In order to enable our engineered bacteria to specifically function at the intestinal lesions of IBD patients, thereby achieving better and more precise therapeutic effects, we designed a colonization system in Saccharomyces cerevisiae. The system consists of two components: the tetrathionate sensor TtrSR and the adhesive protein Als3.
To locate the site of the lesion, we try to find a molecule which can specifically characterize IBD. Current research suggests that thiosulfate and tetrathionate can serve as indicators of intestinal inflammation (Levitt et al., 1999), and the levels of thiosulfate or tetrathionate are directly proportional to the degree of intestinal inflammation. A recent study constructed a tetrathionate sensor in E. coli (Kristina N-M Daeffler, 2017)
fig 1 Schematic of the tetrathionate sensor TtrSR
This system is the TtrSR two-component system (TCS) from the marine bacterium Shewanella halifaxensis HAW-EB4. The TCS consists of TtrS and TtrR. TtrS is 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).
Agglutinin-like sequence protein 3 (Als3) is a cell surface glycoprotein of Candida albicans that plays a significant 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 (Wang Tianming et al., Candida-epithelial interactions, 2023). Since our chassis organism, Saccharomyces cerevisiae, is also a fungus, similar to Candida albicans, expressing Als3 on the surface of S. cerevisiae cells could potentially enable binding to E-cadherin on intestinal epithelial cells, thereby allowing the engineered bacteria to colonize the small intestine.
In the colonization system, we designed to express TtrSR along with Als3 in Saccharomyces cerevisiae. Als3 is located downstream of the ttrB promoter. After expressing this system in S. cerevisiae, when tetrathionate is present in the intestine (at the site of IBD lesions), the tetrathionate sensor is activated, leading to the expression of the Als3 protein. This ultimately enables the engineered bacteria to adhere to intestinal epithelial cells, thereby achieving colonization.
fig 2 Schematic of the colonization system
This system is the TtrSR two-component system (TCS) from the marine bacterium Shewanella halifaxensis HAW-EB4. The TCS consists of TtrS and TtrR. TtrS is 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).
To test the effectiveness of the tetrathionate sensor TtrSR, we designed the corresponding plasmids (Fig.3 ttrR, Fig.4 ttrS). We arranged for EGFP to be expressed downstream of ttrB and validated the effectiveness of TtrSR by measuring the fluorescence intensity of the cells. After successfully transforming the plasmids into Saccharomyces cerevisiae, we selected three well-growing colonies for amplification. (Fig. after transforming)Then, we divided the cells from the same clone into two groups, one group induced with 1mM tetrathionate and the other group without inducer as control group. After 12 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. For more details, please refer to the protocol.
fig 3 ttrR
fig 4 ttrS
To test the effectiveness of Als3, we designed a bacterial attachment experiment. We constructed the corresponding plasmid and after transforming it into Saccharomyces cerevisiae, we used human intestinal sections to test the adhesion effects and differences between S. cerevisiae expressing Als3 and yeast without Als3 expression. For more details, please refer to the protocol.
fig 5 Als3
fig 6 转化后
After acquiring the fluorescence confocal microscopy images, we calculated the ratio of total fluorescence intensity to the number of cells.
fig 7 Confocal images of one group with induction and one without
fig 8 Statistical graph
The results indicate that after induction, the expression of the downstream protein increased, but there was no significant difference compared to the control group as anticipated.
This could be due to compatibility issues that arise when the tetrathionate sensor TtrSR is transferred from E. coli to Saccharomyces cerevisiae. Because of differences between eukaryotic and prokaryotic protein expression and delivery systems, TtrS may not be properly processed and localized to the cell membrane in S. cerevisiae. In subsequent experiments, we optimized the TtrS protein, including codon optimization, structural modeling and prediction (still being modified——, to be added later).
After conducting the attachment experiment, we counted the yeast cells left on the tissue and calculated the ratio of the number of cells to the tissue area.
fig 9 Tissue staining image
fig 8 Light microscopy image
fig 10 Statistical graph
The results indicate that after expressing Als3, the adhesion ability of Saccharomyces cerevisiae to intestinal tissue has significantly improved. It has been demonstrated that Als3 can indeed be used for colonization in Saccharomyces cerevisiae.