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• W3033014932 abstract "Staphylococcus aureus is an important bacterial pathogen that can cause a wide spectrum of diseases in humans and other animals. S. aureus expresses a variety of virulence factors that promote infection with this pathogen. These include cell-surface proteins that mediate adherence of the bacterial cells to host extracellular matrix components, such as fibronectin and fibrinogen. Here, using immunoblotting, ELISA, and surface plasmon resonance analysis, we report that the iron-regulated surface determinant B (IsdB) protein, besides being involved in heme transport, plays a novel role as a receptor for the plasma and extracellular matrix protein vitronectin (Vn). Vn-binding activity was expressed by staphylococcal strains grown under iron starvation conditions when Isd proteins are expressed. Recombinant IsdB bound Vn dose dependently and specifically. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound Vn in a saturable manner, with KD values in the range of 16–18 nm. Binding of Vn to IsdB was specifically blocked by heparin and reduced at high ionic strength. Furthermore, IsdB-expressing bacterial cells bound significantly higher amounts of Vn from human plasma than did an isdB mutant. Adherence to and invasion of epithelial and endothelial cells by IsdB-expressing S. aureus cells was promoted by Vn, and an αvβ3 integrin-blocking mAb or cilengitide inhibited adherence and invasion by staphylococci, suggesting that Vn acts as a bridge between IsdB and host αvβ3 integrin. Staphylococcus aureus is an important bacterial pathogen that can cause a wide spectrum of diseases in humans and other animals. S. aureus expresses a variety of virulence factors that promote infection with this pathogen. These include cell-surface proteins that mediate adherence of the bacterial cells to host extracellular matrix components, such as fibronectin and fibrinogen. Here, using immunoblotting, ELISA, and surface plasmon resonance analysis, we report that the iron-regulated surface determinant B (IsdB) protein, besides being involved in heme transport, plays a novel role as a receptor for the plasma and extracellular matrix protein vitronectin (Vn). Vn-binding activity was expressed by staphylococcal strains grown under iron starvation conditions when Isd proteins are expressed. Recombinant IsdB bound Vn dose dependently and specifically. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound Vn in a saturable manner, with KD values in the range of 16–18 nm. Binding of Vn to IsdB was specifically blocked by heparin and reduced at high ionic strength. Furthermore, IsdB-expressing bacterial cells bound significantly higher amounts of Vn from human plasma than did an isdB mutant. Adherence to and invasion of epithelial and endothelial cells by IsdB-expressing S. aureus cells was promoted by Vn, and an αvβ3 integrin-blocking mAb or cilengitide inhibited adherence and invasion by staphylococci, suggesting that Vn acts as a bridge between IsdB and host αvβ3 integrin. Staphylococcus aureus causes a wide range of opportunistic infections that range from superficial skin infections to life-threatening diseases, including endocarditis, pneumonia, and septicemia (1Tong S.Y. Davis J.S. Eichenberger E. Holland T.L. Fowler Jr., V.G. