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• W3109184006 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. 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. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent nosocomial pathogens that can cause deleterious infections in part due to the emergence of multidrug-resistant strains (1MacDougall C. Harpe S.E. Powell J.P. Johnson C.K. Edmond M.B. Polk R.E. Pseudomonas aeruginosa, Staphylococcus aureus, and fluoroquiniolone use.Emerg. Infect. Dis. 2005; 11: 1197-1210Crossref PubMed Scopus (59) Google Scholar). These pathogens frequently colonize the same sites, including the urinary tract, burn and surgical wounds, and the upper respiratory tract. In patients with the hereditary lung disease cystic fibrosis (CF), P. aeruginosa and S. aureus are the most prevalent bacterial pathogens that colonize the lung, and their co-colonization is associated with poor patient outcomes (2Ahlgren H.G. Benedetti A. Landry J.S. Bernier J. Matouk E. Radzioch D. Lands L.C. Rousseau S. Nguyen D. Clinical outcomes associated with Staphylococcus aureus and Pseudomonas aeruginosa airway infections in adult cystic fibrosis patients.BMC Pulm. Med. 2015; 15: 67Crossref PubMed Scopus (37) Google Scholar). The virulence of these two pathogens in the CF lung and severity of CF lung disease are closely linked to iron homeostasis (3Gifford A.H. Miller S.D. Jackson B.P. Hampton T.H. O'Toole G.A. Stanton B.A. Parker H.W. Iron and CF-related anemia: expanding clinical and biochemical relationships.Pediatr. Pulmonol. 2011; 46: 160-165Crossref PubMed Scopus (26) Google Scholar). In particular, the anti-staphylococcal activity of P. aeruginosa has been shown to be highly dependent on iron availability (4Nguyen A.T. Jones J.W. Ruge M.A. Kane M.A. Oglesby-Sherrouse A.G. Iron depletion enhances production of antimicrobials by Pseudomonas aeruginosa.J. Bacteriol. 2015; 197: 2265-2275Crossref PubMed Scopus (45) Google Scholar). Moreover, as an essential nutrient, iron also plays critical roles in the survival of these organisms and regulates the production of several virulence factors that alter their interactions with the host (5Hammer N.D. Skaar E.P. Molecular mechanisms of Staphylococcus aureus iron acquisition.Annu. Rev. Microbiol. 2011; 65: 129-147Crossref PubMed Scopus (199) Google Scholar, 6Reinhart A.A. Oglesby-Sherrouse A.G. Regulation of Pseudomonas aeruginosa virulence by distinct iron sources.Genes. 2016; 7: 126Crossref PubMed Scopus (23) Google Scholar, 7Minandri F. Imperi F. Frangipani E. Bonchi C. Visaggio D. Facchini M. Pasquali P. Bragonzi A. Visca P. Role of iron uptake systems in Pseudomonas aeruginosa virulence and airway infection.Infect. Immun. 2016; 84: 2324-2335Crossref PubMed Scopus (98) Google Scholar). To support their iron requirements during infection, both P. aeruginosa and S. aureus use several mechanisms to scavenge different forms of iron in the host environment. P. aeruginosa has several iron-uptake strategies to acquire iron from the host, including siderophore-mediated ferric [Fe(III)] uptake, ferrous [Fe(II)] uptake, and heme uptake. P. aeruginosa produces two siderophores, pyoverdine and pyochelin, to obtain Fe(III) in the host (8Budzikiewicz H. Siderophores of the human pathogenic fluorescent pseudomonads.Curr. Top. Med. Chem. 2001; 1: 1-6Crossref PubMed Scopus (22) Google Scholar). Fe(II) can be prevalent in low-oxygen or reducing environments, and P. aeruginosa uses the Feo Fe(II) iron-uptake system to acquire iron in these environments (9Lau C.K. Krewulak K.D. Vogel H.J. Bacterial ferrous iron transport: the Feo system.FEMS Microbiol. Rev. 2015; 40: 273-298Crossref PubMed Scopus (111) Google Scholar). In addition, a class of redox-cycling molecules called phenazines can facilitate Fe(II) uptake by reducing extracellular Fe(III) to Fe(II), which allows for more efficient Fe(II) uptake by the Feo system (10Wang Y. Wilks J.C. Danhorn T. Ramos I. Croal L. Newman D.K. Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron acquisition.J. Bacteriol. 