Author response: Functional membrane microdomains and the hydroxamate siderophore transporter ATPase FhuC govern Isd-dependent heme acquisition in Staphylococcus aureus
2023-04-04

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Sufficient access to transition metals such as iron is essential for bacterial proliferation and their active limitation within host tissues effectively restricts infection. To overcome iron limitation, the invasive pathogen Staphylococcus aureus uses the iron-regulated surface determinant (Isd) system to acquire hemoglobin-derived heme. While heme transport over the cell wall is well understood, its transport over the membrane is hardly investigated. In this study, we show the heme-specific permease IsdF to be energized by the general ATPase FhuC. Additionally, we show that IsdF needs appropriate location within the membrane for functionality. The membrane of S. aureus possesses special compartments (functional membrane microdomains [FMMs]) to organize membrane complexes. We show IsdF to be associated with FMMs, to directly interact with the FMM scaffolding protein flotillin A (FloA) and to co-localize with the latter on intact bacterial cells. Additionally, Isd-dependent bacterial growth required FMMs and FloA. Our study shows that Isd-dependent heme acquisition requires a highly structured cell envelope to allow coordinated transport over the cell wall and membrane and it gives the first example of a bacterial nutrient acquisition system that depends on FMMs. Editor's evaluation In this fundamental manuscript, the authors provide compelling evidence that a housekeeping ATPase is required for heme utilization in the important pathogen Staphylococcus aureus through its interaction with the canonical heme transporter in this organism. The authors convincingly show that this complex associates with functional membrane microdomains and thus establishes a new paradigm for regional localization of the heme transport system in the staphylococci. The work will be of interest to microbiologists, particularly those studying transport for macromolecules. https://doi.org/10.7554/eLife.85304.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Transition metals such as iron, manganese, copper, and zinc are essential trace elements for all kingdoms of life. Due to their redox potential, transition metals can convert between divalent and trivalent states making them ideal to support enzymatic processes. Molecular iron is of major importance in this regard as it is essential for several central metabolic and cellular processes including glycolysis, oxidative decarboxylation, respiration, and DNA replication (Schaible and Kaufmann, 2004). Accordingly, appropriate acquisition of iron is essential for bacterial growth and this dependency is a major target for eukaryotic innate immune strategies to limit bacterial infections. Targeted depletion of human body fluids from trace metals effectively limits bacterial proliferation and is referred to as nutritional immunity (Murdoch and Skaar, 2022; Weinberg, 1975). To overcome host-induced iron starvation, bacterial pathogens have developed a range of strategies to acquire iron during infection (Sheldon et al., 2016). They can secrete siderophores, which possess sufficient affinity toward Fe3+ to extract the ion from host-chelating molecules (e.g. transferrin, lactoferrin) (Miethke and Marahiel, 2007). Staphylococcus aureus secretes the carboxylate-type siderophores staphyloferrin A (SA) and staphyloferrin B (SB), and their iron-saturated forms are acquired by the membrane-located HtsABC and SirABC systems, respectively (Beasley et al., 2009; Cheung et al., 2009). Additionally, S. aureus expresses systems for acquisition of siderophores produced by other bacterial species (xenosiderophores). The FhuBGCD1D2 system enables acquisition of hydroxamate-type siderophores (Sebulsky and Heinrichs, 2001) and the SstABCD system allows acquisition of iron-chelating molecules of the catecholate-type including xenosiderophores, but also human-derived catecholamine stress hormones like epinephrine, norepinephrine, or dopamine (Beasley et al., 2011). Moreover, the FeoAB transporter presumably allows acquisition of inorganic Fe2+(Sheldon and Heinrichs, 2015). In