Contributions of the heme coordinating ligands of the Pseudomonas aeruginosa outer membrane receptor HasR to extracellular heme sensing and transport
2020; Elsevier BV; Volume: 295; Issue: 30 Linguagem: Inglês
10.1074/jbc.ra120.014081
ISSN1083-351X
Autores Tópico(s)Vibrio bacteria research studies
ResumoPseudomonas aeruginosa exhibits a high requirement for iron, which it can acquire via several mechanisms, including the acquisition and utilization of heme. The P. aeruginosa genome encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonas heme utilization (Phu) system. Extracellular heme is sensed via the Has system, which encodes an extracytoplasmic function (ECF) σ factor system. Previous studies have shown that the transfer of heme from the extracellular hemophore HasAp to the outer membrane receptor HasR is required for activation of the σ factor HasI and upregulation of has operon expression. Here, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting, and 13C-heme uptake experiments, we delineated the differential contributions of the extracellular FRAP/PNPNL loop residue His-624 in HasR and of His-221 in its N-terminal plug domain required for heme capture to heme transport and signaling, respectively. Specifically, we show that substitution of the N-terminal plug His-221 disrupts both signaling and transport, leading to dysregulation of both the Has and Phu uptake systems. Our results are consistent with a model wherein heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His-624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Our results provide critical insight into heme release, signaling, and transport in P. aeruginosa and suggest a functional link between the ECF σ factor and Phu heme uptake system. Pseudomonas aeruginosa exhibits a high requirement for iron, which it can acquire via several mechanisms, including the acquisition and utilization of heme. The P. aeruginosa genome encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonas heme utilization (Phu) system. Extracellular heme is sensed via the Has system, which encodes an extracytoplasmic function (ECF) σ factor system. Previous studies have shown that the transfer of heme from the extracellular hemophore HasAp to the outer membrane receptor HasR is required for activation of the σ factor HasI and upregulation of has operon expression. Here, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting, and 13C-heme uptake experiments, we delineated the differential contributions of the extracellular FRAP/PNPNL loop residue His-624 in HasR and of His-221 in its N-terminal plug domain required for heme capture to heme transport and signaling, respectively. Specifically, we show that substitution of the N-terminal plug His-221 disrupts both signaling and transport, leading to dysregulation of both the Has and Phu uptake systems. Our results are consistent with a model wherein heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His-624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Our results provide critical insight into heme release, signaling, and transport in P. aeruginosa and suggest a functional link between the ECF σ factor and Phu heme uptake system. Iron is an essential micronutrient that is required for the survival and virulence of almost all bacterial pathogens. Iron is extremely limited in mammalian hosts because of sequestration by the innate immune system through upregulation of iron storage proteins, downregulation of ferritin, and the secretion of iron-chelating proteins such as lipocalin-2 (1Ganz T. Iron in innate immunity: starve the invaders.Curr. Opin. Immunol. 2009; 21 (19231148): 63-6710.1016/j.coi.2009.01.011Crossref PubMed Scopus (201) Google Scholar). During infection, bacterial pathogens, including Pseudomonas aeruginosa, circumvent iron deficiency by utilizing a variety of iron acquisition systems (2Sheldon J.R. Heinrichs D.E. Recent developments in understanding the iron acquisition strategies of gram positive pathogens.FEMS Microbiol. Rev. 2015; 39 (25862688): 592-63010.1093/femsre/fuv009Crossref PubMed Scopus (136) Google Scholar, 3Huang W. Wilks A. Extracellular heme uptake and the challenge of bacterial cell membranes.Annu. Rev. Biochem. 