Key Points

• Gram-negative bacteria present a significant challenge for drug delivery due to their protective outer membrane.

• The Trojan Horse strategy overcomes this barrier by hijacking the bacterial iron uptake system, conjugating drugs to nutrient-like molecules to disguise them.

• Peptide antibiotics and other therapeutics can be linked to these carriers using click chemistry, enabling targeted delivery into the bacterial cell and reducing toxicity to non-target cells.

Introduction

Antibiotic resistance is one of the most pressing challenges in modern medicine. Both gram-negative and gram-positive pathogens have evolved mechanisms to evade conventional drugs, from almost impermeable cell envelopes and efflux pumps to enzymatic degradation and altered antibiotic targets. In particular, the outer membrane of gram-negative bacteria poses a formidable barrier that often prevents even potent molecules from reaching their site of action. Overcoming this barrier requires innovative strategies that do not rely solely on passive diffusion but instead hijack the bacteria’s own nutrient acquisition pathways.

The Bacterial Cell Envelope

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It is well established that gram-negative bacteria exhibit a high level of resistance to antimicrobial agents due to their complex cell envelope structure, which comprises an inner membrane, a thin peptidoglycan layer, and an outer membrane enriched with lipopolysaccharides (Fig. 1)^[1].

This additional barrier is hard to penetrate and acts as a selective filter that blocks many antibiotics. Therefore, even if a compound has the potential to disrupt bacterial cellular machinery, it is not guaranteed to be effective, since it must first reach the target site. Moreover, reliance on passive transport means that higher doses of the compound must be administered to reliably kill the bacteria, which in turn may increase toxicity for the patient.

This challenge is particularly relevant for the ESKAPE pathogens, a group of highly virulent and antibiotic-resistant bacteria classified by the WHO as a top priority for new therapeutic development. Among them, most are gram-negative, underscoring the challenge in developing effective treatments

Most ESKAPE pathogens are gram-negative, highlighting the urgent need to develop antibiotics capable of effectively targeting and eliminating these organisms (Tab. 1).

In contrast, gram-positive bacteria lack this outer membrane, which makes them generally more accessible to many drugs. However, they compensate with a much thicker peptidoglycan cell wall, often strengthened with teichoic and lipoteichoic acids, which provide structural rigidity and contribute to antibiotic resistance in their own way^[1].

For example, gram-positive pathogens such as Staphylococcus aureus can resist β-lactam antibiotics through the production of altered penicillin-binding proteins, while Enterococcus species employ both intrinsic and acquired mechanisms to withstand glycopeptides like vancomycin^[3][4].

Thus, while gram-negatives are considered especially challenging due to their permeability barrier, gram-positives also present significant obstacles, underscoring the need for strategies that can effectively target both bacterial groups.

The Trojan Horse Strategy

One ingenious strategy developed in recent years is to conjugate antibiotics with molecules that bacteria actively import because they are perceived as beneficial. These molecules can be sugars or siderophores which are compounds that capture iron^[5].

This strategy, known as the Trojan Horse approach, is named after the Greek tale in which soldiers concealed themselves inside a wooden horse to infiltrate Troy. Similarly, antibiotics are “hidden” by molecules like siderophores, the bacterium unknowingly takes up the conjugated antibiotic through its own transport systems, allowing the drug to bypass the bacteria´s protective shielding (Fig. 2)^[6].

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This conjugation is often achieved through click chemistry – a set of reactions that are highly selective, efficient, and rarely occurring in nature. In our project we chose siderophores as our Trojan Horse – an attractive bait for bacteria to take up.

Antibiotics alone have difficulty penetrating the cell envelope of gram-negative bacteria. In contrast, nutrient–antibiotic conjugates are actively transported by the bacterial uptake machinery into at least the periplasmic space, where some antibiotics already exert their activity. In rare cases, the conjugated antibiotic can also be transported further into the cytoplasm.

Bacterial Iron Deficiency & Siderophores

Iron (Fe) is crucial for bacterial growth and multiplication. When bacteria find themselves in an iron-deficient environment, they begin to produce siderophores – specialized iron-scavenging molecules^[7].

