Mechanistic characterization and regulation of the non-redundant phu and has heme uptake systems of Pseudomonas aeruginosa

University Of Maryland Baltimore, Baltimore MD

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Abstract

Pathogenic bacteria require iron for their survival and ability to cause infection. The opportunistic pathogen Pseudomonas aeruginosa has multiple mechanisms by which it can acquire iron, including ferric and ferrous iron uptake systems. However, within the host P. aeruginosa adapts to utilize heme via the heme assimilation (has) and Pseudomonas heme utilization (phu) systems. We have shown through bacterial genetics and biochemical analysis that the Has and Phu systems are non-redundant, with the Has system being required for extracellular heme sensing and signaling and the Phu system the major heme transporter. Furthermore, we have shown the extracellular heme metabolite biliverdin (BV) IXβ post-transcriptionally regulates the heme sensing system at the level of HasAp translation. Phenotypic analysis of allelic strains encoding a HemO that exclusively yields BVIXα (hemOα) or an inactive protein (hemOin) revealed decreased Type IV pili (TFP) motilities (swarming and twitching) and biofilm formation. Comparative proteomics analysis revealed a significant repression of chemosensory pathways and TFP biogenesis. A reduction in TFP was visualized by transmission electron microscopy and quantified by Western blot analysis. Thermal proteome profiling further identified a transcriptional regulator BdrB (Biliverdin dependent regulator responsive to β) that on deletion gave a similar phenotype to the hemO allelic strains. Further analysis revealed direct regulation of the pilJKchpABCD operon by BdrB. We hypothesize that the unique Pa heme metabolite BVIXβ functions as signaling and regulatory molecule integrating the in-host transition to heme utilization as an iron source with the regulatory and chemosensory networks associated with the transition from a planktonic to sessile lifestyle associated with long-term chronic infection. We will test this through; i) elucidating the BVIX post-transcriptional regulatory elements controlling HasAp expression and heme sensing; ii) characterize BdrB and its role in the transition from planktonic to sessile lifestyle; and iii) determine the transcriptional elements regulating the bdrB operon (PA5269-71) and the function of the PA5269 and PA5270 proteins. These studies will advance our understanding of lifestyle adaptations associated with infection and aid in the development of therapeutics effective against both heme utilization and cooperative behaviors essential for survival and persistence within the host.