>
<p>To verify the effectiveness of the tetrathionate sensor TtrSR, we designed the corresponding plasmids. We planed to express EGFP downstream of ttrB and validate the effect of TtrSR by testing the fluorescence intensity of the cells. We used Ura-HIS nutrient-deficient medium to select yeast that had been successfully transformed. Then, we tested the effectiveness of the TtrSR system using confocal microscopy. For more details, please refer to protocol.</p>
<p>Aim:</p>
<p>To confirm the effectiveness of TtrRS in Saccharomyces cerevisiae</p>
<p>To confirm the effectiveness of TtrRS in <i>Saccharomyces cerevisiae</i></p>
<p>Constructs:</p>
<p>pYES2-SV40-ttrS</p>
<p>pESC-SV40-ttrR-PttrB-EGFP</p>
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<h3>Adhesion Protein Als3</h3>
<p>Agglutinin-like sequence protein3 (Als3) is a cell surface glycoprotein of Candida albicans that plays a crucial role in vitro adhesion and biofilm formation. Als3 is essential for the binding of Candida albicans hyphae to various host cell surface receptor proteins, and it induces endocytosis by binding to E-cadherin on epithelial cells <sup>3</sup>. Since our chassis organism, Saccharomyces cerevisiae, is also a fungus like Candida albicans, expressing Als3 on the surface of Saccharomyces cerevisiae cells is expected to enable binding to E-cadherin on the small intestinal epithelium, thereby allowing the engineered bacteria to colonize the small intestine.</p>
<p>To verify the effectiveness of Als3, we designed an adhesion assay experiment.<sup>4</sup> We designed the corresponding plasmids to express Als3 in Saccharomyces cerevisiae and simultaneously used the expression of EGFP for yeast tracking. After transforming the plasmids into Saccharomyces cerevisiae, we selected yeast that had been successfully transformed using Ura nutrient-deficient medium. Then, we verified the effectiveness of Als3 through the adhesion capability assay. For further details, please refer to the protocol</p>
<p>Agglutinin-like sequence protein3 (Als3) is a cell surface glycoprotein of Candida albicans that plays a crucial role in vitro adhesion and biofilm formation. Als3 is essential for the binding of Candida albicans hyphae to various host cell surface receptor proteins, and it induces endocytosis by binding to E-cadherin on epithelial cells <sup>3</sup>. Since our chassis organism, <i>Saccharomyces cerevisiae</i>, is also a fungus like Candida albicans, expressing Als3 on the surface of <i>Saccharomyces cerevisiae</i> cells is expected to enable binding to E-cadherin on the small intestinal epithelium, thereby allowing the engineered bacteria to colonize the small intestine.</p>
<p>To verify the effectiveness of Als3, we designed an adhesion assay experiment.<sup>4</sup> We designed the corresponding plasmids to express Als3 in <i>Saccharomyces cerevisiae</i> and simultaneously used the expression of EGFP for yeast tracking. After transforming the plasmids into <i>Saccharomyces cerevisiae</i>, we selected yeast that had been successfully transformed using Ura nutrient-deficient medium. Then, we verified the effectiveness of Als3 through the adhesion capability assay. For further details, please refer to the protocol</p>
<p>Aim:</p>
<p>To verify the change in adhesion capability of Saccharomyces cerevisiae expressing Als3 to small intestinal cells </p>
<p>To verify the change in adhesion capability of <i>Saccharomyces cerevisiae</i> expressing Als3 to small intestinal cells </p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 7 the result of transformation</p>
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<p>In our colonization system, we designed to express TtrSR and Als3 in Saccharomyces cerevisiae. Als3 is located downstream of the ttrB promoter. After expressing this system in Saccharomyces cerevisiae, when tetrathionate is present in the intestine (i.e., at the site of IBD lesions), the tetrathionate sensor is activated, leading to the expression of Als3 protein. This ultimately allows the engineered bacteria to adhere to the intestinal epithelial cells, thereby achieving colonization. </p>
<p>In our colonization system, we designed to express TtrSR and Als3 in <i>Saccharomyces cerevisiae</i>. Als3 is located downstream of the ttrB promoter. After expressing this system in <i>Saccharomyces cerevisiae</i>, when tetrathionate is present in the intestine (i.e., at the site of IBD lesions), the tetrathionate sensor is activated, leading to the expression of Als3 protein. This ultimately allows the engineered bacteria to adhere to the intestinal epithelial cells, thereby achieving colonization. </p>