2011; 193: 3606-3617Crossref PubMed Scopus (142) Google Scholar). To acquire heme, P. aeruginosa uses a hemophore-dependent heme assimilation system (Has) and a non-hemophore-dependent heme uptake system (Pseudomonas heme uptake [Phu]) (11Ochsner U.A. Johnson Z. Vasil M.L. Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa.Microbiology. 2000; 146: 185-198Crossref PubMed Scopus (222) Google Scholar). The Has system uses the secreted hemophore HasAp to capture extracellular heme, which is then taken up by an outer membrane TonB-dependent receptor, whereas the Phu system uses a TonB-dependent receptor to directly sequester heme (12Huang W. Wilks A. Extracellular heme uptake and the challenge of bacterial cell membranes.Annu. Rev. Biochem. 2017; 86: 799-823Crossref PubMed Scopus (47) Google Scholar). Prior work examining CF lung sputum samples suggested that P. aeruginosa switches from using siderophores to depending more heavily on heme-acquisition and Fe(II)-acquisition strategies as infection progresses (13Konings A.F. Martin L.W. Sharples K.J. Roddam L.F. Latham R. Reid D.W. Lamont I.L. Pseudomonas aeruginosa uses multiple pathways to acquire iron during chronic infection in cystic fibrosis lungs.Infect. Immun. 2013; 81: 2697-2704Crossref PubMed Scopus (73) Google Scholar, 14Nguyen A.T. O'Neill M.J. Watts A.M. Robson C.L. Lamont I.L. Wilks A. Oglesby-Sherrouse A.G. Adaptation of iron homeostasis pathways by a Pseudomonas aeruginosa pyoverdine mutant in the cystic fibrosis lung.J. Bacteriol. 2014; 196: 2265-2276Crossref PubMed Scopus (71) Google Scholar). Two iron-acquisition strategies that are important for S. aureus and have been extensively characterized are siderophore-mediated Fe(III) uptake and heme uptake (5Hammer N.D. Skaar E.P. Molecular mechanisms of Staphylococcus aureus iron acquisition.Annu. Rev. Microbiol. 2011; 65: 129-147Crossref PubMed Scopus (199) Google Scholar). S. aureus produces the siderophores staphyloferrin A and B, which allow the bacterium to compete for Fe(III) with host proteins lactoferrin and transferrin (15Park R.-Y. Sun H.-Y. Choi M.-H. Bai Y.-H. Shin S.-H. Staphylococcus aureus siderophore-mediated iron-acquisition system plays a dominant and essential role in the utilization of transferrin-bound iron.J. Microbiol. 2005; 43: 183-190PubMed Google Scholar). Although these siderophores provide an important iron-acquisition strategy for S. aureus, studies have shown that S. aureus prefers heme, which accounts for ∼80% of the iron in the host, as an iron source (5Hammer N.D. Skaar E.P. Molecular mechanisms of Staphylococcus aureus iron acquisition.Annu. Rev. Microbiol. 2011; 65: 129-147Crossref PubMed Scopus (199) Google Scholar, 16Skaar E.P. Humayun M. Bae T. DeBord K.L. Schneewind O. Iron-source preference of Staphylococcus aureus infections.Science. 2004; 305: 1626-1628Crossref PubMed Scopus (297) Google Scholar). S. aureus is able to access heme by secreting hemolysins, which lyse erythrocytes to release hemoglobin (5Hammer N.D. Skaar E.P. Molecular mechanisms of Staphylococcus aureus iron acquisition.Annu. Rev. Microbiol. 2011; 65: 129-147Crossref PubMed Scopus (199) Google Scholar). Heme-uptake systems, including the iron-regulated surface determinants (Isd) transport system, subsequently import free heme into the bacterium (5Hammer N.D. Skaar E.P. Molecular mechanisms of Staphylococcus aureus iron acquisition.Annu. Rev. Microbiol. 2011; 65: 129-147Crossref PubMed Scopus (199) Google Scholar). Prior work has shown that S. aureus preferentially uses heme and switches to using siderophores only when heme is less available (16Skaar E.P. Humayun M. Bae T. DeBord K.L. Schneewind O. Iron-source preference of Staphylococcus aureus infections.Science. 2004; 305: 1626-1628Crossref PubMed Scopus (297) Google Scholar). The importance of Fe(II) uptake for S. aureus is not understood, but a putative metal uptake system in S. aureus that has homology to other bacterial Feo transporters has been identified and shown to be upregulated in iron-deficient growth conditions (17Ster C. Beaudoin F. Diarra M.S. Jacques M. Malouin F. Lacasse P. Evaluation of some Staphylococcus aureus iron-regulated proteins as vaccine targets.Vet. Immunol. Immunopathol. 