In nature, iron most commonly occurs in two oxidation states: Fe²⁺ (iron(II)) and Fe³⁺ (iron(III)). Fe²⁺ is more soluble and bioavailable but stable only under anaerobic conditions, while Fe³⁺ dominates in oxygen-rich environments but is poorly soluble at neutral pH, quickly forming Fe(OH)₃ or other precipitates^[8].

Because of this microbes cannot directly take up Fe³⁺ unless they use special strategies, like siderophore production. Siderophores act as chemical chelators with very high affinity for iron, also binding insoluble Fe³⁺ and converting it into a form that bacteria can import.

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The general mechanism of siderophore uptake proceeds as follows (Fig. 3):

1. Secretion – Bacteria release siderophores into the environment.

2. Chelation – The siderophore captures Fe³⁺ and forms a soluble Fe³⁺–siderophore complex.

3. Recognition – Specific receptors on the bacterial membrane recognize and bind the complex.

4. Transport – The complex is imported into the cell (via TonB-dependent transporters in gram-negative bacteria or ABC transporters in gram-positives).

5. Release – Inside the cell, Fe³⁺ is reduced to Fe²⁺ (the more soluble form) and released from the siderophore for metabolic use.

Siderophores are secreted under iron-deficient conditions and chelate Fe(III), which is otherwise inaccessible for uptake. The siderophore–iron complex is recognized by substrate-binding proteins and imported via ABC transporters. Once inside, reductases reduce Fe(III) to Fe(II), lowering the affinity of the siderophore for iron and leading to its release. Alternatively, some organisms employ esterases to cleave catecholate rings. In the final step, the siderophore is either reused or degraded.

Siderophore Types and their Chemical Features

Chemically, siderophores are diverse and can be classified into major types based on the functional groups that bind iron^[9].

In our project, we focused on catecholate siderophores, which contain catechol groups that coordinate Fe³⁺ through oxygen atoms. A well-known example is enterobactin from Escherichia coli, considered one of the strongest Fe³⁺ chelators (Fig. 4A).

Other major classes of siderophores include hydroxamates, such as ferrichrome, and carboxylates, such as aerobactin (Fig. 4B and 4C). In addition, some bacteria produce mixed-type siderophores that combine different chelating groups to further enhance iron-binding affinity. A notable example is pyoverdine from Pseudomonas aeruginosa, which also shows fluoresecence (Fig. 4D).

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The Iron Uptake of Pathogenic Bacteria

During infection, pathogenic bacteria compete with mammalian host cells for essential nutrients. Mammals defend themselves against bacteria by actively limiting iron availability in the extracellular space. By depriving bacteria of this essential nutrient the bacteria may die or fail to multiply.

As a counter strategy pathogenic bacteria evolved strategies such as producing additional siderophores or stealing those secreted by other microbes^[10][11].This makes siderophores especially suitable as “Trojan horses” for two reasons:

• Since many pathogens can utilize siderophores from different sources, conjugation to a single siderophore enables targeting of multiple bacterial species.

• Because bacteria are heavily dependent on siderophore-mediated iron uptake, the likelihood of resistance development is significantly reduced.

Together, these factors made us decide to focus on siderophore-mediated drug delivery for our peptide antibiotics as it is a powerful and selective strategy to combat pathogenic bacteria.

Engineering Functional Handles for Tailored Therapeutic Conjugates

When designing new drugs, it is often useful to create a built-in “handle” — a small chemical group that makes the molecule easier to connect with other components (Fig. 5). In drug delivery, such handles allow researchers to link an active compound, for example an antibiotic, to a delivery vehicle such as a siderophore. The principle is similar to adding a universal plug to a device: once the handle is present, a wide variety of chemical “cables” can be attached.

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In our work, we realized this handle by using a functional group compatible with the well-established click chemistry strategy.

Click Chemistry

To conjugate a potential antibiotic with a siderophore, the reaction must be simple, efficient, biocompatible, and highly selective, while avoiding unwanted side reactions in biological systems. Among available bioorthogonal strategies, click chemistry represents the ideal approach due to its robustness, precision, and compatibility with complex biological environments^[12].