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management.Clin. Microbiol. Rev. 2015; 28 (26016486): 603-66110.1128/CMR.00134-14Crossref PubMed Scopus (1642) Google Scholar). Adherence of bacteria to host matrix components is the initial critical event in the pathogenesis of most infections. The extracellular matrix (ECM) essentially consists of macromolecules, such as collagens, proteoglycans, and glycoproteins, that serve as a substrate for the adhesion and migration of tissue cells. These processes involve integrins, a family of heterodimeric cell surface receptors that recognize specific ECM proteins (2Hynes R.O. Naba A. Overview of the matrisome—an inventory of extracellular matrix constituents and functions.Cold Spring Harb. Perspect. Biol. 2012; 4 (21937732): a00490310.1101/cshperspect.a004903Crossref PubMed Scopus (490) Google Scholar, 3Naba A. Clauser K.R. Ding H. Whittaker C.A. Carr S.A. Hynes R.O. The extracellular matrix: tools and insights for the “omics” era.Matrix Biol. 2016; 49 (26163349): 10-2410.1016/j.matbio.2015.06.003Crossref PubMed Scopus (350) Google Scholar). Bacteria, including S. aureus, also utilize the ECM as substrata for their adhesion through a family of cell wall-anchored (CWA) adhesins called MSCRAMMs (microbial surface component recognizing adhesive matrix molecules) that specifically recognize host matrix components (4Foster T.J. Geoghegan J.A. Ganesh V.K. Höök M. Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus.Nat. Rev. Microbiol. 2014; 12 (24336184): 49-6210.1038/nrmicro3161Crossref PubMed Scopus (715) Google Scholar, 5Foster T.J. The MSCRAMM family of cell-wall-anchored surface proteins of gram-positive cocci.Trends Microbiol. 2019; 27 (31375310): 927-94110.1016/j.tim.2019.06.007Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Vitronectin (Vn) is a glycoprotein that is synthesized in the liver and secreted into plasma (6Preissner K.T. Seiffert D. Role of vitronectin and its receptors in haemostasis and vascular remodeling.Thromb Res. 1998; 89 (9610756): 1-2110.1016/S0049-3848(97)00298-3Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) and is also an important component of the ECM (7Leavesley D.I. Kashyap A.S. Croll T. Sivaramakrishnan M. Shokoohmand A. Hollier B.G. Upton Z. Vitronectin—master controller or micromanager?.IUBMB Life. 2013; 65 (24030926): 807-81810.1002/iub.1203PubMed Google Scholar). Vn is found at a high concentration in plasma (200–700 μg/ml) (8Boyd N.A. Bradwell A.R. Thompson R.A. Quantitation of vitronectin in serum: evaluation of its usefulness in routine clinical practice.J. Clin. Pathol. 1993; 46 (7504702): 1042-104510.1136/jcp.46.11.1042Crossref PubMed Scopus (31) Google Scholar, 9Chauhan A.K. Moore T.L. Presence of plasma complement regulatory proteins clusterin (Apo J) and vitronectin (S40) on circulating immune complexes (CIC).Clin. Exp. Immunol. 2006; 145 (16907906): 398-40610.1111/j.1365-2249.2006.03135.xCrossref PubMed Scopus (41) Google Scholar) and is also present in different human tissues (10Berglund L. Björling E. Oksvold P. Fagerberg L. Asplund A. Szigyarto C.A. Persson A. Ottosson J. Wernérus H. Nilsson P. Lundberg E. Sivertsson A. Navani S. Wester K. Kampf C. et al.A genecentric Human Protein Atlas for expression profiles based on antibodies.Mol. Cell. Proteomics. 2008; 7 (18669619): 2019-202710.1074/mcp.R800013-MCP200Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). The N-terminal portion of mature Vn (43 aa residues) consists of a somatomedin B (SMB) domain followed by the classical integrin-binding motif, Arg-Gly-Asp (RGD). Members of the integrin family that engage in Vn binding include integrin α3β1, αvβ3, αvβ5, and α5β1 (11Felding-Habermann B. Cheresh D.A. Vitronectin and its receptors.Curr. Opin. Cell Biol. 1993; 5 (7694604): 864-86810.1016/0955-0674(93)90036-PCrossref PubMed Scopus (339) Google Scholar). The next domain comprises four hemopexin-like domains with putative heme-binding motifs. In addition, Vn has three heparin-binding domains (HBD) spanning residues Vn82–137 (HBD-1), Vn175–219 (HBD-2), and Vn348–361 (HBD-3) (12Schvartz I. Seger D. Shaltiel S. Vitronectin.Int. J. Biochem. Cell Biol. 1999; 31 (10399314): 