Iron metabolism in the opportunistic pathogen Pseudomonas aeruginosa requires two ferritin-like molecules, Pa FtnA and Pa BfrB

Abstract

FtnA is an iron storage protein in Pseudomonas aeruginosa which is capable of storing about 4500 Fe3+ ions in its inner core. According to earlier classifications of this protein Pseudomonas aeruginosa was proposed to have two bacterioferritins coded by two different genes bfrA and bfrB which coexists. Our research was focused on an idea that Pseudomonas aeruginosa have one bacterioferritin and one bacterial ferritin which acts as iron storage proteins instead of a heterogeneous bacterioferritin. To strengthen our hypothesis we first cloned bfrA gene in Pseudomonas aeruginosa and purified Pa FtnA to homogeneity. Crystallization studies were conducted to solve the structure of Pa FtnA protein which reveals that Pa FtnA is composed of 24 identical subunits coordinates together to form a spherical hollow sphere which helps in storage of iron in its inner cavity. Experiments performed in crystallo suggested that iron ions are oxidized at ferroxidase center and taken in to the inner cavity. The most interesting detail found by crystal structure was presence of a third iron site near ferroxidase center at different pH and buffer conditions which is similar to "Site C" in many known bacterial ferritins. Even experiment performed in vitro also showed Fe2+ is oxidized before storing in the inner cavity. Previous observation made by sequence alignment of Pa FtnA with Pa BfrB and other known bacterioferritins showed absence of M52 residue in Pa FtnA which also shows that Pa FtnA is a bacterial ferritin not bacterioferritin as this binds to Heme in Pa BfrB which is a characteristic feature. Earlier studies suggested protein-protein interaction as a key step in releasing iron from iron storage protein BfrB in Pseudomonas aeruginosa. This indicates inhibition of this PPI with inhibitors should prevent the bacteria from releasing iron. In order to apply the developed inhibitor for P. aeruginosa to other pathogenic Gram-negative bacteria, first we need to understand the structure and functional similarity of these pathogenic bacteria in comparison to P. aeruginosa. This can be achieved by doing multiple sequence alignment. This helps us understand the possible structure and function that can form by selected bacteria in comparison to P.aeruginosa. If these bacteria contain similar type of residues at important iron regulation sites than the possible mechanism even in these selected Gram-negative bacteria will be similar to Pseudomonas aeruginosa. Hence we can apply same inhibitors to inhibit PPI even in these bacteria. Presence of these storage proteins is one mode of releasing iron during iron limiting conditions for bacteria to survive, other mode of action is by sequestering iron from host cells by releasing siderophores and hemophores. To study phenotype changes in biofilm formation in Pseudomonas aeruginosa under iron starvation conditions in presence of iron chelators, we choose transposon mutants which were purchased from University of Washington, Manoil lab. Experiments were performed using succinate minimal media in presence and absence of iron chelators with different mutants. Iron chelators such as BP (2,2' Bipyridine) and DTPA (diethylene triamine pentaacetic acid) were used for this study which showed, the mutant in presence of iron chelator have retarded growth whereas mutants and wild type in absence of iron chelators did not show any effects on its biofilm formation. These studies showed release of pyoverdine a type of siderophore by Pseudomonas aeruginosa and also showed release of pyocyanin a phenazine with DTPA. This concludes that initially bacteria tends to release pyoverdine for iron acquisition but when it is not getting enough iron for its growth and under high stress conditions it tends to release phenazine for iron acquisition and start showing a slow growth. We did observe planktonic difference but not phenotype changes with the medium used.