2017; 86 (28426241): 799-82310.1146/annurev-biochem-060815-014214Crossref PubMed Scopus (48) Google Scholar, 4Contreras H. Chim N. Credali A. Goulding C.W. Heme uptake in bacterial pathogens.Curr. Opin. Chem. Biol. 2014; 19 (24780277): 34-4110.1016/j.cbpa.2013.12.014Crossref PubMed Scopus (81) Google Scholar). In response to iron starvation, P. aeruginosa deploys two siderophore systems, pyoverdine (Pvd) and pyochelin (Pch) (5Heinrichs D.E. Young L. Poole K. Pyochelin-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein.Infect. Immun. 1991; 59 (1910015): 3680-368410.1128/IAI.59.10.3680-3684.1991Crossref PubMed Google Scholar, 6Royt P.W. Pyoverdine-mediated iron transport. Fate of iron and ligand in Pseudomonas aeruginosa.Biol. Met. 1990; 3 (2119198): 28-3310.1007/BF01141174Crossref PubMed Scopus (21) Google Scholar), as well as the ferrous uptake system (Feo) (7Hunter R.C. Asfour F. Dingemans J. Osuna B.L. Samad T. Malfroot A. Cornelis P. Newman D.K. Ferrous iron is a significant component of bioavailable iron in cystic fibrosis airways.mBio. 2013; 4 (23963183): e00513-e0055710.1128/mBio.00557-13Crossref Scopus (108) Google Scholar). P. aeruginosa also encodes two nonredundant heme acquisition systems, the Pseudomonas heme uptake (Phu) and heme assimilation (Has) systems, that utilize heme as an iron source (8Ochsner U.A. Johnson Z. Vasil M.L. Genetics and regulation of two distinct haem-uptake systems, phu has, in Pseudomonas aeruginosa.Microbiology. 2000; 146 (10658665): 185-19810.1099/00221287-146-1-185Crossref PubMed Scopus (222) Google Scholar, 9Smith A.D. Wilks A. Differential contributions of the outer membrane receptors PhuR and HasR to heme acquisition in Pseudomonas aeruginosa.J. Biol. Chem. 2015; 290 (25616666): 7756-776610.1074/jbc.M114.633495Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Therefore, bacteria must be able to adapt to utilize the most readily available iron source. One mechanism by which bacteria respond and adapt to extracellular nutrients is through cell surface signaling (CSS) systems, which are coupled to extracytoplasmic function (ECF) σ factors (10Helmann J.D. The extracytoplasmic function (ECF) sigma factors.Adv. Microb. Physiol. 2002; 46 (12073657): 47-11010.1016/S0065-2911(02)46002-XCrossref PubMed Scopus (518) Google Scholar, 11Mascher T. Signaling diversity and evolution of extracytoplasmic function (ECF) sigma factors.Curr. Opin. Microbiol. 2013; 16 (23466210): 148-15510.1016/j.mib.2013.02.001Crossref PubMed Scopus (98) Google Scholar). These alternative σ factors complex with the core RNA polymerase, direct binding to the promoter of a target gene, and activate transcription. P. aeruginosa encodes several iron-responsive ECF σ factors, including PvdS and FpvI, which are coupled to the pyoverdine uptake system (12Potvin E. Sanschagrin F. Levesque R.C. Sigma factors in Pseudomonas aeruginosa.FEMS Microbiol. Rev. 2008; 32 (18070067): 38-5510.1111/j.1574-6976.2007.00092.xCrossref PubMed Scopus (178) Google Scholar, 13Llamas M.A. Mooij M.J. Sparrius M. Vandenbroucke-Grauls C.M. Ratledge C. Bitter W. Characterization of five novel Pseudomonas aeruginosa cell-surface signalling systems.Mol. Microbiol. 2008; 67 (18086184): 458-47210.1111/j.1365-2958.2007.06061.xCrossref PubMed Scopus (71) Google Scholar, 14Llamas M.A. Imperi F. Visca P. Lamont I.L. Cell-surface signaling in Pseudomonas: stress responses, iron transport, and pathogenicity.FEMS Microbiol. Rev. 2014; 38 (24923658): 569-59710.1111/1574-6976.12078Crossref PubMed Scopus (90) Google Scholar). Heme uptake systems associated with ECF σ factor systems are conserved across a number of bacterial pathogens, including Serratia marcescens (15Ghigo J.M. Letoffe S. Wandersman C. A new type of hemophore-dependent heme acquisition system of Serratia marcescens reconstituted in Escherichia coli.J. Bacteriol. 