humans, much of the iron is bound to heme in hemoglobin (Hb), which can be released from erythrocytes by the action of cytolytic toxins (Cassat and Skaar, 2013; Spaan et al., 2015). A well-known mechanism for heme acquisition is the iron-regulated surface determinant (Isd) systems. This was first described for the invasive pathogen S. aureus (Mazmanian et al., 2003). Similar systems have since been identified in Staphylococcus lugdunensis (Heilbronner et al., 2016), Staphylococcus capitis, and Staphylococcus caprea (Sun et al., 2020) as well as in phylogenetically more distant Gram-positive pathogens such as Bacillus anthracis (Gat et al., 2008; Maresso et al., 2006), Bacillus cereus (Abi-Khalil et al., 2015), and Listeria monocytogenes (Klebba et al., 2012; Xiao et al., 2011). All Isd systems contain surface-anchored molecules to extract heme from host hemoproteins and to guide it over the cell wall to a membrane-located ATP-binding cassette (ABC) transporter. In the cytosol, the heme is degraded to release ionic iron. The Isd system of S. aureus is best studied (Sheldon and Heinrichs, 2015). Here, the cell wall-anchored proteins (CWAs) IsdB and IsdH bind Hb and Hb-haptoglobin complexes, respectively. Heme is removed from the hemoproteins by IsdA and IsdB and transferred to IsdC in the cell wall. Heme is then transferred to the heme-specific lipoprotein IsdE on the outer leaflet of the cell membrane. The permease IsdF functions as homodimer and transports heme into the cytosol. There, the two monooxygenases IsdG and IsdI release iron from heme. The passage of heme across the cell wall of S. aureus has been studied in great detail. In contrast, the process of heme transport across the membrane remains somewhat undefined. Importantly, an ATPase is not encoded within the isd operon of S. aureus, raising the question of how the transport of heme is energized. Additionally, it is unclear if heme funneling across the cell wall demands the membrane transporter to be in a specific location within the liquid mosaic of the membrane to ensure effective passage of heme from the CWAs to the lipoprotein IsdE. It is increasingly recognized that bacterial membranes represent highly structured cellular compartments. In this regard, functional membrane microdomains (FMMs) have gained increasing attention in the recent years (Bach and Bramkamp, 2013; Bramkamp and Lopez, 2015; Farnoud et al., 2015; López and Kolter, 2010; Matsumoto et al., 2006; Yokoyama and Matsui, 2020). FMMs are specific domains in the bacterial membrane that, in the case of S. aureus, consist of the polyisoprenoid staphyloxanthin and its derivative lipids. An intrinsic characteristic of FMMs is the structural protein flotillin A (FloA) recruiting proteins to the FMMs and promoting their oligomerization (Bramkamp and Lopez, 2015; López and Kolter, 2010). FMMs have been shown to be crucial to coordinate diverse cellular functions in S. aureus. On one hand, they control activity of cell wall biosynthetic enzymes such as PBP2a and are essential for expression of resistance to β-lactam antibiotics in methicillin-resistant S. aureus (MRSA) (García-Fernández et al., 2017). On the other hand, several membrane-associated processes such as the secretion of type VII secretion effector proteins (Mielich-Süss et al., 2017) or the RNase Rny depend on FloA (Koch et al., 2017). In this study, we have used bacterial two-hybrid assays to show direct interaction between the iron-responsive ATPase FhuC and the heme permease protein IsdF. Additionally, we have shown that Hb-dependent growth requires FhuC activity, suggesting that FhuC energizes heme transport in S. aureus. Moreover, the heme permease IsdF was shown to be associated with FMMs, to interact directly with FloA and to co-localize with the latter in the bacterial envelope. Accordingly, Hb-dependent proliferation was reliant on the correct formation of FMMs. Finally, we demonstrated that appropriate FMM formation is also required for Isd-dependent proliferation of S. lugdunensis, suggesting that FMM-dependent structuring of the membrane is crucial for the functionality of Isd systems in staphylococci. Results FhuC is crucial for heme-dependent proliferation of S. aureus The Isd system facilitates the acquisition of