<p>Through our efforts, we have obtained some experimental results to support our system design. We divided the colonization system into the TtrRS system and the Als3 protein and tested their functionality through a series of wet lab experiments. Finally, we plan to integrate them to test the effectiveness of the complete colonization system. You can see our experimental results in the following content.</p>
<h3>Tetrathionate sensor TtrSR</h3>
<p>After successfully transforming the pYES2-SV40-ttrS and pESC-SV40-ttrR-PttrB-EGFP plasmids into Saccharomyces cerevisiae, we selected three colonies with good conditions for amplification. Then, the same clone was divided into two groups, one group was induced with 2mM tetrathionate, and the other group was used as a control without inducer. After 24 hours of induction, an appropriate amount of bacterial solution was prepared into temporary slides and the fluorescence intensity was tested using a fluorescence confocal microscope. After obtaining the fluorescence confocal microscopy images, we calculated the ratio of total fluorescence intensity to the number of cells. The images were processed using Image J, and statistical graphs were plotted using GraphPad Prism.</p>
<p>After successfully transforming the pYES2-SV40-ttrS and pESC-SV40-ttrR-PttrB-EGFP plasmids into <i>Saccharomyces cerevisiae</i>, we selected three colonies with good conditions for amplification. Then, the same clone was divided into two groups, one group was induced with 2mM tetrathionate, and the other group was used as a control without inducer. After 24 hours of induction, an appropriate amount of bacterial solution was prepared into temporary slides and the fluorescence intensity was tested using a fluorescence confocal microscope. After obtaining the fluorescence confocal microscopy images, we calculated the ratio of total fluorescence intensity to the number of cells. The images were processed using Image J, and statistical graphs were plotted using GraphPad Prism.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 10 Fluorescence confocal microscopy imaging results (s: control group without inducer; s+: K<sub>2</sub>O<sub>6</sub>S<sub>4</sub> added)</p>
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<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 11 Statistical results of tetrathionate induction experiment (s: control group without inducer; s+: K<sub>2</sub>O<sub>6</sub>S<sub>4</sub> added)</p>
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<p>The results showed that after tetrathionate induction, the expression of the downstream protein EGFP increased, but there was no significant difference compared to the control group. Meanwhile, in the S+ group, although EGFP was expressed, the fluorescence was weak, which means the expression level was low</p>
<p>To explore the reasons for the insignificant induction difference and the low expression level, we conducted further literature research and group discussions. We finally concluded that this might be due to compatibility issues that arise when the tetrathionate sensor TtrSR is transplanted from E. coli to Saccharomyces cerevisiae. Due to differences in protein expression and delivery systems between eukaryotic and prokaryotic cells, TtrS may not effectively function in Saccharomyces cerevisiae due to issues with signal peptides, transmembrane domains, etc., which may prevent it from properly localizing to the cell membrane. Additionally, the expression levels of TtrS and TtrR in Saccharomyces cerevisiae may be insufficient. During the literature research, we found that some researches have achieved the application of two-component systems in eukaryotic systems through codon optimization and other manipulations. <sup>5,6</sup> In following experiments, we plan to optimize the TtrS protein, including codon optimization, signal peptide prediction and optimization, membrane localization prediction, and transmembrane domain optimization, in order to enable TtrRS to function better in Saccharomyces cerevisiae.</p>
<p>To explore the reasons for the insignificant induction difference and the low expression level, we conducted further literature research and group discussions. We finally concluded that this might be due to compatibility issues that arise when the tetrathionate sensor TtrSR is transplanted from E. coli to <i>Saccharomyces cerevisiae</i>. Due to differences in protein expression and delivery systems between eukaryotic and prokaryotic cells, TtrS may not effectively function in <i>Saccharomyces cerevisiae</i> due to issues with signal peptides, transmembrane domains, etc., which may prevent it from properly localizing to the cell membrane. Additionally, the expression levels of TtrS and TtrR in <i>Saccharomyces cerevisiae</i> may be insufficient. During the literature research, we found that some researches have achieved the application of two-component systems in eukaryotic systems through codon optimization and other manipulations. <sup>5,6</sup> In following experiments, we plan to optimize the TtrS protein, including codon optimization, signal peptide prediction and optimization, membrane localization prediction, and transmembrane domain optimization, in order to enable TtrRS to function better in <i>Saccharomyces cerevisiae</i>.</p>