539-54410.1016/S1357-2725(99)00005-9Crossref PubMed Scopus (287) Google Scholar, 13Stanley K.K. Homology with hemopexin suggests a possible scavenging function for S-protein/vitronectin.FEBS Lett. 1986; 199 (2422056): 249-25310.1016/0014-5793(86)80489-6Crossref PubMed Scopus (13) Google Scholar, 14Liang O.D. Rosenblatt S. Chhatwal G.S. Preissner K.T. Identification of novel heparin-binding domains of vitronectin.FEBS Lett. 1997; 407 (9166893): 169-17210.1016/S0014-5793(97)00330-XCrossref PubMed Scopus (40) Google Scholar) (Fig. S1A). Vn is present in the organism in different conformational states: as the native, folded monomer (65–75 kDa, the 65-kDa form being derived from the proteolytic cleavage in the C-terminal region of the protein) in plasma/serum and as a multimeric unfolded form in the ECM (6Preissner K.T. Seiffert D. Role of vitronectin and its receptors in haemostasis and vascular remodeling.Thromb Res. 1998; 89 (9610756): 1-2110.1016/S0049-3848(97)00298-3Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 15Seiffert D. Evidence that conformational changes upon the transition of the native to the modified form of vitronectin are not limited to the heparin binding domain.FEBS Lett. 1995; 368: 155-15910.1016/0014-5793(95)00630-RCrossref PubMed Scopus (27) Google Scholar, 16Izumi M. Yamada K.M. Hayashi M. Vitronectin exists in two structurally and functionally distinct forms in human plasma.Biochim. Biophys. Acta. 1989; 990 (2465025): 101-10810.1016/S0304-4165(89)80019-4Crossref PubMed Scopus (81) Google Scholar). Conformational change from the monomeric to the activated, multimeric state is promoted by exposure of Vn to agents, such as urea, or binding to physiological ligands, such as the thrombin-antithrombin complex and the membrane attack complex. Vn conformational activation reveals a number of cryptic sites, including the full exposure of the heparin-binding site at the C-terminal domain of the protein (17Stockmann A. Hess S. Declerck P. Timpl R. Preissner K.T. Multimeric vitronectin. Identification and characterization of conformation-dependent self-association of the adhesive protein.J. Biol. Chem. 1993; 268 (7693680): 22874-22882Abstract Full Text PDF PubMed Google Scholar, 18Zhuang P. Li H. Williams J.G. Wagner N.V. Seiffert D. Peterson C.B. Characterization of the denaturation and renaturation of human plasma vitronectin. II. Investigation into the mechanism of formation of multimers.J. Biol. Chem. 1996; 271 (8663085): 14333-1434310.1074/jbc.271.24.14333Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and cell-binding motif (RGD) (19Lynn G.W. Heller W.T. Mayasundari A. Minor K.H. Peterson C.B. A model for the three-dimensional structure of human plasma vitronectin from small-angle scattering measurements.Biochemistry. 2005; 44 (15641781): 565-57410.1021/bi048347sCrossref PubMed Scopus (36) Google Scholar, 20Seiffert D. Smith J.W. The cell adhesion domain in plasma vitronectin is cryptic.J. Biol. Chem. 1997; 272 (9153222): 13705-1371010.1074/jbc.272.21.13705Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Vn binds the terminal complement C5b-7 complex. It occupies the metastable membrane binding site and thereby inhibits membrane insertion of the complex (21Milis L. Morris C.A. Sheehan M.C. Charlesworth J.A. Pussell B.A. Vitronectin-mediated inhibition of complement: evidence for different binding sites for C5b-7 and C9.Clin. Exp. Immunol. 1993; 92 (7682159): 114-11910.1111/j.1365-2249.1993.tb05956.xCrossref PubMed Scopus (74) Google Scholar). It also binds C9 and directly inhibits C9 polymerization (21Milis L. Morris C.A. Sheehan M.C. Charlesworth J.A. Pussell B.A. Vitronectin-mediated inhibition of complement: evidence for different binding sites for C5b-7 and C9.Clin. Exp. Immunol. 