1997; 179 (9171402): 3572-357910.1128/jb.179.11.3572-3579.1997Crossref PubMed Scopus (141) Google Scholar, 16Biville F. Cwerman H. Letoffe S. Rossi M.S. Drouet V. Ghigo J.M. Wandersman C. Haemophore-mediated signalling in Serratia marcescens: a new mode of regulation for an extra cytoplasmic function (ECF) sigma factor involved in haem acquisition.Mol. Microbiol. 2004; 53 (15306027): 1267-127710.1111/j.1365-2958.2004.04207.xCrossref PubMed Scopus (41) Google Scholar), Bordetella pertussis (17Vanderpool C.K. Armstrong S.K. Heme-responsive transcriptional activation of Bordetella bhu genes.J. Bacteriol. 2003; 185 (12533466): 909-91710.1128/jb.185.3.909-917.2003Crossref PubMed Scopus (51) Google Scholar), and P. aeruginosa (8Ochsner U.A. Johnson Z. Vasil M.L. Genetics and regulation of two distinct haem-uptake systems, phu has, in Pseudomonas aeruginosa.Microbiology. 2000; 146 (10658665): 185-19810.1099/00221287-146-1-185Crossref PubMed Scopus (222) Google Scholar). The P. aeruginosa Has system senses heme through the interaction of a secreted extracellular hemophore, HasAp, which scavenges and releases heme to the outer membrane receptor HasR (Fig. 1A). Capture of the heme by the N-terminal plug domain of HasR results in inactivation of the anti-σ factor HasS and release of the σ factor HasI. HasI then binds to the hasR promoter, recruits the core RNA polymerase, and upregulates transcription of the has operon (Fig. 1A). Simultaneously, heme released to HasR is transported through the receptor by the TonB-dependent coupling of the proton-motive force of the cytoplasmic membrane. The Has system does not encode a periplasmic transport system, so heme is sequestered and translocated to the cytoplasm by the PhuT-PhuUV periplasmic ATP-dependent binding cassette (ABC) transport system (Fig. 1A). Within the cytoplasm, heme is trafficked to the iron-dependent heme oxygenase (HemO) by the cytoplasmic heme-binding protein PhuS. HemO catalyzes the oxidative cleavage of heme to release CO, iron, and the heme metabolites biliverdin IXβ (BVIXβ) and IXδ (BVIXδ) (18Ratliff M. Zhu W. Deshmukh R. Wilks A. Stojiljkovic I. Homologues of neisserial heme oxygenase in gram-negative bacteria: degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa.J. Bacteriol. 2001; 183 (11591684): 6394-640310.1128/JB.183.21.6394-6403.2001Crossref PubMed Scopus (181) Google Scholar). In addition to heme-dependent transcriptional activation of the Has system, the hasAp transcript is subject to posttranscriptional regulation by the heme metabolites BVIXβ and/or BVIXδ (19Mourino S. Giardina B.J. Reyes-Caballero H. Wilks A. Metabolite-driven regulation of heme uptake by the biliverdin IXbeta/delta-selective heme oxygenase (HemO) of Pseudomonas aeruginosa.J. Biol. Chem. 2016; 291 (27493207): 20503-2051510.1074/jbc.M116.728527Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The several layers of transcriptional and posttranscriptional regulation over the Has system allow the bacteria to rapidly fine-tune its response to changes in the host environment (19Mourino S. Giardina B.J. Reyes-Caballero H. Wilks A. Metabolite-driven regulation of heme uptake by the biliverdin IXbeta/delta-selective heme oxygenase (HemO) of Pseudomonas aeruginosa.J. Biol. Chem. 2016; 291 (27493207): 20503-2051510.1074/jbc.M116.728527Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 20Dent A.T. Mourino S. Huang W. Wilks A. Post-transcriptional regulation of the Pseudomonas aeruginosa heme assimilation system (Has) fine-tunes extracellular heme sensing.J. Biol. Chem. 2019; 294 (30593511): 2771-278510.1074/jbc.RA118.006185Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). We have further shown the P. aeruginosa Has and Phu systems have nonredundant roles, where the Has system is primarily required for sensing, whereas the Phu system is the major transporter (9Smith A.D. Wilks A. Differential contributions of the outer membrane receptors PhuR and HasR to heme acquisition in Pseudomonas aeruginosa.J. Biol. Chem. 2015; 290 (25616666): 7756-776610.1074/jbc.M114.633495Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The significance of heme sensing and uptake to P. aeruginosa pathogenesis is evident in the fact that clinical isolates from patients with chronic lung infection adapt to utilize