Is the GehD Lipase from Staphylococcus epidermidis a Collagen Binding Adhesin?

Abstract

The opportunistic human pathogenStaphylococcus epidermidis is the major cause of nosocomial biomaterial infections. S. epidermidis has the ability to attach to indwelling materials coated with extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and collagen. To identify the proteins necessary for S. epidermidisattachment to collagen, we screened an expression library using digoxigenin-labeled collagen as well as two monoclonal antibodies generated against the Staphylococcus aureuscollagen-adhesin, Cna, as probes. These monoclonal antibodies recognize collagen binding epitopes on the surface of S. aureus andS. epidermidis cells. Using this approach, we identified GehD, the extracellular lipase originally found in S. epidermidis 9, as a collagen-binding protein. Despite the monoclonal antibody cross-reactivity, the GehD amino acid sequence and predicted structure are radically different from those of Cna. The mature GehD circular dichroism spectra differs from that of Cna but strongly resembles that of a mammalian cell-surface collagen binding receptor, known as the α1 integrin I domain, suggesting that they have similar secondary structures. The GehD protein is translated as a preproenzyme, secreted, and post-translationally processed into mature lipase. GehD does not have the conserved LPXTG C-terminal motif present in cell wall-anchored proteins, but it can be detected in lysostaphin cell wall extracts. A recombinant version of mature GehD binds to collagens type I, II, and IV adsorbed onto microtiter plates in a dose-dependent saturable manner. Recombinant, mature GehD protein and anti-GehD antibodies can inhibit the attachment of S. epidermidis to immobilized collagen. These results provide evidence that GehD may be a bi-functional molecule, acting not only as a lipase but also as a cell surface-associated collagen adhesin. The opportunistic human pathogenStaphylococcus epidermidis is the major cause of nosocomial biomaterial infections. S. epidermidis has the ability to attach to indwelling materials coated with extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and collagen. To identify the proteins necessary for S. epidermidisattachment to collagen, we screened an expression library using digoxigenin-labeled collagen as well as two monoclonal antibodies generated against the Staphylococcus aureuscollagen-adhesin, Cna, as probes. These monoclonal antibodies recognize collagen binding epitopes on the surface of S. aureus andS. epidermidis cells. Using this approach, we identified GehD, the extracellular lipase originally found in S. epidermidis 9, as a collagen-binding protein. Despite the monoclonal antibody cross-reactivity, the GehD amino acid sequence and predicted structure are radically different from those of Cna. The mature GehD circular dichroism spectra differs from that of Cna but strongly resembles that of a mammalian cell-surface collagen binding receptor, known as the α1 integrin I domain, suggesting that they have similar secondary structures. The GehD protein is translated as a preproenzyme, secreted, and post-translationally processed into mature lipase. GehD does not have the conserved LPXTG C-terminal motif present in cell wall-anchored proteins, but it can be detected in lysostaphin cell wall extracts. A recombinant version of mature GehD binds to collagens type I, II, and IV adsorbed onto microtiter plates in a dose-dependent saturable manner. Recombinant, mature GehD protein and anti-GehD antibodies can inhibit the attachment of S. epidermidis to immobilized collagen. These results provide evidence that GehD may be a bi-functional molecule, acting not only as a lipase but also as a cell surface-associated collagen adhesin. microbial surface components recognizing adhesive matrix molecules phosphate-buffered saline monoclonal antibody (Ab) bovine serum albumin enzyme-linked immunosorbent assay Staphylococcus epidermidis is now recognized as an important nosocomial pathogen. In the past 20 years it has emerged as a frequent cause of infections associated with indwelling devices such as catheters, artificial heart valves, and orthopedic implants (1Garrett D.O. Jochimsen E. Murfitt K. Hill B. McAllister S. Nelson P. Spera R.V. Sall R.K. Tenover F.C. Johnston J. Zimmer B. Jarvis W.R. Infect. Control. Hosp. Epidemiol. 