Coupling gold nanoparticles to siderophores increases specificity. Siderophores hijack bacterial iron uptake pathways, allowing for targeted entry into bacteria—particularly Gram-negative pathogens—while minimizing accidental uptake by healthy human cells.

Gallium is indeed much closer in molecular weight and structure to iron than gold is, but we reject the idea of working with gallium for a few reasons. First of all, gallium-siderophore complexes are already in development by both academic researchers [13] and iGEM teams [14]. Also, since gallium disrupts cellular processes such as DNA and protein synthesis, energy production and mitochondrial function, bacteria could still have the possibility of developing resistance before they die [13]. We want them to be as unaware as possible they’ve been hacked before they die.

The general process of siderophores’ internalization has been annotated and characterized in both Gram-negative and Gram-positive bacteria [4]. In Gram-negative species, such as Klebsiella pneumoniae, this process involves multiple steps to transport the siderophores from the extracellular environment, across the periplasm, and into the cytoplasm..

First, since siderophores exceed the molecular weight of 600 Daltons, they cannot cross the outer membrane by passive diffusion through its trimeric β-barrel proteins termed “porins”. They therefore must involve an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In normal conditions, Fe³⁺ is cleaved off of siderophores by an esterase or a reductase in the cytoplasm. As the siderophore has only a low affinity for Fe²⁺, it is either degraded or recycled back to the extracellular space to scavenge for new Fe³⁺ ions [4].

Whether these mechanisms are modified or halted due to the addition of a polyethylene glycol and an AuNP remains unknown.

For more details, check out our Modelling page!

This is a valid concern. While we have not observed this in our research and found no reports of it in the literature, we acknowledge the possibility. To reduce this risk, we plan to functionalize the complex with polyethylene glycol (PEG), a strategy known to improve biocompatibility and reduce immune recognition. However, this plan remains theoretical as polyethylene glycol may generate osmotic stress to the TonB receptor, impairing the necessary conformational change for siderophore uptake [15]. For more details, see our Implementation page.

It's true that Gram-positive bacteria have a less complex cell envelope, potentially making them more accessible. However, compared to Gram-negative bacteria, less is known about iron uptake in Gram-positive bacteria, making them a less suitable target for our purposes as it would have been harder to design something that is actually taken up by the bacteria [13].

Besides, Gram-negative bacteria—such as K. pneumoniae-pose a greater clinical challenge due to their multiple resistance mechanisms and impermeable outer membrane. Our strategy leverages siderophores to exploit Gram-negative iron transport systems, offering a novel way to bypass their defenses. This targeted approach is especially valuable for tackling drug-resistant Gram-negative infections, which are a major public health threat.

Choosing a bacterium with the ability to recognize multiple siderophores increases the probability of it successfully uptaking the aerobactin-gold nanoparticle conjugate because its receptors are less specific to one particular molecular conformation. Aerobactin also accounts for more than 90% of the total siderophores produced by hypervirulent K. pneumoniae (hvKP), a strain with the capacity to induce devastating sequelae such as meningitis and endophthalmitis in the immunocompetent host, and that has already started building resistance to extended-spectrum β-lactamases (ESBLs) and carbapenemases [16]. Finally, aerobactin was chosen because it is part of the NRPS-independent siderophore (NIS) synthetase pathway [17]. This essentially means that it relies on smaller, simpler, and more modular enzymes with accessible substrates, unlike the giant, complex NRPS systems, making it easier to clone.

Yes, that’s one of the major limitations of our project. We don’t yet know whether the relevant reductases and esterases will be able to cleave our complex. If they cannot, a backlog could form, killing the bacteria. Alternatively, if the bacteria detect saturation, they might stop importing additional complexes. In that case, too few gold nanoparticles may accumulate inside the cell to ensure its destruction once the NIR laser is activated. The critical threshold for such a backlog is still unknown.

If it’s any consolation, Ha et al. tested a similar approach on P. aeruginosa (a Gram-negative bacterium) using a different siderophore–gold nanoparticle complex, and achieved a 96% kill rate without damaging adjacent healthy mouse skin tissue [8]. This suggests that, even without fully understanding the underlying mechanisms, the strategy can be effective against Gram-negative bacteria. However, it is important to note that 4% of the bacteria survived, raising the risk of eventual resistance, and that in the case of Gram-positive bacteria, the treatment actually worsened outcomes.