heme while decreasing their ability to biosynthesize pyoverdine (21Nguyen 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 (24727222): 2265-227610.1128/JB.01491-14Crossref PubMed Scopus (73) Google Scholar). In an attempt to dissect the mechanism of heme signaling and transport by the P. aeruginosa Has system, we have employed site-directed mutagenesis, allelic exchange, quantitative PCR (qPCR), immunoblotting, and 13C-heme uptake studies to determine the contributions of the heme coordinating ligands of HasR (Fig. 1). Previous studies employing similar approaches with variants of the extracellular hemophore HasAp showed heme release to HasR is required to activate the cell surface signaling cascade (20Dent A.T. Mourino S. Huang W. Wilks A. Post-transcriptional regulation of the Pseudomonas aeruginosa heme assimilation system (Has) fine-tunes extracellular heme sensing.J. Biol. Chem. 2019; 294 (30593511): 2771-278510.1074/jbc.RA118.006185Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The P. aeruginosa HasR protein has significant sequence identity (50%) to that of S. marcescens. The S. marcescens HasAs-HasR structure revealed His-603 of the FRAP/PNPNL loop (so called for the conserved amino acid motifs in the heme receptors) and His-189 of the N-terminal plug as the heme coordinating residues of HasR (Fig. 1B). Following heme release and dissociation of HasAp, heme coordination through His-603 was proposed to close off the extracellular milieu from the HasR heme transport channel. Despite structural determination of the HasAs-HasR complex, the molecular mechanism of heme signaling and transport has remained elusive. Here, we show the corresponding FRAP/PNPNL loop residue in P. aeruginosa HasAp His-624 is required for heme transport (Fig. 1B). In contrast, the N-terminal plug His-221 is critical for both heme signaling and transport. Based on the HasAs-HasR structure, Ile-671, located on extracellular loop L8, was proposed to prevent heme backsliding to HasAp. We further show that the corresponding Ile-694 in P. aeruginosa HasR is also essential for heme transport (Fig. 1B). Interestingly, heme bound to HasAp is not accessible to the Phu system, as shown by the significant growth defect in the transport-deficient HasR mutants when supplemented with holo-HasAp rather than heme. Interestingly, the N-terminal plug hasRH221R strain, which is unable to either activate the CSS cascade or transport heme, revealed a global dysregulation in both the Has and Phu system, indicating a direct or indirect link between the ECF σ factor HasI and the Phu heme uptake system. The expression profiles of the hasR allelic strains were first analyzed under iron-depleted conditions. The hasR variant strains all show a growth profile similar to that of PAO1 WT (Fig. 2A). For the hasR loop mutant strains, the hasR mRNA levels at each time point were all within 2-fold of the parent PAO1 strain, with negligible differences in protein expression (Fig. 3, A and B, and Fig. S1A). The data confirm the HasR loop variants are folded and stable. Similarly, the hasAp mRNA and protein levels for the hasR loop variants were very similar to those in the PAO1 WT strain (Fig. 3, C and D, and Fig. S1B). In contrast to the hasR loop variants, the N-terminal plug hasRH221R strain showed a significant increase in HasR protein at the later time points (Fig. 3B and Fig. S1A). To confirm that the expression of the Phu system was not compromised in the hasR variant strains, we analyzed the mRNA and protein levels by qPCR and Western blotting, respectively. The loop hasRH624A, H624Y, and I694G variants showed mRNA and protein profiles similar to that of the PAO1 WT strain (Fig. 4). Interestingly, the relative mRNA levels of phuR in the hasRH221R strain revealed a significant increase over the PAO1 WT (Fig. 4A). Furthermore, this increase in relative mRNA levels translated to protein expression (Fig. 4B). Overall, the data suggest that the hasRH221R variant, even under low-iron conditions, has a phenotype distinct from that of the hasR loop variants.Figure 3Relative HasR and HasAp mRNA and protein levels