heme from Hb. Heme membrane transport involves an ABC transporter consisting of the heme-specific lipoprotein (IsdE) and the transmembrane protein IsdF (Grigg et al., 2007; Mazmanian et al., 2003). Interestingly, an ATPase to energize membrane transport is not encoded within the S. aureus isd operon. Similarly, the operons encoding the SA and SB membrane transport systems (HtsABC and SirABC) lack intrinsic ATPases and both systems were previously shown to be energized by FhuC (Beasley et al., 2009; Speziali et al., 2006). This ATPase is encoded within the fhuCBG operon allowing hydroxamate siderophore transport. Thus, FhuC appears to act as a housekeeping ATPase for iron acquisition in S. aureus. In order to determine if FhuC is required for heme acquisition by Isd, we studied growth of fhuC mutants in iron-limited medium. We found that a S. aureus USA300 JE2 fhuC::Erm mutant, obtained from the Nebraska transposon mutant library (Fey et al., 2013), showed significantly reduced growth when human Hb (hHb) was supplied as a sole source of nutrient iron (Figure 1A). This deficit was rescued by addition of iron(II) sulfate (Figure 1B), suggesting that FhuC energizes heme transport across the membrane. To investigate this further, we created an isogenic deletion mutant of fhuC (ΔfhuC) in S. aureus strain Newman. Again, the fhuC-deficient strain showed a pronounced growth deficit in the presence of Hb. Importantly, plasmid-based expression of FhuC restored Hb-dependent growth (Figure 1C). This supports the idea that FhuC is needed for Isd-dependent heme acquisition. Figure 1 Download asset Open asset FhuC is needed for hemoglobin (Hb)-dependent proliferation of S. aureus. One-hundred μl (A, B) or 500 μl (C) of bacterial cultures were grown with human Hb or FeSO4 as the sole source of iron in 96-well (A, B) or 48-well (C) format and OD600 was monitored over time. For reasons of clarity, values taken every 2 hr are displayed. (A, B) S. aureus USA300 JE2 wild type (WT) and USA300 JE2 fhuC::Erm. Means and SD of six experiments are shown. (C) S. aureus Newman ΔfhuC was complemented using a plasmid expressing FhuC from the native promotor (pRB473:fhuC). Newman WT pRB473, ΔfhuC pRB473, and ΔfhuC pRB473:fhuC. Means and SD of three experiments are shown. (A, C) Statistical analysis comparing the WT strains and the fhuC mutants was performed using GraphPad Prism 9 Student's unpaired t-test. **p<0.01, ***p<0.001. Figure 1—source data 1 FhuC is needed for hemoglobin (Hb)-dependent proliferation of S. aureus. https://cdn.elifesciences.org/articles/85304/elife-85304-fig1-data1-v2.zip Download elife-85304-fig1-data1-v2.zip Conserved amino acids in the coupling helix of IsdF promote the interaction with FhuC We used the bacterial adenylate cyclase two-hybrid system (BACTH) to determine whether FhuC physically interacts with the Isd permease IsdF. In BACTH, putatively interacting proteins are fused to domains of an adenylate cyclase. Interaction of the two proteins reconstitutes adenylate cyclase which allows expression of β-galactosidase (LacZ). Co-expression of FhuC with its native permease FhuB as well as with IsdF generated distinct positive signals (Figure 2A). In contrast, co-expression of FhuC with the iron-independent permease MntB (manganese uptake) did not result in detectable LacZ activity (Figure 2A). Quantification of LacZ activity confirmed the plate-based screening (Figure 2—figure supplement 1A). This suggests that FhuC energizes the heme transport system IsdEF and structural characteristics within the permease must allow discrimination of FhuC targets and non-targets. Figure 2 with 1 supplement see all Download asset Open asset FhuC interacts directly with IsdF. (A, D, E) Escherichia coli BTH101 was co-transformed with pUT18C:fhuC and pKT25 vectors expressing permeases of interest. Where protein-protein interactions occur, the T25 and T18 catalytic domains of adenylate cyclase dimerize forming an active enzyme which produces cAMP. This activates LacZ expression leading to X-Gal degradation and blue signals on indicator plates. (A) Positive control, leucine zippers (zip). Negative control, empty vectors (pKT25+pUT18C) (-). (B) Schematic representation of the IsdF topology prediction using TOPCONS. The amino acids