Compete

Background

The first step in which most UTIs occur is UPEC adhering to the epithelial cells of the urinary tract. To address recurrent UTIs, we aimed to compete with UPEC for adhesion sites on epithelial cells, thereby preventing UPEC from invading the cells and establishing an intracellular biofilm. Additionally, we sought to endow our engineered bacteria with additional advantages for long-term microbiota establishment, as elaborated in our description.

Idea 1: Expressing FimH in Lactobacillus crispatus

Our initial concept was to equip L. crispatus with FimH, the same adhesin used by UPEC, to manifest competition and also promote better adhesion of L. crispatus to gain a microbiota advantage.

However, we quickly abandoned this idea, recognizing FimH as a potent pathogenicity factor [1]. Studies indicated that FimH was crucial for UPEC invasion, with FimH-deficient UPEC being unable to invade cells, while beads coated with FimH were internalized [1]. This arises from FimH's ability to bind to various receptors, including Integrins, integral membrane proteins that normally link the extracellular matrix to the actin cytoskeleton [1]. Considering these findings, we understood the potential risk of inducing pathogenicity by expressing FimH in the L. crispatus. Putting safety and ethics as main priorities in our project, we decided to not research this idea further.

Idea 2: Secreting FimH for Competitive Advantage

Our next idea was to secrete FimH by L. crispatus, thus competing with UPEC for adhesion sites, while ensuring the L. crispatus microbiota remains non-pathogenic. To learn more about that see our future plans page.

Recognizing that competition alone might not suffice through consulting Prof. Yoram Riter (see human practices page), and that a significant FimH dose might be necessary to truly compete with UPEC, we aimed to enhance the effectiveness of our solution by incorporating additional mechanisms to attack UPEC. However, concerns arose regarding the metabolic strain on our bacteria, as FimH is a relatively large protein, composed of 279 amino acids, that would need to be produced constitutively to combat UPEC effectively [2]. This led us to the following idea.

Idea 3: Secreting Only the Active Domain of FimH

In response to these concerns, we explored the possibility of secreting only the active domain of FimH (FimHL), consisting of the N-terminal mannoside-binding lectin domain [2], [3]. Research demonstrated that FimHL alone exhibited ligand-binding affinity about two orders of magnitude higher than that of full-length FimH, due to reduced ligand-release tendency allowing for longer ligand binding [3]. This adaptation could provide enhanced protection against UPEC.

Considering these finding we believe that this option, of secreting only a part of FimH, would allow for our solution to be both safe and effective.

Finally, we were able to settle on our overall design: a strong constitutive promoter, a secretion peptide, and FimHL.

Bacillus subtilis part design

FimH has many alleles, with slight variations between them, we chose to take the sequence of FimH30 from E. coli ST131 because it is a potent version of the adhesin and it is a common variant in the literature [5], [6].

We took the genetic sequence for FimH from the FimTyper database corresponding to amino acids 1-163, since those compose the FimHL domain [3], [5], [6].For the remaining components, we chose known and tested parts from the iGEM registry, as detailed on our parts page.

L. crispatus part design

As seen in figure 2, our design composed of three elements:

To learn more about how our design was executed in the lab please see our results page.