2010; 136: 311-318Crossref PubMed Scopus (19) Google Scholar). In response to bacterial infection, the host uses iron-sequestering host-defense proteins, which are capable of limiting Fe(III), Fe(II), and heme at infection sites. These proteins include lactoferrin, lipocalin-2, haptoglobin, hemopexin, and calprotectin (CP) and serve to starve invading bacterial pathogens of iron as part of the metal-withholding innate immune response (12Huang W. Wilks A. Extracellular heme uptake and the challenge of bacterial cell membranes.Annu. Rev. Biochem. 2017; 86: 799-823Crossref PubMed Scopus (47) Google Scholar, 18Cassat J.E. Skaar E.P. Iron in infection and immunity.Cell Host Microbe. 2013; 13: 509-519Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 19Becker K.W. Skaar E.P. Metal limitation and toxicity at the interface between host and pathogen.FEMS Microbiol. Rev. 2014; 38: 1235-1249Crossref PubMed Scopus (123) Google Scholar, 20Nakashige T.G. Zhang B. Krebs C. Nolan E.M. Human calprotectin is an iron-sequestering host-defense protein.Nat. Chem. Biol. 2015; 11: 765-771Crossref PubMed Scopus (129) Google Scholar, 21Zygiel E.M. Nolan E.M. Exploring iron withholding by the innate immune protein human calprotectin.Acc. Chem. Res. 2019; 52: 2301-2308Crossref PubMed Scopus (7) Google Scholar). CP (S100A8/S100A9 hetero-oligomer, MRP9/MRP14 oligomer) is a host-defense protein that sequesters divalent first-row transition metal ions, including Mn(II), Fe(II), Ni(II), and Zn(II) (20Nakashige T.G. Zhang B. Krebs C. Nolan E.M. Human calprotectin is an iron-sequestering host-defense protein.Nat. Chem. Biol. 2015; 11: 765-771Crossref PubMed Scopus (129) Google Scholar, 21Zygiel E.M. Nolan E.M. Exploring iron withholding by the innate immune protein human calprotectin.Acc. Chem. Res. 2019; 52: 2301-2308Crossref PubMed Scopus (7) Google Scholar, 22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 23Zygiel E.M. Nolan E.M. Transition metal sequestration by the host-defense protein calprotectin.Annu. Rev. Biochem. 2018; 87: 621-643Crossref PubMed Scopus (53) Google Scholar). CP is released from white blood cells at infection sites, where it is capable of competing with microbial pathogens for nutrient metal ions (23Zygiel E.M. Nolan E.M. Transition metal sequestration by the host-defense protein calprotectin.Annu. Rev. Biochem. 2018; 87: 621-643Crossref PubMed Scopus (53) Google Scholar). Prior work revealed a broad-spectrum antimicrobial activity of CP, which is attributed to its ability to chelate multiple nutrient metal ions and thereby prevent microbial acquisition of these nutrients (20Nakashige T.G. Zhang B. Krebs C. Nolan E.M. Human calprotectin is an iron-sequestering host-defense protein.Nat. Chem. Biol. 2015; 11: 765-771Crossref PubMed Scopus (129) Google Scholar, 23Zygiel E.M. Nolan E.M. Transition metal sequestration by the host-defense protein calprotectin.Annu. Rev. Biochem. 2018; 87: 621-643Crossref PubMed Scopus (53) Google Scholar, 24Corbin B.D. Seeley E.H. Raab A. Feldmann J. Miller M.R. Torres V.J. Anderson K.L. Dattilo B.M. Dunman P.M. Gerads R. Caprioli R.M. Nacken W. Chazin W.J. Skaar E.P. Metal chelation and inhibition of bacterial growth in tissue abscesses.Science. 2008; 319: 962-965Crossref PubMed Scopus (577) Google Scholar, 25Brophy M.B. Hayden J.A. Nolan E.M. Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin.J. Am. Chem. Soc. 2012; 134: 18089-18100Crossref PubMed Scopus (100) Google Scholar). From the standpoint of iron in infection and immunity, CP is the only known Fe(II)-sequestering host-defense protein (20Nakashige T.G. Zhang B. Krebs C. Nolan E.M. Human calprotectin is an iron-sequestering host-defense protein.Nat. Chem. Biol. 2015; 11: 765-771Crossref PubMed Scopus (129) Google Scholar, 21Zygiel E.M. Nolan E.M. Exploring iron withholding by the innate immune protein human calprotectin.Acc. Chem. Res. 2019; 52: 2301-2308Crossref PubMed Scopus (7) Google Scholar, 22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In the current work, we evaluate how the presence of heme affects responses of P. aeruginosa and S. aureus to Fe(II) withholding by CP. This topic is of significant interest because CP colocalizes with P. aeruginosa and S. aureus in the CF lung and its levels correlate with disease severity (26Wakeman C.A. Moore J.L. Noto M.J. Zhang Y. Singleton M.D. Prentice B.M. Gilston B.A. Doster R.S. Gaddy J.A. Chazin W.J. Caprioli R.M. Skaar E.P. The innate immune protein calprotectin promotes Pseudomonas aeruginosa and Staphylococcus aureus interaction.Nat. Commun. 