marking the transmembrane domains (TM) are shown with the conserved A213 and G217 indicated by yellow asterisks. Truncations are indicated in red. (C) IsdF structure prediction using Alphafold with visualization using PyMOL. The coupling helix is shown in red with the conserved A231 and G217 in yellow. The N-terminus is in cyan and the C-terminus in green. (D) Bacterial adenylate cyclase two-hybrid (BACTH) analysis of FhuC and IsdF with single and double amino acid substitutions as well as with the MntB_CHisdF fusion protein (coupling helix IsdF). (E) BACTH analysis of FhuC and truncated IsdF derivatives. Figure 2—source data 1 FhuC interacts directly with IsdF. https://cdn.elifesciences.org/articles/85304/elife-85304-fig2-data1-v2.zip Download elife-85304-fig2-data1-v2.zip ATPases and their respective permeases display Q-loops and coupling helixes, respectively, to mediate their interaction (Hollenstein et al., 2007; Wen and Tajkhorshid, 2011). We speculated that conserved motifs within the coupling helixes of iron compound permeases might promote their energization by FhuC. To investigate this, we modeled the topology of the permease IsdF using TOPCONS (Tsirigos et al., 2015) and Alphafold (Jumper et al., 2021; Varadi et al., 2022). This resulted in prediction of 10 transmembrane helices. Both the N- and C-termini of the protein as well as four loops were proposed to be located in the cytoplasm (Figure 2B, Figure 2—figure supplement 1B). The third cytoplasmic loop (aa 204–217) contained the cytosolic coupling helix (Figure 2B+C). To identify motifs that might determine energization by FhuC, we aligned the sequences of the coupling helixes of all putative FhuC interaction partners (IsdF, FhuB, FhuG, SirB, SirC, HtsB, HtsC), as well as of MntB, using Clustal Omega (Madeira et al., 2022). This analysis showed that within all putative coupling helixes only a single glycine residue (G217 in IsdF) is conserved. Additionally, a single alanine residue (A213 in IsdF) was conserved in all iron compound permeases but not in MntB (Figure 2B+C, Figure 2—figure supplement 1C). Many coupling helices contain an EAAxxxGxxxxxxxxxIxLP (EAA) motif (ter Beek et al., 2014). We speculated that the AxxxG motif identified in the iron permeases might be important to mediate interaction with FhuC. Exchange of the alanine 213 to the bulky amino acid phenylalanine (A213F) did not prevent interaction with FhuC. The same was true when glycine 217 was exchanged for phenylalanine (G217F). However, combination of both substitutions abrogated the interaction (Figure 2D). These results show the importance of A213 and G217 for the recognition of FhuC, but they do not explain how MntB and IsdF are discriminated. To examine this, we inserted the full-length coupling helix of IsdF at the appropriate position within MntB. Interestingly, the resulting fusion protein remained negative in BACTH analysis with FhuC (Figure 2D), suggesting that the sequence and structure of the coupling helix alone is not sufficient to allow FhuC to discriminate between IsdF and MntB. To further investigate this, we created a series of C-terminal truncations of IsdF (Figure 2B+E). Deletion of the C-terminal cytoplasmic domain (short_1) did not affect the interaction with FhuC. However, truncation at the fourth cytosolic loop (short_2) abrogated the interaction and the same was true for additional truncations lacking more extended C-terminal fragments (short_3/short_4) (Figure 2E). These data indicate that besides the coupling helix, the C-terminal part of IsdF is crucial for appropriate formation of the IsdF-FhuC complex. IsdF is located within FMMs and interacts with FloA FMMs and the associated structuring protein FloA promote the correct structure of membrane-associated proteins and protein complexes in S. aureus (Bramkamp and Lopez, 2015). Proteomic profiles of FMM-membrane fractions have been recorded previously and FMM-associated proteins were identified (García-Fernández et al., 2017). Revisiting these datasets showed that a high number of iron uptake-associated proteins are enriched in the FMM-containing membrane fraction including the heme transport system IsdEF and the siderophore transporters Fhu, Hts, and Sir. To investigate further the subcellular localization