1993; 92 (7682159): 114-11910.1111/j.1365-2249.1993.tb05956.xCrossref PubMed Scopus (74) Google Scholar, 22Attia A.S. Ram S. Rice P.A. Hansen E.J. Binding of vitronectin by the Moraxella catarrhalis UspA2 protein interferes with late stages of the complement cascade.Infect. Immun. 2006; 74 (16495531): 1597-161110.1128/IAI.74.3.1597-1611.2006Crossref PubMed Scopus (78) Google Scholar). Several bacterial species interact with host cell-bound multimeric Vn, facilitating adherence to epithelial cells and artificial surfaces (23Singh B. Su Y.-C. Riesbeck K. Vitronectin in bacterial pathogenesis: a host protein used in complement escape and cellular invasion.Mol. Microbiol. 2010; 78 (20807208): 545-56010.1111/j.1365-2958.2010.07373.xCrossref PubMed Scopus (130) Google Scholar). Simultaneous interaction of Vn with an integrin and bacterial surface proteins results in the formation of a bridge between bacteria and host cells. This leads to internalization of bacteria, as exemplified by Streptococcus pneumoniae (24Bergmann S. Lang A. Rohde M. Agarwal V. Rennemeier C. Grashoff C. Preissner K.T. Hammerschmidt S. Integrin-linked kinase is required for vitronectin-mediated internalization of Streptococcus pneumoniae by host cells.J. Cell Sci. 2009; 122 (19118218): 256-26710.1242/jcs.035600Crossref PubMed Scopus (99) Google Scholar) or Pseudomonas aeruginosa (25Buommino E. Di Domenico M. Paoletti I. Fusco A. De Gregorio V. Cozza V. Rizzo A. Tufano M.A. Donnarumma G. AlphaVBeta5 integrins mediates Pseudomonas fluorescens interaction with A549 cells.Front. Biosci. 2014; 19 (24389192): 408-41510.2741/4215Crossref PubMed Scopus (10) Google Scholar), resulting in downstream signaling events (24Bergmann S. Lang A. Rohde M. Agarwal V. Rennemeier C. Grashoff C. Preissner K.T. Hammerschmidt S. Integrin-linked kinase is required for vitronectin-mediated internalization of Streptococcus pneumoniae by host cells.J. Cell Sci. 2009; 122 (19118218): 256-26710.1242/jcs.035600Crossref PubMed Scopus (99) Google Scholar). Staphylococci contain several Vn-binding proteins, including the autolysins AtlE and Aae from S. epidermidis and the homologous proteins AtlA and Aaa from S. aureus (26Heilmann C. Hussain M. Peters G. Götz F. Evidence for autolysin‐mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface.Mol. Microbiol. 1997; 24 (9220008): 1013-102410.1046/j.1365-2958.1997.4101774.xCrossref PubMed Scopus (540) Google Scholar, 27Heilmann C. Thumm G. Chhatwal G.S. Hartleib J. Uekötter A. Peters G. Identification and characterization of a novel autolysin (Aae) with adhesive properties from Staphylococcus epidermidis.Microbiology. 2003; 149 (14523110): 2769-277810.1099/mic.0.26527-0Crossref PubMed Scopus (139) Google Scholar). Also, the multifunctional autolysin AtlL from Staphylococcus lugdunensis interacts with Vn (28Hussain M. Steinbacher T. Peters G. Heilmann C. Becker K. The adhesive properties of the Staphylococcus lugdunensis multifunctional autolysin AtlL and its role in biofilm formation and internalization.Int. J. Med. Microbiol. 2015; 305 (25515664): 129-13910.1016/j.ijmm.2014.11.010Crossref PubMed Scopus (31) Google Scholar). Atl autolysins have a similar modular organization (signal peptide, propeptide, amidase activity, three major repeats, R1 to R3, and glucosaminidase activity), share a high degree of sequence similarity, and are functionally interchangeable (29Zoll S. Schlag M. Shkumatov A.V. Rautenberg M. Svergun D.I. Götz F. Stehle T. Ligand-binding properties and conformational dynamics of autolysin repeat domains in staphylococcal cell wall recognition.J. Bacteriol. 