<h3>Adhesion Protein Als3</h3>
<p>We used human small intestinal sections to test the adhesion effects and differences between Saccharomyces cerevisiae expressing Als3 and yeast without Als3 expression. The yeast expressing Als3 was transformed with the pYES2-SV40-ALS3-EGFP plasmid, while the yeast without Als3 expression was also transformed with plasmid that could express EGFP, allowing for the observation of Saccharomyces cerevisiae. The human small intestinal sections were purchased from Zhongke Guanghua Company. After the adhesion assay, we stained the sections with Hoechst 33342. This staining allowed the small intestinal cells to emit blue fluorescence. Therefore, we could observe the small intestinal tissue through the blue fluorescence, while the green fluorescence marked the position of Saccharomyces cerevisiae. We counted the yeast cells adhered to the tissue and calculated the ratio of cell number to tissue area. The images were processed using Image J, and statistical graphs were plotted using GraphPad Prism.</p>
<p>We used human small intestinal sections to test the adhesion effects and differences between <i>Saccharomyces cerevisiae</i> expressing Als3 and yeast without Als3 expression. The yeast expressing Als3 was transformed with the pYES2-SV40-ALS3-EGFP plasmid, while the yeast without Als3 expression was also transformed with plasmid that could express EGFP, allowing for the observation of <i>Saccharomyces cerevisiae</i>. The human small intestinal sections were purchased from Zhongke Guanghua Company. After the adhesion assay, we stained the sections with Hoechst 33342. This staining allowed the small intestinal cells to emit blue fluorescence. Therefore, we could observe the small intestinal tissue through the blue fluorescence, while the green fluorescence marked the position of <i>Saccharomyces cerevisiae</i>. We counted the yeast cells adhered to the tissue and calculated the ratio of cell number to tissue area. The images were processed using Image J, and statistical graphs were plotted using GraphPad Prism.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 13 <b>Statistical results of quantification of attachment experiment</b> (Als3: the attachment of saccharomyces cerevisiae expressing Als3; WT: the attachment of wild-type saccharomyces cerevisiae, control groups)</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 13 <b>Statistical results of quantification of attachment experiment</b> (Als3: the attachment of <i>Saccharomyces cerevisiae</i> expressing Als3; WT: the attachment of wild-type <i>Saccharomyces cerevisiae</i>, control groups)</p>
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<p>The results showed that after expressing Als3, there was a significant difference in the number of Saccharomyces cerevisiae adhering per unit area of small intestinal epithelium compared to the wild-type group, with an increase in adhesion. This indicates that Als3 can enhance the adhesion ability of Saccharomyces cerevisiae to small intestinal tissue to some extent. However, during the observation of the images, we found that the green fluorescence of Saccharomyces cerevisiae was not stable, with some yeast showing weak or even no fluorescence. Additionally, there was spontaneous fluorescence within the small intestinal tissue, which could affect the counting of yeast cells. </p>
<p>The results showed that after expressing Als3, there was a significant difference in the number of <i>Saccharomyces cerevisiae</i> adhering per unit area of small intestinal epithelium compared to the wild-type group, with an increase in adhesion. This indicates that Als3 can enhance the adhesion ability of <i>Saccharomyces cerevisiae</i> to small intestinal tissue to some extent. However, during the observation of the images, we found that the green fluorescence of <i>Saccharomyces cerevisiae</i> was not stable, with some yeast showing weak or even no fluorescence. Additionally, there was spontaneous fluorescence within the small intestinal tissue, which could affect the counting of yeast cells. </p>
<p>Therefore, in subsequent experiments, we plan to use cell dyes such as Trypan Blue for more uniform and accurate staining. Moreover, to better test the adhesion effect of the engineered bacteria, we plan to conduct further in vivo experiments using mice. Furthermore, since it seems that expressing Als3 does not significantly increase the number of adherent cells, we also plan to compare or optimize using other adhesion proteins or systems.</p>
