How the Presence of Hemin Affects the Expression of the Different Iron Uptake Pathways in Pseudomonas aeruginosa Cells

Abstract

Iron is an essential nutriment for almost all organisms, but this metal is poorly bioavailable.During infection, bacteria access iron from the host by importing either iron or heme.Pseudomonas aeruginosa, a Gram-negative pathogen, secretes two siderophores, pyoverdine (PVD) and pyochelin (PCH), to access iron and is also able to use many siderophores produced by other microorganisms (called xenosiderophores). To access heme, P. aeruginosa uses three distinct uptake pathways, named Has, Phu, and Hxu. We previously showed that P. aeruginosa expresses the Has and Phu heme uptake systems, as well as the PVD-and PCH-dependent ironuptake pathways, in iron-restricted growth conditions, using proteomic and RT-qPCR approaches. Here, using the same approaches, we show that physiological concentrations of hemin in the bacterial growth medium result in the repression of the expression of the proteins of the PVD-and PCH-dependent iron-uptake pathways, leading to less production of these two siderophores. This indicates that the pathogen adapts its phenotype to use hemin as an iron source rather than produce PVD and PCH to access iron. Moreover, the presence of both hemin and a xenosiderophore resulted in (i) the strong induction of the expression of the proteins of the added xenosiderophore-uptake pathway, (ii) repression of the PVD-and PCH-dependent iron-uptake pathways, and (iii) no effect on the expression levels of the Has, Phu, or Hxu systems, indicating that bacteria use both xenosiderophores and heme to access iron.

Profiling the Heme‐Binding Proteomes of Bacteria Using Chemical Proteomics

“…Ferri-siderophore transporters, such as FoxA, FptA, FpvA and FpvB have been previously reported to be downregulated in the presence of exogenous heme, enabling adaptation to prioritise iron acquisition through heme rather than iron uptake pathways. [60] Accord-ingly, in this work, these transporters were found to be significantly downregulated by treatment with either heme or heme-based photoaffinity probe 4 (Figure 2e). Furthermore, upregulation of catalase KatA expression and increased catalase activity were both observed with heme and heme probe treatment (Figure 2b,f).…”

“…However, TonB heme transporters HasR, HxuA and PhuR are downregulated in the presence of heme at 5 μM (higher than the typical availability of heme in eukaryotic cells, estimated to be between 0.025 and 0.3 μM [64] ), but only HasR and HxuA were concomitantly downregulated by photoaffinity probe 4 . Ferri‐siderophore transporters, such as FoxA, FptA, FpvA and FpvB have been previously reported to be downregulated in the presence of exogenous heme, enabling adaptation to prioritise iron acquisition through heme rather than iron uptake pathways [60] . Accordingly, in this work, these transporters were found to be significantly downregulated by treatment with either heme or heme‐based photoaffinity probe 4 (Figure 2e).…”

“…Heme proteome profiling was also investigated using photoaffinity probe 4 under different conditions, such as during the exponential phase, after 4 h nutrient starvation, and after growth solely in chemically defined minimal media (Figure 4c), as growth stage and nutrient availability are known to influence protein expression, [60,63,[72][73][74] and could therefore enable the identification of heme-binding proteins which were previously undetectable. Across the suite of six probes and seven different labelling conditions, a total of 57 different PAO1 heme-binding proteins were identified (Figure 4f).…”

Untersuchung des Häm‐bindenden bakteriellen Proteoms mittels chemischer Proteomik

“…Als Nächstes wurde die Eignung der Sonden für Experimente in Pseudomonas aeruginosa analysiert, einem pathogenen gramnegativen Bakterium mit antibiotikaresistenten Stämmen, die dazu geführt haben, dass es von der WHO (Weltgesundheitsorganisation) als prioritäres Pathogen eingestuft wurde [59] . P. aeruginosa kann Häm direkt aufnehmen über das sekretierte Hämophor HasAp [17, 60, 61] . Die klinische Bedeutung des Verständnisses der Häm‐Aufnahme wurde durch eine Studie an Mukoviszidose‐Patienten unterstrichen, die zeigte, dass Mutationen, die sich in P. aeruginosa während der Infektion des Wirts entwickelten, die die Aufnahme von externem Häm als einzige Eisenquelle ermöglichten, Wachstumsvorteile verschafften und zu einer chronischen Infektion beitrugen [62] .…”

“…Die TonB‐Häm‐Transporter HasR, HxuA und PhuR werden jedoch in Gegenwart von 5 μM Häm herunterreguliert (höher als die typische Verfügbarkeit von Häm in eukaryotischen Zellen, die auf 0.025 bis 0.3 μM geschätzt wird [64] ), jedoch lediglich HasR und HxuA wurden auch durch die Photoaffinitätssonde 4 herunterreguliert. Von Ferri‐Siderophor‐Transportern wie FoxA, FptA, FpvA und FpvB wurde bereits berichtet, dass sie in Gegenwart von exogenem Häm herunterreguliert werden, was die Anpassung an den Eisenerwerb durch Häm gegenüber der Eisenaufnahme ermöglicht [60] . Dementsprechend wurde in dieser Arbeit festgestellt, dass diese Transporter entweder durch die Behandlung mit Häm oder mit der auf Häm basierenden Photoaffinitätssonde 4 signifikant herunterreguliert werden (Abbildung 2e).…”