Mobilization of Iron Stored in Bacterioferritin Is Required for Metabolic Homeostasis in Pseudomonas aeruginosa

Abstract

Iron homeostasis offers a significant bacterial vulnerability because pathogens obtain essential iron from their mammalian hosts, but host-defenses maintain vanishingly low levels of free iron. Although pathogens have evolved mechanisms to procure host-iron, these depend on well-regulated iron homeostasis. To disrupt iron homeostasis, our work has targeted iron mobilization from the iron storage protein bacterioferritin (BfrB) by blocking a required interaction with its cognate ferredoxin partner (Bfd). The blockade of the BfrB–Bfd complex by deletion of the bfd gene (Δbfd) causes iron to irreversibly accumulate in BfrB. In this study we used mass spectrometry and NMR spectroscopy to compare the proteomic response and the levels of key intracellular metabolites between wild type (wt) and isogenic ΔbfdP. aeruginosa strains. We find that the irreversible accumulation of unusable iron in BfrB leads to acute intracellular iron limitation, even if the culture media is iron-sufficient. Importantly, the iron limitation and concomitant iron metabolism dysregulation trigger a cascade of events that lead to broader metabolic homeostasis disruption, which includes sulfur limitation, phenazine-mediated oxidative stress, suboptimal amino acid synthesis and altered carbon metabolism.

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity

“…The dpsL gene (PA4880) is expressed under iron-replete conditions and is regulated by the small regulatory RNAs PrrF1 and PrrF2 ( Wilderman et al, 2004 ). In agreement, proteomic analysis of the P. aeruginosa iron starvation response imposed by limiting iron in the media ( Nelson et al, 2019 ), or by irreversibly trapping iron in bacterioferritin ( Punchi Hewage et al, 2020 ), results in low abundance of Pa DpsL. The transcriptional response of dpsL to environmental iron levels is also in agreement with the structure of Pa DpsL, which harbors ferroxidase centers to catalyze the oxidation of Fe 2+ and an interior cavity that can compartmentalize Fe 3+ .…”

“…The ftnA and bfrB genes are scattered in the P. aeruginosa genome and are regulated differently: bfrB transcription is stimulated by iron ( Palma et al, 2003 ), whereas the ftnA gene is under control of the Anr transcription regulator, which functions to enable the adaptation of P. aeruginosa cells to microoxic and anoxic growth ( Hammond et al, 2015 ). Mobilization of Fe 3+ stored in Bfr, which is required for metabolic homeostasis in P. aeruginosa ( Punchi Hewage et al, 2020 ), requires binding of a ferredoxin (Bfd), which transfers electrons to the Fe 3+ mineral in the Bfr interior cavity for subsequent mobilization of Fe 2+ to the cytosol ( Weeratunga et al, 2009 ; Yao et al, 2012 ). Blocking the Bfr-Bfd complex in a P. aeruginosa Δ bfd mutant leads to an irreversible accumulation of iron in Bfr ( Eshelman et al, 2017 ), and concomitant intracellular iron limitation, dysregulated carbon and sulfur metabolism, depleted amino acid biosynthesis ( Punchi Hewage et al, 2020 ), and inability to mature and maintain biofilms ( Soldano et al, 2020 ).…”

“…Mobilization of Fe 3+ stored in Bfr, which is required for metabolic homeostasis in P. aeruginosa ( Punchi Hewage et al, 2020 ), requires binding of a ferredoxin (Bfd), which transfers electrons to the Fe 3+ mineral in the Bfr interior cavity for subsequent mobilization of Fe 2+ to the cytosol ( Weeratunga et al, 2009 ; Yao et al, 2012 ). Blocking the Bfr-Bfd complex in a P. aeruginosa Δ bfd mutant leads to an irreversible accumulation of iron in Bfr ( Eshelman et al, 2017 ), and concomitant intracellular iron limitation, dysregulated carbon and sulfur metabolism, depleted amino acid biosynthesis ( Punchi Hewage et al, 2020 ), and inability to mature and maintain biofilms ( Soldano et al, 2020 ). The significant consequences of blocking the Bfr-Bfd complex on P. aeruginosa fitness motivated the discovery of small molecule inhibitors of the BfrB-Bfd complex ( Punchi Hewage et al, 2019 ).…”

Text-Mining to Identify Gene Sets Involved in Biocorrosion by Sulfate-Reducing Bacteria: A Semi-Automated Workflow

“…They postulated that sufficient iron is needed for the development of biofilm in Pseudomonas aeruginosa , and blocking the bacterioferritin–ferredoxin complex can lead to irreversible accumulation of unusable iron in Bfr , leading to acute cytosolic iron limitation. Therefore, deficiency of cytosolic iron can be observed in the planktonic and biofilm-embedded cells when the Bfr-Bfd complex is blocked, which results in poor biofilm development even in culture conditions with adequate iron [ 86 , 87 ]. Moreover, Qi et al suggested that gene DVU2571 encoding for ferrous iron transport protein B (feoB) is required under high iron concentrations to maintain the integrity of D. vulgaris biofilm [ 88 ].…”

Shared and host-specific transcriptomic response of Pseudomonas donghuensis P482 to the exudates of tomato (Dicot) and maize (Monocot) shed light on the host-adaptive traits in the promiscuous root colonizing bacteria

“…While the expression of fecR genes for the detection of xenosiderophores and the heme acquisition genes was downregulated as part of the shared response of P482 to exudates, the expression of bfr gene encoding bacterioferritin was upregulated (1.87 and 2.67 log 2 FC for tomato and maize, respectively). Bacterioferritin is a cytosolic iron storage protein shown to have a crucial role in maintaining iron homeostasis in Pseudomonas aeruginosa (48, 49).…”