Project Description

The problem

Urinary Tract Infections (UTIs) are a prevalent clinical condition characterized by an imbalance in the microbiome [3]. In 2019, there were approximately 404.61 million UTI cases worldwide, leading to 236,790 deaths in both women and men [4]. The annual economic burden in the United States alone is estimated to be between $340 and $450 million [4].

These infections underscore a marked gender disparity, as premenopausal women are 20-40 times more susceptible to UTIs compared to men of the same age ​[3]​. Notably, there are distinct variations in the composition of bladder microbiota between men and women. The specific components of the microbiota in each gender can significantly influence the likelihood of UTI occurrence, with deviations from healthy women's or men's microbiome components potentially elevating the risk for UTIs ​[5].

Hypotheses based on anatomical differences, such as the shorter distance between the anus and the urethral opening in females and the longer urethra in males, lack strong experimental support [5] . Nevertheless, it's important to note that male infants under 6 months of age are about twice as likely to have an initial UTI compared to female infants [5].

UTIs are a significant health concern, with about 60% of women experiencing at least one UTI in their lifetime [6]. The highest incidence occurs between the ages of 18 and 39, typically due to increased sexual activity [6]. Recurrent UTIs are common, with about 20-30% experiencing a confirmed recurrence within six months after the initial infection [6].

UTI pathology

UTIs occur when pathogenic bacteria cause an infection in the urinary system [7]. The infection starts in the ureter and can reach all the way to the kidneys in severe cases that require more extensive medical intervention and even hospitalization [8]. The infection may cause physical pain and discomfort, affecting the daily lives of humans globally. Those afflicted with UTI require frequent visits to the bathroom, interrupting sleeping, working, and engaging in social activities, leading to loneliness or depression [9]. Despite the embarrassment that might arise from discussing this issue, we believe it is important to discuss it with sensitivity to women’s needs [2].

The bacteria uropathogenic E. coli (UPEC) is the most common cause of bacterial UTIs [7]. These pathogenic bacteria arrive from the natural microbiome of the gut [7] . They reach the ureter and then climb up the urinary tract (step 1) [10]. When they arrive at the bladder, they strongly adhere to the epithelial cells by specialized structures known as fimbriae (step 2) [10]. Among these, the dominant default adherence fimbria is type 1 fimbria, which is equipped with a binding protein called 'FimH' located at its tip [11].

Urothelial cells in the bladder have a protective feature called uroplakin plaques [11]. These plaques are composed of uroplakin membrane proteins that contain the sugar mannose [11]. The bacteria, UPEC, sticks to the mannose using its FimH protein [11]. When these bacteria enter the bladder, FimH binds to the mannose on the uroplakin and other receptors, which triggers a signaling process that helps the bacteria invade the bladder (step 3) [11].

After the invasion, fimH adhesin protein allows UPEC to proliferates and creates a dense biofilm (step 5), leading to the infection[10].

In response to UTIs, the primary treatment approach for UTIs, as advised by urologists we've consulted , predominantly involves the use of antibiotics. In some cases, antibacterial medications like Hyprex or hormonal treatments administered vaginally with estrogen may be considered.

When it comes to recurrent UTIs, a common course of action involves the continuous administration of low-dose antibiotics, which can span over several months. The extensive use of antibiotics for UTIs in the United States, estimated to be around 15% of all prescribed antibiotics, raises concerns about antibiotic resistance among urinary tract pathogens [12]. This in turn complicates the management of UTIs, emphasizing the pressing need for the development of novel antimicrobial agents to combat pathogenic E. coli UTIs [1].

The current treatment of antibiotics faces significant ineffectiveness in combating urinary tract infections (UTIs) due to two primary reasons: intracellular UPEC and biofilm formation [1].

Biofilm formation presents a formidable challenge within infectious diseases, where UPEC's ability to create biofilms contributes to recurrent UTIs [1]. These biofilm structures are resistant to antibiotics, and, paradoxically, subinhibitory antibiotic concentrations may even enhance their formation [1].

In addition to the biofilm challenge, the treatment's effectiveness is hindered by UPEC's capacity to invade epithelial cells, making it hard for antibiotics to reach them. This adds complexity to the treatment process [13].

In brief, the existing approach to treating UTIs, particularly recurrent cases, proves largely ineffective and may lead to long-term issues such as antibiotic resistance. This highlights the necessity of addressing this issue and providing an effective and viable treatment for UTIs [1],[13].

Through in-depth research on this subject, we have uncovered the significant role played by the urinary tract's microbiome in preserving a balanced bacterial environment and preventing the establishment of pathogenic bacteria within the tract. A crucial factor enabling the pathogenic E. coli to take hold in the tract initially is an imbalance in the microbiome.

Furthermore, some naturally occurring bacteria in the female microbiome, including Lactobacillus crispatus (L. crispatus), possess inherent mechanisms to reduce the presence of pathogenic E. coli that have already infiltrated the epithelial cells [14].

Exploring L. crispatus in Microbiome Dynamics of UTIs

The female bladder microbiome plays a crucial role in the occurrence of UTIs [1],[14]. Among the various microorganisms residing in the urinary tract, Lactobacillus crispatus, a gram-positive bacterium, is a natural member of the urinary tract microbiota [15]. Evidence suggests that women harboring higher levels of L. crispatus in their microbiota exhibit reduced susceptibility to recurrent UTIs compared to those with lower L. crispatus abundance [1],[14]. This evidence indicates that L. crispatus emerges as a pivotal player in defense against recurrent UTIs [1],[14].

This remarkable bacterium employs several defense mechanisms to safeguard against UTIs [14]. One of L. crispatus 's unique mechanisms is its ability to combat intracellular UPEC. It accomplishes this by acidifying vesicles that house UPEC, thereby preventing the pathogen from escaping into the cell's cytosol [14].

One notable challenge in working with L. crispatus is its infrequent use in research, resulting in limited understanding and available resources. Additionally, this bacterium has a relatively long doubling time of about 110 minutes, which can slow down experimentation and research progress[16].

Our Solution

Contrary to a long-held clinical belief, it is now understood that the urine of healthy individuals is not sterile, and this revelation has expanded the scope of therapeutic tools rooted in biosynthetic biology [17].

Our project's objective is to develop an innovative biosynthesis tool to achieve an effective, long-term solution for rUTIs and prevent the emergence of antibiotic resistance.

Acknowledging L. crispatus's inherent strengths, we recognize that its wild-type version alone cannot effectively prevent recurrent UTIs [18]. To address this, we are enhancing it through synthetic biology, as elaborated in the Design pages: Compete, Attack, Protect and Integrate. Our aim is to engineer L. crispatus, a natural bacterium in a healthy bladder microbiome, to compete with UPEC and disrupt its adhesion to epithelial cells, a pivotal step in UTI pathology [14].

We intend to add antimicrobial peptides, targeting specifically UPEC without harming the rest of the bacteria in the microbiome.

To tackle this challenge, we developed tools for efficient gene insertion into L. crispatus, emphasizing safety by incorporating arabinose as a kill-switch inducer.

Our solution has the potential to not only revolutionize UTI treatment but also pave the way for future novel microbiome engineering applications using L. crispatus as a new chassis.

Due to the challenges that can arise when using L. crispatus, we decided to work simultaneously with a simpler gram-positive bacterium, Bacillus subtilis, as a model organism to test our systems [19]. This allows us to take advantage of its more favorable characteristics for experiments.

This can be a valuable biosynthesis tool for addressing other conditions and diseases linked to the female microbiome, including those such as Bacterial Vaginosis and Endometriosis [20] ,[21].