Heme protects Pseudomonas aeruginosa and Staphylococcus aureus from calprotectin-induced iron starvation

Abstract

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacterial pathogens that cause severe infections in immunocompromised individuals and patients with cystic fibrosis. Both P. aeruginosa and S. aureus require iron to infect the mammalian host. To obtain iron, these pathogens may rely on siderophore-mediated ferric iron uptake, ferrous iron uptake, or heme uptake at different points during infection. The preferred iron source depends on environmental conditions, including the presence of iron-sequestering host-defense proteins. Here, we investigate how the presence of heme, a highly relevant iron source during infection, affects bacterial responses to iron withholding by the innate immune protein calprotectin (CP). Prior work has shown that P. aeruginosa is starved of iron in the presence of CP. We report that P. aeruginosa upregulates expression of heme uptake machinery in response to CP. Furthermore, we show that heme protects P. aeruginosa from CP-mediated inhibition of iron uptake and iron-starvation responses. We extend our study to a second bacterial pathogen, S. aureus , and demonstrate that CP also inhibits iron uptake and induces iron-starvation responses by this pathogen. Similarly to P. aeruginosa , we show that heme protects S. aureus from CP-mediated inhibition of iron uptake and iron-starvation responses. These findings expand our understanding of microbial responses to iron sequestration by CP and highlight the importance of heme utilization for bacterial adaptation to host iron-withholding strategies.

The Human Innate Immune Protein Calprotectin Elicits a Multimetal Starvation Response in Pseudomonas aeruginosa

“…1C ; Table S1) following CP treatment showed significantly more interactions than if they were a random collection of genes from the genome, indicating a biological relationship between the proteins in each analysis (Table S2). In agreement with recent studies showing that CP induces an Fe starvation response in P. aeruginosa ( 10 , 45 ), proteins involved in pyoverdine-mediated Fe(III) uptake, in the Feo Fe(II)-import system, and in heme-dependent Fe acquisition were upregulated in response to CP, whereas proteins involved in phenazine biosynthesis (PhzE, PhzD, PhzC, PhzM, and PhzH) and secretion (MexH and MexI) were downregulated (Fig. S1).…”

“…S3). Despite the robust Zn starvation response to CP observed here and in previous work ( 38 ), only negligible changes in cell-associated Zn levels were observed for PAO1 and PA14 grown in metal-replete CDM supplemented with 10 μM CP ( 10 , 45 ).…”

“…We selected CDM for these studies because it allows for control over the metal concentrations and sources (e.g., nonheme versus heme Fe), and it is amino acid rich, similar to what is found in CF sputum ( 44 ). Prior whole-cell metal analyses in metal-replete CDM revealed that treatment of PA14 with CP results in a significant decrease in cell-associated Fe and negligible change in cell-associated Mn, Ni, Cu, and Zn ( 45 ). PA14 was grown for 8 h, which afforded growth to early stationary phase ( 10 ).…”

“…Although CP has the ability to withhold Mn, levels of known Mn cofactored proteins (HutG, GpmI, and UbiD) and predicted Mn uptake proteins (MntH1 and MntH2) in PA14 were not significantly affected by CP treatment. Prior work revealed that the ability of CP to reduce cell-associated Mn in P. aeruginosa is medium dependent ( 10 , 45 ). Whereas CP treatment reduced cell-associated Mn levels when PA14 and PAO1 were grown in a mixture of Tris buffer and tryptic soy both (Tris:TSB) ( 10 ), a negligible change in cell-associated Mn was observed when these strains were treated with CP in metal-replete CDM ( 45 ).…”

“…Prior work revealed that the ability of CP to reduce cell-associated Mn in P. aeruginosa is medium dependent ( 10 , 45 ). Whereas CP treatment reduced cell-associated Mn levels when PA14 and PAO1 were grown in a mixture of Tris buffer and tryptic soy both (Tris:TSB) ( 10 ), a negligible change in cell-associated Mn was observed when these strains were treated with CP in metal-replete CDM ( 45 ). This medium effect may explain the lack of a putative Mn starvation response in this proteomics analysis.…”