In our iGEM project, we employed click chemistry as a key tool for the site-specific functionalization of our engineered test compound, Chaiaphumine, within our biosynthetic pipeline. This approach enabled the conjugation of Chaiaphumine to two distinct molecular tags designed for different applications, demonstrating the versatility and practical utility of this chemistry in peptide modification. One of these molecular tags is a siderophore, which was incorporated to implement a Trojan horse–based approach, enabling targeted uptake of the antibiotic by bacterial cells. The most widely used click reaction is the copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). In this reaction, an azide group reacts with a terminal alkyne to form a stable 1,2,3-triazole linkage, catalyzed by Cu(I) ions (Fig. 6).

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This reaction proceeds under mild conditions, yields minimal by-products, and offers exceptional chemoselectivity, making it ideally suited for biological and pharmaceutical applications^[12].

A key advantage of click chemistry is its broad applicability. It allows the covalent attachment of diverse functional molecules to NRPS-derived peptides. Beyond siderophores, potential conjugation partners include heme, sugars, nucleobases, vitamins, lipids, and fatty acids. Such modular coupling strategies enable targeted delivery of antimicrobial peptides and can be extended to therapeutic applications against cancer, fungal pathogens, parasites, and agricultural pests.

By integrating click chemistry into our biosynthetic workflow, we combined the specificity of enzymatic peptide synthesis with the versatility of chemical modification, creating a powerful platform for the rational design of multifunctional bioactive compounds.

NRPS Engineering & Trojan Horse Strategy

In our project, we aim to discover new antibiotics through NRPS Engineering. To enhance the efficacy of our peptide drugs—particularly against Gram-negative pathogens—we combine the Trojan horse strategy with click chemistry.

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By engineering NRPS, a canonical amino acid can be replaced with a non-proteinogenic or unnatural amino acid. If the substituted residue carries a distinctive chemical group, such as an azide, the resulting peptide contains a reactive site (Fig. 7). This site can be selectively modified in subsequent reactions, for example via click chemistry.

During the design, we evaluated whether to incorporate an azide or an alkyne moiety into the nonribosomal peptide. Given the documented success of incorporating azide-bearing amino acids into NRPS, we chose to incorporate the azide into the peptide while assigning the alkyne to the siderophore^[13].

Specifically in our project, we introduced the amino acid with the azide handle into the peptide by NRPS Engineering. We exchanged a part of the NRPS with the reported domains from the literature and successfully incorporated the handle into the peptide. This modification provides the peptide with the azide group required for conjugation with a nutrient of choice, effectively creating a “Trojan Horse” system for selective delivery.

For details on how we implemented this strategy, visit the Drug Delivery section in the Results page.

Challenges of the Trojan Horse Strategy

A potential concern with siderophore–antibiotic conjugates is that bacteria may develop resistance by modifying their uptake systems. However, bacteria typically possess multiple, siderophore uptake pathways, allowing them to import iron-bound siderophores through more than one mechanism. This redundancy ensures survival under iron limitation and reduces the chance that a single mutation blocks siderophore–antibiotic uptake. This makes siderophores robust delivery vectors that are less prone to rapid resistance development than conventional antibiotics.

Another challenge is that the size of some siderophores could make their conjugates too bulky for efficient import. We consulted an expert and literature on this topic.

Although some siderophores are large, size does not seem limiting. Literature and expert consultation show even bulky conjugates, like daptomycin–fimsbactin, are imported effectively^[14]. Given the relatively small size of our peptides, cellular import is anticipated to occur without significant limitations.

Conclusion

By integrating the inherent modularity of NRPS with the specificity of modern chemical methods, our approach facilitates the synthesis of tailored therapeutic conjugates in which the active drug is covalently linked to targeting or delivery moieties. This study demonstrates how the Trojan Horse strategy can be employed to develop effective interventions against hard-to-reach bacterial targets.

For details on NRP–siderophore conjugation, see the wet lab results page. To follow the DBTL iterations that led to our final construct, visit the engineering page.