Genetic and Chemical Intervention of the BfrB:Bfd Interaction Dysregulate Iron Homeostasis in Pseudomonas aeruginosa and Affect its Broader Metabolism

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is accountable for multiple types of infections, including pneumonia, wound, burn, and urinary tract infections. P. aeruginosa is an emerging threat in the hospital environments and preferentially found in comorbid illnesses. Current treatments for the P. aeruginosa infections recommend the use of combination therapy, a β-lactam with an aminoglycoside or a fluoroquinolone. Also, resistance to such procedures is rapidly emerging. Moreover, P. aeruginosa has been given critical priority for anti-infective development in the 2017 World Health Organization (WHO) report on prioritizing pathogens to guide the discovery of new antibiotics. Iron metabolism in bacteria has gained much more attention as a potential target for antibiotic development. Regulation of iron homeostasis is crucial for cells to have enough iron for growth while avoiding iron-induced toxicity. Iron homeostasis involves iron uptake, storage, and mobilization. Two iron storage molecules co-exist in P. aeruginosa, FtnA, and BfrB. Previous in-vitro studies established the importance of the bacterioferritin associated ferredoxin (Bfd) in iron mobilization from BfrB. The X-ray crystallographic structure and biochemical characterization of the BfrB:Bfd complex identified the hot spot residues of the interaction. The residues E81, E85, and L68 in BfrB, and M1, Y2, and L5 in Bfd majorly contribute to the binding energy. Mutating E81 and L68 to alanine completely inhibited iron mobilization from BfrB. The insights gathered from these in-vitro studies were used in this work to interrogate the importance of the BfrB:Bfd interaction in P. aeruginosa cells. In this work, P. aeruginosa, wild type (PAO1), Δbfd, bfrB(E81A/L68A), and ΔbfrB cells were used to study the significances of the BfrB:Bfd interaction in P. aeruginosa cells. A Native-PAGE method was optimized to image iron in BfrB in P. aeruginosa lysates. This advanced technique allowed us to identify BfrB as the main iron storage protein of P. aeruginosa. The same method was used to image iron storage in BfrB and its subsequent mobilization in P. aeruginosa cells (wild type, Δbfd, and bfrB(E81A/L68A)). The intact BfrB:Bfd interaction capable wild type cells showed maximum accumulation of iron during the early stationary phase, and iron mobilization from BfrB during the late stationary phase. In contrast, the Δbfd and bfrB(E81A/L68A) mutants demonstrated irreversible accumulation of iron in BfrB. Due to the irreversible flux of iron into BfrB, the Δbfd and bfrB(E81A/L68A) mutants developed low cytosolic free iron levels and a high total iron to free iron ratio. The consequences of the genetic blockade of the BfrB:Bfd interaction was further investigated by comparing the expression proteomes of the wild type and Δbfd mutant cells. The iron homeostasis dysregulation affected the iron-dependent and independent metabolic processes in the Δbfd mutant cells. Proteins involved in the TCA cycle, amino acid biosynthesis, oxidative stress regulation, and respiratory chain were under-represented in the Δbfd mutant cells. On the other hand, proteins involved in the iron acquisition systems (pyoverdine, pyochelin, Heme iron acquisition), sulfur assimilation, quorum sensing were over-represented in the Δbfd mutant cells. These findings show that iron homeostasis dysregulation can affect the broader metabolism of P. aeruginosa cells. The chemical intervention of the BfrB:Bfd interaction in P.aeruginosa cells was carried out with small molecule inhibitors, analogs of 4-aminoisoindoline-1,3-dione. These analogs acted on their target, BfrB in P. aeruginosa cells. Moreover, the analogs elicited a concentration-dependent growth retardation, and a pyoverdine hyper-production as a result of cytosolic iron deprivation. Analog-treated cells experienced an irreversible accumulation of iron in BfrB and high total iron content in the treated cells. Hence, the developed 4-aminoisoindoline-1,3-dione derivatives were potent to dysregulate iron homeostasis in P. aeruginosa cells. The BfrB:Bfd inhibitors also potentiated the activity of fluoroquinolones in P. aeruginosa (PA01), and in two cystic fibrosis isolates, MR3B and MR60. The growth impairment of the cystic fibrosis and urinary tract clinical isolates of P. aeruginosa, and Acinetobacter baumannii (AB5075) demonstrate the potential widespread application of the developed 4-aminoisoindoline-1,3-dione derivatives in blocking the BfrB:Bfd interaction. These findings strongly support the suitability of inhibiting the BfrB:Bfd interaction as a novel target in anti-infective development.