for the hasR allelic strains under iron-depleted conditions. A, hasR mRNA isolated at 0, 2, 4, and 6 h following growth in M9 minimal media. mRNA values represent the mean from three biological experiments, each performed in triplicate and normalized to PAO1 WT at the same time point. Error bars represent the standard deviations from three independent experiments performed in triplicate. The indicated p values were normalized to mRNA levels of PAO1 WT at the same time point: *, p < 0.05. B, Western blot analysis of PAO1 WT and the hasR allelic strains. For HasR, total protein (5 μg) was loaded in each well. RNApolα was used as a loading control. Normalized density (n = 3) was performed for three separate biological replicates. The indicated p values were normalized to PAO1 at the same time point: *, p < 0.05; **, p < 0.005. C, hasAp mRNA analyzed as in panel A. D, Western blot analysis of HasAp as in panel B. Extracellular supernatant (4 μl) was loaded and run on the automated Wes capillary Western system as described in Experimental procedures. Representative Western blot images are shown in the supporting information (Fig. S1).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Relative phuR mRNA and protein levels for the hasR allelic strains under iron-depleted conditions. A, mRNA isolated at 0, 2, 4, and 6 h following growth in M9 minimal media. mRNA values represent the means from three biological experiments, each performed in triplicate and normalized to PAO1 WT at the same time point. Error bars represent the standard deviations from three independent experiments performed in triplicate. The indicated p values were normalized to mRNA levels of PAO1 WT at the same time point: *, p < 0.05; **, p < 0.005. B, normalized density (n = 3) was determined on Western blots for three separate biological replicates. The indicated p values were normalized to PAO1 WT at the same time point: *, p < 0.05; **, p < 0.005. C, representative Western blotting of PAO1 WT and the hasR allelic strains. Total protein (5 μg) was loaded in each well. RNApolα was used as a loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The growth of the hasRH624A, hasH624Y, hasI694G, and hasRH221R strains was monitored following supplementation with either 1 μm heme or 1 μm holo-HasAp. All of the mutant strains were competent to utilize free heme as an iron source compared with the growth rates in iron-depleted media (Fig. 2A). However, when heme was supplied in the form of holo-HasAp, a significant inhibition in growth was observed for all of the variant strains compared with that of PAO1 WT (Fig. 2B). These results suggest that heme, but not heme complexed to HasAp, is accessible to the hasR variant strains via the Phu heme uptake system. qPCR analysis of phuR on heme supplementation showed that the relative mRNA levels of the hasR loop variants are similar to that of PAO1 WT at all time points (Fig. S2). However, the relative mRNA levels in the hasRH221R variant are 10-fold higher than those of PAO1 at all time points, consistent with the profile under iron-depleted conditions. The PhuR protein levels for PAO1 WT and all of the hasR variant strains follow a similar profile, where the protein is induced at 2 h, followed by a decrease at 4 h and a subsequent increase again at 6 h. This pattern of expression we interpret as a shock response on the addition of free heme, as we do not observe this effect when heme is complexed to HasAp. However, despite statistically significant differences in protein expression levels, as determined by Western blotting, it is clear that all of the hasR variant strains express PhuR protein within 2-fold of the level observed for PAO1 WT (Fig. S2, B and C). The ability of the hasR variant strains to utilize heme but not holo-HasAp was confirmed by isotopically labeled 13C-heme uptake and inductively coupled plasma mass spectrometry (ICP-MS) experiments. Supernatants were collected 4 h following supplementation of PAO1 WT or the hasR allelic strains with either 13C-heme or 13C-heme-HasAp. The resulting BVIX metabolites were extracted and quantified by