2012; 194 (22609916): 3789-380210.1128/JB.00331-12Crossref PubMed Scopus (53) Google Scholar). R1-R2 repeats are critical for autolysin binding to Vn (30Kohler T.P. Gisch N. Binsker U. Schlag M. Darm K. Völker U. Zähringer U. Hammerschmidt S. Repeating structures of the major staphylococcal autolysin are essential for the interaction with human thrombospondin 1 and vitronectin.J. Biol. Chem. 2014; 289 (24371140): 4070-408210.1074/jbc.M113.521229Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Moreover, the major autolysin, Atl, mediates S. aureus internalization via direct interaction with host heat shock protein Hsc70 (31Hirschhausen N. Schlesier T. Schmidt M.A. Götz F. Peters G. Heilmann C. A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor.Cell. Microbiol. 2010; 12 (20642807): 1746-176410.1111/j.1462-5822.2010.01506.xCrossref PubMed Scopus (99) Google Scholar). Studies on S. aureus adhesion to and invasion of host cells have been performed with bacteria grown in rich medium containing iron (4Foster T.J. Geoghegan J.A. Ganesh V.K. Höök M. Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus.Nat. Rev. Microbiol. 2014; 12 (24336184): 49-6210.1038/nrmicro3161Crossref PubMed Scopus (715) Google Scholar). In contrast, in vivo S. aureus has restricted access to iron, and the lack of available iron leads to the upregulation of a number of genes, among which are those that encode surface determinant (Isd) proteins (32Hammer N.D. Skaar E.P. Molecular mechanisms of Staphylococcus aureus iron acquisition.Annu. Rev. Microbiol. 2011; 65 (21639791): 129-14710.1146/annurev-micro-090110-102851Crossref PubMed Scopus (200) Google Scholar). The Isd system contains nine proteins whose expression is coordinately upregulated under iron-depleted conditions (33Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.A. Schneewind O. Passage of heme-iron across the envelope of Staphylococcus aureus.Science. 2003; 299 (12574635): 906-90910.1126/science.1081147Crossref PubMed Scopus (447) Google Scholar, 34Skaar E.P. Schneewind O. Iron-regulated surface determinants (Isd) of Staphylococcus aureus: stealing iron from heme.Microbes Infect. 2004; 6 (15101396): 390-39710.1016/j.micinf.2003.12.008Crossref PubMed Scopus (156) Google Scholar, 35Dryla A. Gelbmann D. Von Gabain A. Nagy E. Identification of a novel iron regulated staphylococcal surface protein with haptoglobin‐haemoglobin binding activity.Mol. Microbiol. 2003; 49 (12823809): 37-5310.1046/j.1365-2958.2003.03542.xCrossref PubMed Scopus (121) Google Scholar, 36Clarke S.R. Wiltshire M.D. Foster S.J. IsdA of Staphylococcus aureus is a broad spectrum, iron‐regulated adhesin.Mol. Microbiol. 2004; 51 (14982642): 1509-151910.1111/j.1365-2958.2003.03938.xCrossref PubMed Scopus (117) Google Scholar). The primary role of Isd proteins is to capture heme from hemoglobin (Hb) and transport it into the cell (32Hammer N.D. Skaar E.P. Molecular mechanisms of Staphylococcus aureus iron acquisition.Annu. Rev. Microbiol. 2011; 65 (21639791): 129-14710.1146/annurev-micro-090110-102851Crossref PubMed Scopus (200) Google Scholar). These include IsdA, IsdB, IsdC, and IsdH, which are anchored to cell wall peptidoglycan by sortases and are exposed on the cell surface (37Maresso A.W. Schneewind O. Iron acquisition and transport in Staphylococcus aureus.Biometals. 2006; 19 (16718604): 193-20310.1007/s10534-005-4863-7Crossref PubMed Scopus (91) Google Scholar, 38Grigg J.C. Ukpabi G. Gaudin C.F. Murphy M.E. Structural biology of heme binding in the Staphylococcus aureus Isd system.J. Inorg. Biochem. 