Bacterial virulence is closely associated with nutrient acquisition, which is essential for growth and proliferation of pathogens. Metal ions constitute essential nutrients, and the regulation of bacterial metal ion homeostasis within the host environment plays a pivotal role; however, unbound essential metal ions exhibit low bioavailability. For instance, the low solubility of Fe(OH)3 (Ksp = 6.3 x 10-38) at pH 7.4 would result in an insufficient quantity of iron for bacteria to grow, thus bacteria rely on targeting the hosts’ labile iron reserves through synthesis of endogenous, hydrophilic metallophores that are internalized using ATP-dependent bacterial transmembrane shuttles. These metallophores also retain affinity for non-essential xenometal ions with identical charge, comparable ionic radius and chemical hardness to the essential metal ion. For instance, trivalent metal ions with similar ionic radius to high spin Fe3+ (0.78 Å), such as Ga3+ (0.76 Å), Sc3+ (0.87 Å) and In3+ (0.93 Å) are transported to the bacterial peri- and cytoplasm when coordinated by bacterial iron-metallophores such as enterobactin or desferioxamine. These xenometals cannot be utilized for desired biological functions; recent strategies to utilize bacterial metal homeostasis pathways to deliver therapeutics has resulted in renewed interest in xenometals as alternative antibiotics. In bacteria, iron’s cytoplasmic fate and influence on gene and protein regulation is well-understood; however, xenometal homeostasis and utilization, especially in light of differential pH-dependent speciation behavior, remains rudimentary. To this end, we seek to investigate the following questions: (1) Are M3+-metallophore complexes efficiently recognized and transported across bacterial membranes? Size, hardness and Lewis acidity of metal ions influence their coordination complex structure. Substantial divergence from the parent Fe3+ complex results in diminished transport efficiency. We will study xenometal complex speciation under physiological conditions and employ a photoreactive tagging strategy to identify transmembrane shuttle protein interaction. (2) (How) Does M3+ release from metallophores proceed in absence of accessible redox events? Fe3+ is released by reduction to Fe2+ and enzymatic degradation of the metallophore induced by Fe2+-dependent proteins. The xenometals of interest, Ga3+, Sc3+ and In3+, do not have accessible redox events under physiological conditions. We will employ a radiochemical labeling strategy to track their metallophore-mediated uptake and identify metabolites. (3) What is the fate of M3+ xenometals in the cytoplasm and their influence on protein expression? The fate of non-redox active xenometals, once they reach the bacterial cytoplasm, including their effect on the bacterial protein expression is not well understood but hold the key to their growth inhibitory activity. We will combine radiochemical tagging strategies with mass spectrometry isolate and identify xenometal-target proteins. We will assess and quantitate the change in bacterial metabolites following exposure to different xenometal- metallophore complexes, which will inform on altered bacterial metabolism.

Bacterial virulence is closely associated with nutrient acquisition, which is essential for growth and proliferation of pathogens; metal ions constitute essential nutrients, and thus the regulation of bacterial metal ion homeostasis within the host environment plays a pivotal role for survival. Non-essential xenometal ions with identical charge, comparable ionic radius and chemical hardness to essential metal ions can take advantage of and critically disrupt essential metal homeostasis in bacteria by to-date unknown mechanisms. Here, we seek to uncover how the high spin Fe3+ mimics Ga3+, Sc3+, and In3+ are transported to the bacterial cytoplasm and interact with the bacterial proteo- and metabolome.