of IsdF, we generated a S. aureus Newman strain that constitutively expresses IsdF with a triple FLAG tag. The cell membrane was isolated and non-ionic detergents were used to collect FMMs, which accumulate in the detergent-resistant membrane (DRM) fraction compared to the detergent-sensitive membrane (DSM) fraction (Bramkamp and Lopez, 2015; Brown, 2002; Shah and Sehgal, 2007). The abundance of proteins in general appeared to be slightly increased in the DSM fraction (Figure 3A). In contrast, quantitative western blotting showed that 80% of the IsdF signal was detected in the DRM fraction (Figure 3B). This confirms that IsdF is predominantly associated with the DRM fraction and suggests that IsdF is localized within FMMs. Figure 3 with 1 supplement see all Download asset Open asset IsdF localizes within functional membrane microdomains (FMMs) and interacts directly with flotillin A (FloA). S. aureus Newman FloA-His pRB474:isdF-3xFLAG membranes were isolated and (A, B) separated into detergent-resistant membrane (DRM) and detergent-sensitive membrane (DSM) fractions or (C) solubilized with 1% n-dodecyl-β-D-maltopyranosid (DDM) overnight and co-precipitated using Ni-NTA affinity chromatography. (A) Coomassie blue-stained gel of DRM and DSM fractions (equal amounts loaded). (B) Immunoblot analysis and quantification of IsdF in DRM and DSM fractions using anti-FLAG antibody and LI-COR infrared technology. An example of the IsdF-3xFLAG bands in the DRM and DSM fractions is shown. Quantification of signals were calculated as a percentage of the total signal (DRM+DSM). Means and SD of three independent experiments are shown. (C) Co-precipitation analysis using anti-FLAG antibody for detection of IsdF-3xFLAG (pRB474:isdF-3xFLAG) that co-eluted with FloA-His or FloA WT. An example of the IsdF-3xFLAG bands that co-eluted with FloA-His and FloA WT are shown. Quantification of FloA-His IsdF-3xFLAG signals in immunoblots was normalized to FloA WT strain signals (set to 1). Means and SD of four independent experiments are shown. Statistical analysis (Student's unpaired t-test) was performed using GraphPad Prism 8. Figure 3—source data 1 IsdF localizes within functional membrane microdomains (FMMs) and interacts directly with flotillin A (FloA). https://cdn.elifesciences.org/articles/85304/elife-85304-fig3-data1-v2.zip Download elife-85304-fig3-data1-v2.zip It was previously postulated that the structuring protein FloA recruits target proteins to FMMs (Bramkamp and Lopez, 2015; Lopez and Koch, 2017). Therefore, we investigated if this is also true for IsdF. We co-expressed IsdF-triple FLAG with the FloA WT protein or with FloA tagged with hexa-histidine in S. aureus Newman and performed Ni-NTA affinity chromatography. Prior to Ni-NTA chromatography, IsdF was equally abundant in both strains showing equal expression (Figure 3—figure supplement 1). However, IsdF was three times more abundant in the Ni-NTA of the FloA-His expressing strain (Figure This suggests that IsdF interacts with FloA which allows of IsdF-triple FLAG were detected within the of the FloA WT expressing suggesting interaction of IsdF or FloA with the Ni-NTA FloA and IsdF are coordinated in intact We used to investigate the localization of FloA and IsdF in intact of S. aureus Newman. The IsdF protein was tagged by FloA was to The latter was by addition of described FloA signals distinct at the bacterial surface FMMs (Figure Interestingly, IsdF also in (Figure However, indicated that the IsdF and FloA signals did not but to be in We this by the between of IsdF and FloA in of (Figure This showed a in of were separated by a single or by two in and of respectively. The number of within a with increasing that were further were we found that signals from plasmid were in a FloA expressing strain but in a mutant (Figure of the mutant with the functional WT by restored IsdF signals to WT (Figure These data that FloA is crucial for the appropriate and of IsdF within the membrane of S. aureus. Figure Download asset Open asset A (FloA) is crucial for of IsdF in the membrane of S. aureus. (A) of a of S. aureus Newman 1 (B) Quantification of the of and The of to the was with The shows the of show and SD of three independent number of was for (C) An example of a