2010; 104 (19853304): 341-34810.1016/j.jinorgbio.2009.09.012Crossref PubMed Scopus (102) Google Scholar). Each protein contains a structurally conserved near iron transporter (NEAT) motif(s) that binds Hb and heme. IsdA and IsdC contain one NEAT domain each, whereas IsdB and IsdH contain two and three NEAT domains, respectively. The NEAT domains adopt a beta sandwich fold that consists of two five-stranded antiparallel beta sheets (39Pilpa R.M. Fadeev E.A. Villareal V.A. Wong M.L. Phillips M. Clubb R.T. Solution structure of the NEAT (NEAr Transporter) domain from IsdH/HarA: the human hemoglobin receptor in Staphylococcus aureus.J. Mol. Biol. 2006; 360 (16762363): 435-44710.1016/j.jmb.2006.05.019Crossref PubMed Scopus (78) Google Scholar). Fig. S1B shows the organization and primary sequence comparisons between the seven known NEAT domains in S. aureus. Sequence homologs of this class of proteins are found in a number of important human pathogens, such as S. lugdunensis, (40Zapotoczna M. Heilbronner S. Speziale P. Foster T.J. Iron-regulated surface determinant (Isd) proteins of Staphylococcus lugdunensis.J. Bacteriol. 2012; 194 (23002220): 6453-646710.1128/JB.01195-12Crossref PubMed Scopus (34) Google Scholar, 41Farrand A.J. Haley K.P. Lareau N.M. Heilbronner S. McLean J.A. Foster T. Skaar E.P. An iron-regulated autolysin remodels the cell wall to facilitate heme acquisition in Staphylococcus lugdunensis.Infect. Immun. 2015; 83 (26123800): 3578-358910.1128/IAI.00397-15Crossref PubMed Scopus (14) Google Scholar, 42Heilbronner S. Monk I.R. Brozyna J.R. Heinrichs D.E. Skaar E.P. Peschel A. Foster T.J. Competing for iron: duplication and amplification of the isd locus in Staphylococcus lugdunensis HKU09-01 provides a competitive advantage to overcome nutritional limitation.PLoS Genet. 2016; 12: e100624610.1371/journal.pgen.1006246Crossref PubMed Scopus (11) Google Scholar), Listeria monocytogenes (43Newton S.M. Klebba P.E. Raynaud C. Shao Y. Jiang X. Dubail I. Archer C. Frehel C. Charbit A. The svpA‐srtB locus of Listeria monocytogenes: Fur‐mediated iron regulation and effect on virulence.Mol. Microbiol. 2005; 55 (15661014): 927-94010.1111/j.1365-2958.2004.04436.xCrossref PubMed Scopus (56) Google Scholar), Bacillus anthracis (44Skaar E.P. Gaspar A.H. Schneewind O. Bacillus anthracis IsdG, a heme-degrading monooxygenase.J. Bacteriol. 2006; 188 (16428411): 1071-108010.1128/JB.188.3.1071-1080.2006Crossref PubMed Scopus (93) Google Scholar), and Streptococcus pyogenes (45Andrade M.A. Ciccarelli F.D. Perez-Iratxeta C. Bork P. NEAT: a domain duplicated in genes near the components of a putative Fe3+ siderophore transporter from Gram-positive pathogenic bacteria.Genome Biol. 2002; 3 (research0047-1) (12225586)10.1186/gb-2002-3-9-research0047Crossref PubMed Google Scholar). IsdA, IsdB, and IsdH of S. aureus are known to have other biological functions. IsdA interacts with an array of host proteins (36Clarke S.R. Wiltshire M.D. Foster S.J. IsdA of Staphylococcus aureus is a broad spectrum, iron‐regulated adhesin.Mol. Microbiol. 2004; 51 (14982642): 1509-151910.1111/j.1365-2958.2003.03938.xCrossref PubMed Scopus (117) Google Scholar) and confers resistance to the innate defenses of the human skin (46Clarke S.R. Mohamed R. Bian L. Routh A.F. Kokai-Kun J.F. Mond J.J. Tarkowski A. Foster S.J. The Staphylococcus aureus surface protein IsdA mediates resistance to innate defenses of human skin.Cell. Host Microbe. 2007; 1 (18005699): 199-21210.1016/j.chom.2007.04.005Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). IsdH plays a role in the evasion of phagocytosis as a result of accelerated degradation of C3b (47Visai L. Yanagisawa N. Josefsson E. Tarkowski A. Pezzali I. Rooijakkers S.H.M. Foster T.J. Speziale P. Immune evasion by Staphylococcus aureus conferred by iron-regulated surface determinant protein IsdH.Microbiology. 2009; 155: 667-67910.1099/mic.0.025684-0Crossref PubMed Scopus (49) Google Scholar). IsdB binds to platelets via direct interaction with the platelet integrin GPIIb/IIIa and also promotes S. aureus adherence to and internalization by nonphagocytic human cells (48Zapotoczna M. Jevnikar Z. Miajlovic H. Kos J. Foster T.J. Iron-regulated surface determinant B (IsdB) promotes Staphylococcus aureus adherence to and internalization by non-phagocytic human cells.Cell Microbiol. 