2016; 7: 11951Crossref PubMed Scopus (58) Google Scholar, 27Lyczak J.B. Cannon C.L. Pier G.B. Lung infections associated with cystic fibrosis.Clin. Microbiol. Rev. 2002; 15: 194-222Crossref PubMed Scopus (1106) Google Scholar). Importantly, this study represents a step toward addressing the chemical complexity of infection sites by evaluating how a biologically relevant iron source affects bacterial responses to Fe(II) sequestration by CP. We report that P. aeruginosa upregulates expression of heme-uptake machinery in response to CP. Furthermore, we demonstrate that heme alleviates the inhibition of P. aeruginosa iron uptake and iron-starvation responses that CP causes (Fig. 1). We also examine the effect of CP on S. aureus iron homeostasis, providing the first comprehensive evaluation of whether CP induces iron starvation in this bacterial pathogen. We report that eight S. aureus strains, including CF clinical isolates, exhibit decreased iron uptake after CP treatment. Moreover, our results uncover that CP induces iron-starvation responses in S. aureus. Similarly to our observations for P. aeruginosa, heme protects S. aureus from both inhibition of iron uptake and iron starvation by CP. Taken together, these findings reveal the importance of heme for adaptation of P. aeruginosa and S. aureus to Fe(II) withholding by CP. P. aeruginosa uses heme uptake machinery during infection, and in some cases, preferentially utilizes heme over other iron sources (14Nguyen A.T. O'Neill M.J. Watts A.M. Robson C.L. Lamont I.L. Wilks A. Oglesby-Sherrouse A.G. Adaptation of iron homeostasis pathways by a Pseudomonas aeruginosa pyoverdine mutant in the cystic fibrosis lung.J. Bacteriol. 2014; 196: 2265-2276Crossref PubMed Scopus (71) Google Scholar, 28Marvig R.L. Damkiær S. Khademi S.M.H. Markussen T.M. Molin S. Jelsbak L. Within-host evolution of Pseudomonas aeruginosa reveals adaptation toward iron acquisition from hemoglobin.mBio. 2014; 5e00966-14Crossref PubMed Scopus (89) Google Scholar). Because CP can sequester Fe(II) and thereby lower the availability of non-heme iron, we questioned whether P. aeruginosa resists CP by upregulating expression of heme-acquisition machinery to obtain heme iron. We analyzed the effect of CP on transcriptional changes in hasA and hasR, two components of the Has heme uptake system, and phuS, a component of the Phu heme uptake system. Expression of hasA, hasR, and phuS was increased in cultures grown in the presence of CP (Fig. 2), indicating that P. aeruginosa upregulates heme-acquisition machinery in response to CP. Based on this observation, we reasoned that P. aeruginosa may adapt to CP by utilizing heme to rescue cellular iron levels. We analyzed cellular metal inventory by inductively coupled plasma–mass spectrometry (ICP–MS) to determine the effect of heme supplementation on iron uptake during growth of P. aeruginosa in the absence or presence of CP. These studies were performed with CP and/or CP-Ser, which is a CP variant with two Cys→Ser point mutations [S100A8(C42S)/S100A9(C3S)]. CP-Ser was originally used in coordination chemistry studies to avoid potential complications of the two Cys residues (25Brophy M.B. Hayden J.A. Nolan E.M. Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin.J. Am. Chem. Soc. 2012; 134: 18089-18100Crossref PubMed Scopus (100) Google Scholar). It has been commonly used in metal-binding and antimicrobial studies of CP and displays comparable activity to CP (25Brophy M.B. Hayden J.A. Nolan E.M. Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin.J. Am. Chem. Soc. 2012; 134: 18089-18100Crossref PubMed Scopus (100) Google Scholar). P. aeruginosa PAO1 and PA14 cultures were grown in a chemically-defined medium (CDM) with or without a 5 μM heme supplement. We chose to perform these studies using CDM, which contains ∼300 nM manganese, ∼5 μM iron, and ∼6 μM zinc because it is a defined medium that lacks heme (29Nakashige T.G. Zygiel E.M. Drennan C.L. Nolan E.M. Nickel sequestration by the host-defense protein human calprotectin.J. Am. Chem. Soc. 2017; 139: 8828-8836Crossref PubMed Scopus (52) Google Scholar). We also performed select experiments with Tris:tryptic soy broth (TSB), which contains ∼150 nM manganese, ∼4 μM iron, and ∼5 μM zinc (Table S1). This undefined medium contains low levels of heme but serves as a point of comparison based on its use in prior studies of CP and P. aeruginosa (22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Regardless of the medium composition, we observed a decrease in cell-associated iron content for cultures of both PAO1 and PA14 that were grown in the presence of CP, in agreement with our prior work (Fig. 3, A–C) (22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In contrast, for cultures grown in the presence of both heme and CP, we observed iron levels comparable with the untreated cultures and the cultures treated with only heme (Fig. 3, A–C). These data suggest that when heme is available, P. aeruginosa will use it as an iron source and thereby resist the consequences of Fe(II) withholding by CP (Fig. 3, A–C). In addition, when we used a heme-uptake knockout (KO) strain of P. aeruginosa PAO1 (ΔhasRphuR) in this experiment, we found that the presence of heme did not affect inhibition of iron uptake by CP when heme could not be used (Fig. 3D). Cell-associated manganese for PA14 grown in Tris:TSB was also reduced by CP, consistent with our prior results for PAO1 in this medium (Fig. S3A) (22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). For PAO1 and PA14 grown in CDM, CP did not reduce manganese uptake, indicating that medium conditions may affect manganese withholding by CP (Figs. S1A and S2A). We observed negligible changes in cell-associated nickel, copper, and zinc in PAO1 and PA14 from CP treatment, consistent with our prior study (Figs S1, C–E, S2, C–E, and S3, C–E) (22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The presence of heme did not alter the effect of CP on cell-associated manganese, nickel, copper, or zinc in either CDM or Tris–TSB (Figs. S1, A and C–E, S2, A and C–E, and S3, A and C–E). We next questioned how CP affects cellular iron uptake if heme is the only iron source available to P. aeruginosa. We grew P. aeruginosa PAO1 in CDM prepared without iron salt and supplemented with 5 μM heme in the absence or presence of CP and measured cell-associated iron levels. When heme was the only iron source, CP treatment afforded an increase in iron uptake relative to the heme-only condition (Fig. 3A). We reason that the increase in iron uptake by P. aeruginosa when grown with CP and heme is attributable to the enhanced expression of heme uptake machinery caused by CP (Fig. 2). To probe whether heme uptake accounted for the observed increase in cell-associated iron in cultures treated with heme and CP, we used the stable 57Fe isotope to prepare CDM, allowing us to simultaneously track the uptake of heme iron (56Fe; 91.75% natural abundance) and non-heme iron (57Fe; 95% isotopic purity) using ICP–MS. For cultures treated with heme and CP, we observed a significantly higher 56Fe:57Fe ratio (∼2.7) than cultures treated with heme alone (∼1.0) (Fig. 3E). Thus, when CP is present in culture, P. aeruginosa prioritizes heme as a source of iron. Building upon our current observations, we hypothesized that heme utilization protects P. aeruginosa from undergoing CP-induced iron-starvation responses. We therefore examined select markers of iron-starvation responses in P. aeruginosa cultures treated with CP and/or heme. P. aeruginosa upregulates production of the siderophore pyoverdine in response to CP (22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We analyzed pyoverdine levels in culture supernatants grown in the absence or presence of CP-Ser and heme and found that in comparison with the cultures treated with CP-Ser where pyoverdine was observed in culture supernatants, cultures treated with CP-Ser and heme had