Inhibiting Iron Mobilization from Bacterioferritin in <i>Pseudomonas aeruginosa</i> Impairs Biofilm Formation Irrespective of Environmental Iron Availability

Although iron is essential for bacteria, the nutrient presents problems of toxicity and solubility. Bacteria circumvent these problems with the aid of iron storage proteins where Fe3+ is deposited and, when necessary, mobilized as Fe2+ for metabolic requirements. In Pseudomonas aeruginosa, Fe3+ is compartmentalized in bacterioferritin (BfrB), and its mobilization as Fe2+ requires specific binding of a ferredoxin (Bfd) to reduce the stored Fe3+. Blocking the BfrB-Bfd complex leads to irreversible iron accumulation in BfrB and cytosolic iron deprivation. Consequently, given the intracellular iron sufficiency requirement for biofilm development, we hypothesized that blocking the BfrB-Bfd interaction in P. aeruginosa would impair biofilm development. Our results show that planktonic and biofilm-embedded cells where the BfrB-Bfd complex is blocked exhibit cytosolic iron deficiency, and poorly developed biofilms, even in iron-sufficient culture conditions. These results underscore inhibition of the BfrB-Bfd complex as a rational target to dysregulate iron homeostasis and possibly control biofilms

Optimization of Host Cell-Compatible, Antimicrobial Peptides Effective against Biofilms and Clinical Isolates of Drug-Resistant Bacteria

Here, we describe the continued synthetic molecular evolution of a lineage of host-compatible antimicrobial peptides (AMP) intended for the treatment of wounds infected with drug-resistant, biofilm-forming bacteria. The peptides tested are variants of an evolved AMP called d-amino acid CONsensus with Glycine Absent (d-CONGA), which has excellent antimicrobial activities in vitro and in vivo. In this newest generation of rational d-CONGA variants, we tested multiple sequence–structure–function hypotheses that had not been tested in previous generations. Many of the peptide variants have lower antibacterial activity against Gram-positive or Gram-negative pathogens, especially variants that have altered hydrophobicity, secondary structure potential, or spatial distribution of charged and hydrophobic residues. Thus, d-CONGA is generally well tuned for antimicrobial activity. However, we identified a variant, d-CONGA-Q7, with a polar glutamine inserted into the middle of the sequence, that has higher activity against both planktonic and biofilm-forming bacteria as well as lower cytotoxicity against human fibroblasts. Against clinical isolates of Klebsiella pneumoniae, innate resistance to d-CONGA was surprisingly common despite a lack of inducible resistance in Pseudomonas aeruginosa reported previously. Yet, these same isolates were susceptible to d-CONGA-Q7. d-CONGA-Q7 is much less vulnerable to AMP resistance in Gram-negative bacteria than its predecessor. Consistent with the spirit of synthetic molecular evolution, d-CONGA-Q7 achieved a critical gain-of-function and has a significantly better activity profile