2013; 15 (23279065): 1026-104110.1111/cmi.12097Crossref PubMed Scopus (42) Google Scholar). The objective of the current study was to investigate in more detail the binding of Vn to S. aureus cells. We show that cells expressing IsdB specifically bind to Vn and analyze the nature and the biological consequences of this interaction. In preliminary experiments, we tested the capture of Vn by S. aureus strain SH1000 grown to stationary phase in rich brain heart infusion (BHI) or iron-restricted Roswell Park Memorial Institute 1640 (RPMI) medium with or without FeCl3. Bacteria grown in RPMI showed a higher ability to capture Vn than those grown in iron-rich medium or in RPMI supplemented with FeCl3, suggesting that binding of strain SH1000 depends on proteins induced by iron starvation. Interestingly, a protein A-deficient (spa) mutant showed a Vn-binding profile overlapping that of the WT strain, suggesting that Vn binding to the bacterial surface is not related to protein A expression (Fig. 1, A and B). To further analyze the interaction of Vn with S. aureus, SH1000 spa organisms grown in BHI and RPMI medium were immobilized onto microtiter wells and allowed to interact with increasing amounts of soluble Vn (Fig. 1C). Under both conditions, Vn bound to bacteria in a dose-dependent and saturable fashion. RPMI-grown bacteria captured significantly larger amounts of Vn, suggesting that S. aureus cells express a larger number of receptors on their surface or that novel receptors were induced when grown in iron starvation. To compare the Vn binding potential of different S. aureus strains, spa mutants of the S. aureus laboratory strains Newman, SH1000, and 8325-4 and the clinical strain USA300 grown in BHI or RPMI medium to the stationary phase were immobilized in microtiter wells and tested for binding to soluble Vn. All the strains grown in RPMI medium showed a higher ability to bind Vn when grown under iron starvation conditions than in iron-rich medium (Fig. 1D). To identify the surface component(s) involved in Vn binding, SH1000 spa cells grown in BHI or RPMI medium were digested with lysostaphin and the released material subjected to SDS-PAGE under reducing conditions and far Western blotting. The nitrocellulose membrane was incubated with Vn, and the bound ligand was detected with anti-Vn IgG. A strong signal corresponding to a molecule of 75 kDa was noted in protein from cells grown in RPMI medium, whereas no significant signal was detected in material released from cells grown in BHI medium (Fig. 2, lanes 1 and 2). To further investigate this issue, proteins were separated by SDS-PAGE under reducing conditions and visualized by staining with Coomassie brilliant blue (Fig. 2, lane 3). A candidate protein of 75 kDa was excised from the stained gel, digested with trypsin, and analyzed by MS. The database search unequivocally identified IsdB as the potential Vn-binding protein of S. aureus SH1000 (Fig. S2A). Since S. aureus SH1000 spa cells grown to stationary phase in BHI medium bound Vn significantly less than when grown in RPMI medium (Fig. 1), we analyzed the expression of the isdB gene in cells grown both in BHI or RPMI medium to mid-exponential and stationary phases of growth by quantitative RT-PCR (qRT-PCR). Expression of isdB in cells grown to stationary phase in RPMI medium was about 5-fold higher than that in cells grown in BHI medium or cells grown to mid-exponential phase in either medium (Fig. S2B). This finding was validated by comparing the expression levels of the IsdB protein by bacteria grown to mid-exponential and