LC-MS/MS. When supplemented with 13C-heme, the hasR variant strains were all capable of taking up and degrading heme, as judged by the similar levels of 13C-BVIXβ and 13C-BVIXδ compared that of PAO1 WT (Fig. 5A). Consistent with the ability to utilize exogenously added 13C-heme, ICP-MS analysis confirmed the intracellular iron content of the hasR variant strains is similar to that of PAO1 WT (Fig. 5B). Taken together, the data suggest that in the absence of a functional Has system, free heme is readily acquired by the major transporter PhuR. In contrast, the hasR variant strains were unable to utilize the 13C-heme–HasAp complex as an iron source, as indicated by the lack of 13C-BVIXβ and BVIXδ metabolites (Fig. 6A). Concomitant with the decreased ability to utilize extracellular heme, we see a slight but significant increase in endogenously derived BVIX (12C-heme) metabolites. Such redistribution of endogenous heme iron is consistent with the lower growth rate and dysregulation of iron homeostasis (Fig. 2B). The inability to utilize heme bound to HasAp was further confirmed on measurement of the intracellular iron levels by ICP-MS (Fig. 6B). The hasR variant strains all showed decreased intracellular iron content, which is particularly noticeable for the H221R N-terminal plug mutant. Analysis of phuR mRNA levels in the hasR loop variants are within 2-fold of those for PAO1 WT (Fig. S3). Western blotting further confirmed protein levels mirror those of PAO1 (Fig. S3, B and C). However, as can be seen from the qPCR analysis, the relative mRNA levels for all time points are significantly higher for the hasRH221R plug variant (Fig. S3). This difference is not solely because of the decreased iron levels, as iron depletion is also observed for the hasR loop variants (Fig. 6B). Furthermore, we observed a similar mRNA profile for the hasR variant strains in cultures supplemented with heme, where the intracellular iron levels were identical to that of PAO1 WT (Fig. 4B). However, the increase in mRNA levels when supplemented with holo-HasAp is not reflected in significant changes in protein levels (Fig. S3). Taken together, the data clearly show heme bound to HasAp, in the absence of a functional HasR transporter, is not accessible to PhuR. This is consistent with previous studies in which we have shown the Has and Phu systems have distinct nonredundant roles in heme signaling and transport, respectively (9Smith A.D. Wilks A. Differential contributions of the outer membrane receptors PhuR and HasR to heme acquisition in Pseudomonas aeruginosa.J. Biol. Chem. 2015; 290 (25616666): 7756-776610.1074/jbc.M114.633495Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). We next sought to determine the contributions of the extracellular loop and N-terminal plug residues on activation of the CSS cascade. HasI-dependent transcriptional activation of hasR and hasAp following supplementation with heme or holo-HasAp was monitored over time by qPCR analysis and Western blotting. As previously reported for supplementation of PAO1 WT with 1 μm heme, following an initial decrease in mRNA, we observed an increase in hasR mRNA levels over time as extracellular HasAp levels accumulate (Fig. 7A). The hasAp mRNA profiles mirror those of hasR; however, as previously reported, the relative mRNA levels are greater than those of hasR because of increased mRNA stability following processing of the hasRAp transcript (Fig. 7B) (20Dent A.T. Mourino S. Huang W. Wilks A. Post-transcriptional regulation of the Pseudomonas aeruginosa heme assimilation system (Has) fine-tunes extracellular heme sensing.J. Biol. Chem. 2019; 294 (30593511): 2771-278510.1074/jbc.RA118.006185Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Western blot analysis of HasR and HasAp protein levels are consistent with the respective mRNA profiles (Fig. 8).Figure 8Relative HasR and HasAp protein levels for the hasR allelic strains under heme-supplemented conditions. A, HasR protein at 0, 2, 4, and 6 h following growth in M9 minimal media supplemented with 1 μm heme. Total protein (5 μg) was loaded in each well. RNApolα was used as a