negligible pyoverdine levels (Fig. 4A). These data indicate that heme prevents CP from inducing a siderophore-mediated iron-starvation response. P. aeruginosa also downregulates expression of antR, which encodes an activator of an anthranilate catabolic pathway that is regulated by iron via ferric uptake regulator (Fur)-controlled PrrF sRNAs, in response to CP (22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). AntR directly controls transcription of iron-containing proteins; thus, when iron availability is low, PrrF sRNAs inhibit antR translation as part of an iron-sparing response (30Oglesby A.G. Farrow 3rd, J.M. Lee J.-H. Tomaras A.P. Greenberg E.P. Pesci E.C. Vasil M.L. The influence of iron on Pseudomonas aeruginosa physiology: a regulatory link between iron and quorum sensing.J. Biol. Chem. 2008; 283: 15558-15567Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). We evaluated activity of an antR translational reporter in P. aeruginosa PAO1 (PAO1/PantR-‘lacZ-SD) grown in the absence or presence of CP and heme (31Djapgne L. Panja S. Brewer L.K. Gans J.H. Kane M.A. Woodson S.A. Oglesby-Sherrouse A.G. The Pseudomonas aeruginosa PrrF1 and PrrF2 small regulatory RNAs promote 2-alkyl-4-quinolone production through redundant regulation of the antR mRNA.J. Bacteriol. 2018; 200e00704-17Crossref PubMed Scopus (19) Google Scholar). We observed lower levels of antR reporter activity in cultures grown in the presence of CP than in its absence, in agreement with our prior work (22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In cultures grown in the presence of heme and CP, the observed levels of antR reporter activity were comparable with those of the untreated and heme-treated cultures and significantly higher than those of CP-treated cultures (Fig. 4B). Thus, the presence of heme prevents CP from inducing a PrrF-regulated iron-starvation response. The redox-cycling function of phenazines serves a number of purposes for P. aeruginosa biology, including promotion of Fe(II) uptake by the Feo system (10Wang Y. Wilks J.C. Danhorn T. Ramos I. Croal L. Newman D.K. Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron acquisition.J. Bacteriol. 2011; 193: 3606-3617Crossref PubMed Scopus (142) Google Scholar). We previously found that both CP treatment and iron-deficient conditions inhibit phenazine production by P. aeruginosa (22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Based on our current results, we questioned whether heme utilization would recover phenazine production by P. aeruginosa in the presence CP. We quantified the concentrations of two phenazines, phenazine-1-carboxylate (PCA) and pyocyanin (PYO), in supernatants of P. aeruginosa PA14 cultures grown in the absence or presence of heme and CP-Ser (Fig. 5). In agreement with our prior data (22Zygiel E.M. Nelson C.A. Brewer L.K. Oglesby-Sherrouse A.G. Nolan E.M. The innate immune protein human calprotectin induces iron starvation responses in Pseudomonas aeruginosa.J. Biol. Chem. 2019; 294: 3549-3562Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), we observed a significant decrease in levels of both PCA and PYO for cultures treated with CP-Ser (Fig. 5, A and B). For cultures grown in the presence of heme, CP-Ser caused a similar decrease in phenazine levels. This result differs from the heme-mediated recovery of siderophore- and PrrF-controlled iron-starvation responses caused by CP. It is possible that CP affects distinct regulatory systems that inhibit phenazine production even when other iron-regulated responses are recovered by heme. Indeed, phenazine production can be mod" @default.
• W3109184006 created "2020-12-07" @default.
• W3109184006 creator A5009719245 @default.
• W3109184006 creator A5048710700 @default.
• W3109184006 creator A5050876141 @default.
• W3109184006 creator A5066305106 @default.
• W3109184006 creator A5077804116 @default.
• W3109184006 date "2021-01-01" @default.
• W3109184006 modified "2023-10-05" @default.
• W3109184006 title "Heme protects Pseudomonas aeruginosa and Staphylococcus aureus from calprotectin-induced iron starvation" @default.
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