stationary phases in BHI and RPMI media by Western immunoblotting. While IsdB was virtually undetectable in material released from bacteria grown to both the mid-exponential and stationary phases in BHI medium, the protein was abundant in material released from bacteria grown to the stationary phase in RPMI medium (Fig. S2C). To study IsdB in isolation from other S. aureus CWA proteins, a strain of L. lactis expressing IsdB from a gene cloned into the plasmid vector pNZ8037 was used. To validate the expression of IsdB from the bacterium, L. lactis pNZ8037::isdB and the isogenic strain carrying the empty vector were treated with mutanolysin and lysozyme, and the released material subjected to far Western blotting. A Vn binding protein of 75 kDa was detected (along with an approximately 60-kDa protein, which may be a breakdown product), whereas the material released from L. lactis (pNZ8037) lacked reactivity (Fig. 3A). Moreover, the lactococci were immobilized onto microtiter wells, and binding of soluble Vn was examined by ELISA. Significantly higher binding of Vn to L. lactis (pNZ8037::isdB) was observed compared to that of L. lactis harboring the empty vector (Fig. 3B). To investigate the specificity of Vn binding to IsdB, recombinant IsdB NEAT1-NEAT2 protein was immobilized onto microtiter wells and tested for binding to extracellular matrix proteins, including fibrinogen, fibronectin, collagen, and Vn. Only Vn bound to the surface-coated IsdB, whereas no binding of the other proteins was observed (Fig. 4A). To localize the Vn-binding region within the IsdB protein, the NEAT1 and NEAT2 domains were expressed in E. coli and employed in binding studies. First, the binding of soluble Vn to immobilized recombinant NEAT1 and NEAT2 was determined by ELISA. Vn bound dose dependently and saturably to both NEAT1 and NEAT2 fragments and with a binding profile resembling that exhibited by intact full-length IsdB NEAT1-NEAT2 (Fig. 4B). Surface plasmon resonance (SPR) experiments were performed to compare the binding of Vn to those of immobilized IsdB NEAT1-NEAT2 and single NEAT1 and NEAT2 fragments. Vn displayed a high binding activity, as indicated by the high response values and the slow dissociation of the IsdB-Vn complexes upon removal of the ligand. The best fit of the data points was obtained with the Langmuir isotherm equation describing a one-site binding model. From this analysis, we obtained dissociation constant (KD) values of 17.85 ± 3.51 nm, 16.73 ± 3.02 nm, and 16.21 ± 2.79 nm for IsdB NEAT1-NEAT2/Vn, NEAT1/Vn, and NEAT2/Vn, respectively (Fig. 5). All these findings show that full-length IsdB contains two separate binding sites that interact with nearly identical high affinities for Vn. To investigate whether IsdB competes with glycosaminoglycans for binding to Vn, the effect of heparin, heparan sulfate, and chondroitin sulfate on Vn binding to immobilized IsdB NEAT1-NEAT2 was studied. In contrast to heparan sulfate and chondroitin sulfate, heparin dose dependently inhibited the IsdB-Vn interaction, suggesting that IsdB interacts with the heparin-binding domain(s) of the protein (Fig. S3A). To" @default.
• W3033014932 created "2020-06-12" @default.
• W3033014932 creator A5003945619 @default.
• W3033014932 creator A5018841163 @default.
• W3033014932 creator A5023919296 @default.
• W3033014932 creator A5049873457 @default.
• W3033014932 creator A5069055076 @default.
• W3033014932 creator A5074636544 @default.
• W3033014932 date "2020-07-01" @default.
• W3033014932 modified "2023-10-01" @default.
• W3033014932 title "The iron-regulated surface determinant B (IsdB) protein from Staphylococcus aureus acts as a receptor for the host protein vitronectin" @default.
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