Attack

Background

In pursuit of a more targeted approach to combat uropathogenic Escherichia coli (UPEC) and enhance the efficacy of our solution, we directed our focus toward the intricate iron acquisition mechanisms employed by this pathogen. Metal ions serve as indispensable cofactors for approximately 40% of enzymes with well-characterized structures, and in many instances, these metal cofactors play a direct role in catalyzing redox reactions [1]. Given the great importance of metal ions, bacteria, including UPEC, have evolved diverse strategies to secure iron from their surroundings, particularly in the context of pathogenic infections, where they scavenge iron from the host [1].

One such mechanism involves the synthesis and transport of siderophores, small chelator molecules with a high affinity for iron. Bacteria secrete these siderophores into their surroundings, and subsequently, upon binding iron, they are actively transported back into the bacterial cell via dedicated transporter proteins [1]. It is noteworthy that the urinary tract typically represents a low-iron environment. When UPEC transitions from its gut habitat to the urinary tract, it undergoes a metabolic shift towards aerobic respiration, intensifying its demand for metal ions [1].

UPEC, along with other pathogenic microorganisms, possesses the capability to produce siderophores with iron-binding affinities surpassing those of host proteins [1].

General design

Idea 1: Inhibition of Siderophore Entry to Disrupt UPEC Iron Acquisition

As previously explained, siderophores permeate bacterial cells via specialized transporters and our objective was to obstruct this process. Notably, UPEC, being a gram-negative bacterium, possesses both an outer membrane and an inner membrane. To access the cytoplasm, molecules must bypass both of these barriers [2].

The utilization of transporters necessitates energy, typically derived from the proton motive force generated by the charge differential across the two sides of the membrane. However, maintaining a charge differential across the outer membrane poses a challenge for gram-negative bacteria [2]. Within the inner membrane, the protein TonB, in conjunction with ExbB and ExbD, constitutes a complex capable of energizing the outer membrane to activate embedded transporters [2]. These transporters are commonly referred to as TonB-dependent transporters (TBDTs). Initially, we considered inhibiting the TonB system; however, we quickly recognized that this approach was overly broad for our objectives. The TonB system is ubiquitous among gram-negative bacteria, encompassing not only pathogens but also non-pathogenic species. Furthermore, it serves as a means for bacteria to acquire a diverse array of nutrients, including carbohydrates and vitamins, rather than exhibiting specificity for iron or siderophores [2], [3].

Idea 2: Inhibiting Siderophore Entry to Disrupt UPEC Iron Acquisition

The human body employs different mechanisms to restrict iron accessibility to pathogens, among which is the action of Lipocalin-2 (LCN2), also known as Neutrophil Gelatinase-Associated Lipocalin (NGAL) [4]. LCN2 can effectively sequester siderophores, forming complexes that are incapable of entering bacterial cells, thereby rendering iron inaccessible to UPEC [4], [5].

However, this approach was ultimately dismissed due to the intricate challenges associated with achieving post-translational modifications of NGAL, a glycoprotein, in bacterial systems [5]. Moreover, it should be noted that NGAL exhibits affinity not only for siderophores but also for various other molecules, such as steroids and vitamins [5].

Idea 3: "Trojan Horse" Strategy - Engineering a Synthetic Siderophore Conjugated with an Antimicrobial Peptide to Target UPEC

An intriguing concept we explored was the development of a "Trojan horse" strategy, producing a siderophore fused with an antimicrobial peptide. The rationale behind this approach was to exploit the inherent systems of UPEC, whereby the bacteria would unwittingly facilitate their own demise. By producing a siderophore-antimicrobial peptide chimera, we aimed to specifically target bacteria reliant on siderophores for iron acquisition.

Regrettably, this approach was ultimately deemed infeasible due to the intricate nature of siderophore biosynthesis, which entails a complex and multifaceted pathway governed by several genes [6].

As such, the practical challenges associated with engineering an artificial siderophore system, coupled with the need for precise regulation of antimicrobial peptide activity, led to the abandonment of this particular strategy.

Idea 4: Targeting UPEC Directly with Antimicrobial Peptides

With the intention of directly combating UPEC, we delved into the realm of antimicrobial peptides (AMPs), a class of peptides, from varied natural or synthetic sources, characterized by their potent antimicrobial properties. It's worth noting that while the discovery of over 3,000 AMPs has occurred, a mere seven have received FDA approval [7]. Many AMPs fail prior to or during clinical trials, which indicates that many of them are not safe for humans [7].

Recognizing the difficulty in finding the ideal AMP for our application, we wanted to enhance safety by using an inducible promoter system that is controlled by the concentrations of iron. It means that in the lack of iron the bacteriocin, Colicin D, which is specific to E.coli, should be expressed and attack the UPEC, and in the presence of high concentrations of iron it should be repressed.

Due to unavoidable circumstances and the constraints imposed by the Home Front Command, as well as time limitations associated with iGEM, the consideration of using GFP instead of Colicin D as a proof of concept was delayed, with the intention to begin work after the wiki freeze.

The system was based on Evry 2013 iGEM project [8].

Part design

Antimicrobial peptide - Colicin D

Colicins are a group of bacteriocins produced by E. coli to eliminate their rival microorganisms. Among these colicins, Colicin D is a sizable protein with a molecular weight of 75 kDa, it is not harmful to humans, comprising three distinct regions: a translocation domain, a receptor-binding domain, and a cytotoxic domain. Notably, the cytotoxic domain is responsible for the specific cleavage of the anticodon loop in all four tRNA^Arg isoacceptors. This action disrupts the vital process of protein synthesis, ultimately causing cellular demise [9].