of S. aureus Newman and 1 (D) Quantification of of cells. The shows the and SD of three independent Data was normalized to the respective Statistical was using unpaired Student's with Figure data 1 A (FloA) is crucial for of IsdF in the membrane of S. aureus. Download FloA and FMMs are needed for bacterial growth using Hb as the sole iron source The functionality of membrane-associated protein complexes in S. aureus requires both FMMs and FloA (García-Fernández et al., et al., et al., 2017). Due to the location of IsdF within FMMs, we that FMMs be for iron acquisition. we this using a USA300 JE2 mutant from the Nebraska transposon mutant in the presence of FeSO4 was not by this (Figure supplement but a growth was when Hb was supplied as the sole source of nutrient iron. of the mutant with the functional WT by restored growth (Figure These data support the idea that of IsdF is needed for heme acquisition. Figure with 1 supplement see all Download asset Open asset A (FloA) and functional membrane microdomains (FMMs) are needed for proliferation with were grown in iron-limited μl in 96-well 500 μl in 48-well (A) of S. aureus USA300 JE2 and in the presence of 1 human hemoglobin For reasons of clarity, values hr are displayed. Means and SD of four experiments are shown. (B) of S. aureus Newman and were grown in the presence of 1 hr are displayed. Means and SD of three experiments are shown. Newman WT (C) and ΔfhuC (D) were grown in the presence of 10 acid and taken every 2 hr are displayed. Means and SD of four experiments are shown. Statistical Student's by for was performed using GraphPad Prism Statistical Student's unpaired was performed using GraphPad Prism 8. **p<0.01, ***p<0.001. Figure data 1 A (FloA) and functional membrane microdomains (FMMs) are needed for proliferation with Download we the importance of FMMs for heme acquisition also for S. aureus Newman. We created three mutant strains lacking the Isd system FloA and which results in FMM to the to polyisoprenoid et al., 2012; López and Kolter, 2010). All mutants showed WT of growth in the presence of FeSO4 (Figure supplement but a growth deficit in the presence of Hb (Figure Additionally, of FMM by acid and Kolter, reduced Hb-dependent growth (Figure Importantly, the reduced growth of the fhuC mutant was not further by the addition of (Figure suggesting that iron acquisition and not of were for the growth in the WT In did not affect growth in the presence of FeSO4 (Figure supplement All these experiments that functional FMMs are crucial for Hb by S. aureus. We also found proteins of the siderophore acquisition systems Hts, and Fhu, to be enriched in the of FMM-membrane fractions (García-Fernández et al., 2017). Therefore, we investigated if FloA is also for siderophore acquisition. We created mutants of all three systems in S. aureus Newman and compared growth of the mutants to that of the mutant in the presence of or the hydroxamate siderophores and a source of SA and of S. aureus USA300 JE2 only and S. aureus USA300 JE2 only was respectively. All mutants showed WT in the presence of FeSO4 (Figure supplement The and mutants showed growth in the presence of SA and SB (Figure respectively, and to in the presence of and (Figure The fhuC mutant showed growth with all siderophores (Figure These data are in with datasets (Beasley et al., 2009; Cheung et al., 2009; et al., Speziali et al., 2006). However, the mutant did not show growth compared to the WT strain the (Figure This data suggests that in to acquisition of acquisition of siderophores is not on membrane structuring by FMMs. Figure with 1 supplement see all Download asset Open asset using siderophores is independent of functional membrane microdomains Newman and were grown in the presence of of S. aureus USA300 staphyloferrin of USA300 JE2 staphyloferrin B) (C) or were grown in 500 μl of iron-limited in 48-well plates. For reasons of clarity, values hr (A, B) or hr are displayed. Means and SD of three experiments are shown. Statistical Student's by for was performed using GraphPad Prism Figure data 1 using siderophores is independent of functional membrane microdomains Download not depend on FloA and FMMs A major between the heme membrane transporter IsdEF and all siderophore transport systems is