loading control. Normalized density (n = 3) was performed on Western blots for three separate biological replicates. B, as in panel A but for HasAp. Extracellular supernatant (4 μl) was loaded for analysis on the automated Wes capillary system as described in Experimental procedures. Normalized density (n = 3) was corrected for differences in OD600 for three biological replicates. Error bars represent the standard deviations from three independent experiments performed in triplicate. Indicated p values for the hasR allelic strains normalized to PAO1 WT at the same time point: *, p < 0.05; **, p < 0.005. C, representative Western blotting of PAO1 WT and the hasR allelic strains for HasR and digital HasAp images generated by capillary Western blot analysis on an automated Wes system.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The hasR loop variants show a similar profile but slightly lower hasR and hasAp mRNA and protein levels compared with those of the WT (Figure 7, Figure 8). The increase in relative mRNA levels on addition of heme suggests that despite an inability to transport heme, they retain the ability to activate the CSS cascade (Fig. 7). In contrast, the hasRH221R strain showed no heme-dependent transcriptional activation of hasR and hasAp (Fig. 7). This lack of transcriptional activation is not solely the result of decreased HasAp levels in the extracellular medium, as we observed significant protein levels by Western blotting (Fig. 8). Previous studies from our laboratory have shown hasAp is subject to positive posttranscriptional regulation by the heme metabolites BVIXβ and/or BVIXδ (19Mourino S. Giardina B.J. Reyes-Caballero H. Wilks A. Metabolite-driven regulation of heme uptake by the biliverdin IXbeta/delta-selective heme oxygenase (HemO) of Pseudomonas aeruginosa.J. Biol. Chem. 2016; 291 (27493207): 20503-2051510.1074/jbc.M116.728527Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 20Dent A.T. Mourino S. Huang W. Wilks A. Post-transcriptional regulation of the Pseudomonas aeruginosa heme assimilation system (Has) fine-tunes extracellular heme sensing.J. Biol. Chem. 2019; 294 (30593511): 2771-278510.1074/jbc.RA118.006185Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Therefore, the discrepancy between mRNA and protein levels can be rationalized by the fact that heme actively taken up by the Phu system is further metabolized to BVIXβ and BVIXδ. We further analyzed the transcriptional activation of the has operon on supplementation with a fixed concentration of holo-HasAp. In these studies, we utilized a previously characterized functional but truncated HasAp to distinguish, by Wes analysis, the supplemented holo-HasAp from the endogenously produced protein (19Mourino S. Giardina B.J. Reyes-Caballero H. Wilks A. Metabolite-driven regulation of heme uptake by the biliverdin IXbeta/delta-selective heme oxygenase (HemO) of Pseudomonas aeruginosa.J. Biol. Chem. 2016; 291 (27493207): 20503-2051510.1074/jbc.M116.728527Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). In contrast to the lag on addition of heme (∼4 h; Fig. 7), addition of holo-HasAp to PAO1 immediately activates transcription from the hasR promoter (Fig. 9A). For PAO1 WT, transcriptional activation peaks at the earlier 2-h time point and is maintained over 6 h. In contrast, the hasR loop variants show decreased transcriptional activation at the early time points. The lag in transcriptional activation for the loop variants is further manifested at the protein level, where significantly higher HasR expression is observed for PAO1 WT at the earlier time points (Fig. 10A). Whereas the hasAp mRNA levels for the hasR loop variants follow a trend similar to those of hasR (Fig. 9B), we do not detect any endogenous HasAp protein (Fig. 10B). This lack of protein expression can be rationalized by the inability of the hasR loop variants to take up heme, leading to lower intracellular BVIX levels (Fig. 6A) and decreased HasAp translational efficiency. For the N-terminal plug hasRH221R strain, we observed no transcriptional activation of the hasR operon, as shown by
Referência(s)