1999; 20: 167-170Crossref PubMed Scopus (96) Google Scholar). In certain populations such as low birth weight infants and immuno-compromised patients S. epidermidis can be a prominent source of morbidity and mortality (2de Silva G.D.I. Justice A. Wilkinson A.R. Buttery J. Herbert M. Day N.P.J. Peacock S.J. Clin. Infect. Dis. 2001; 33: 1520-1528Crossref PubMed Scopus (28) Google Scholar). The molecular mechanisms of pathogenesis of S. epidermidisdisease are not well understood, but as with most infections, bacterial adherence to host surfaces is recognized as the first crucial step in the infection process and a prerequisite for colonization. A two-step process of S. epidermidis adherence is often described in which the first step is bacterial attachment to the biomaterial, and the second step includes microbial proliferation, intercellular adhesion, and biofilm formation. Almost all S. epidermidisstrains are able to attach to native abiotic surfaces (3Hogt A.H. Dankert J. Feijen J. J. Biomed. Mater. Res. 1986; 20: 533-545Crossref PubMed Scopus (135) Google Scholar, 4Pascual A. Fleer A. Westerdaal N.A. Verhoef J. Eur. J. Clin. Microbiol. 1986; 5: 518-522Crossref PubMed Scopus (117) Google Scholar, 5Tojo M. Yamashita N. Goldmann D.A. Pier G.B. J. Infect Dis. 1988; 157: 713-722Crossref PubMed Scopus (222) Google Scholar, 6Muller E. Hubner J. Gutierrez N. Takeda S. Goldmann D.A. Pier G.B. Infect. Immun. 1993; 61: 551-558Crossref PubMed Google Scholar). However, any foreign material implanted into the human body is quickly coated with various plasma proteins such as fibrinogen, fibronectin, and vitronectin (7Herrmann M. Vaudaux P., D. Pittet R.A. Lew P.D. Schumacher- Perdreau F. Peters G. Waldvogel F.A. J. Infect. Dis. 1988; 158: 693-701Crossref PubMed Scopus (429) Google Scholar, 8Yu J. Montelius M.N. Paulsson M. Gouda I. Larm O. Montelius L. Ljungh A. Biomaterials. 1994; 15: 805-814Crossref PubMed Scopus (36) Google Scholar), and Staphylococcus aureus, which is also a common cause of biomaterial centered infections, appears to adhere to this protein coat via adhesins of the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)1 type. Analysis of the adherence behavior of S. epidermidissuggests that this organism also expresses MSCRAMMs. In fact, a gene encoding a fibrinogen binding MSCRAMM (sdrG, also calledfbe) was cloned and sequenced from S. epidermidis(9Nilsson M. Frykberg L. Flock J.I. Lindberg M. Guss B. Infect. Immun. 1998; 66: 2666-2673Crossref PubMed Google Scholar). SdrG, a 119-kDa MSCRAMM, has a structural organization similar to the clumping factor (ClfA) from S. aureus and specifically recognizes the N-terminal region of the fibrinogen Bβ chain (10Davis S.L. Gurusiddappa S. McCrea K.W. Perkins S. Höök M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In addition, the autolysin AtlE, necessary for S. epidermidisattachment to polystyrene, was shown to specifically bind to biotin-labeled vitronectin (11Heilmann C. Hussain M. Peters G. Gotz F. Mol. Microbiol. 1997; 24: 1013-1024Crossref PubMed Scopus (574) Google Scholar). These data indicate that S. epidermidis, similarly to S. aureus, may express specific MSCRAMMs that mediate cell attachment to host protein-conditioned surfaces. In the present communication, we report that the GehD (12Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Microbiology. 2000; 146: 1419-1427Crossref PubMed Scopus (59) Google Scholar) lipase binds to collagen type I, II, and IV and may mediate the adherence ofS. epidermidis cells to immobilized collagens. We identified GehD probing a S. epidermidis expression library with labeled collagen type I and monoclonal antibodies generated against theS. aureus collagen-binding protein, Cna. Staphylococcal lipases have been implicated as possible virulence factors in localized infections such as absesses (13Hedström S.A. Acta Pathol. Microbiol. Scand. Sect. B Microbiol. 1975; 83: 285-292Google Scholar, 14Hedström S.A. Nilsson-Ehle P. Acta Pathol. Microbiol. Scand. Sect. B Microbiol. 1983; 91: 169-173Google Scholar, 15Rollof J. Hedström S.A. Nilsson-Ehle P. Acta Pathol. Mocrobiol. Immunol. Scand. Sect. B Microbiol. 1987; 95: 109-113PubMed Google Scholar), and there is evidence that they are highly expressed during infection in a murine model (16Lowe A.M. Beattie D.T. Deresiewicz R.L. Mol. Microbiol. 1998; 27: 967-976Crossref PubMed Scopus (164) Google Scholar). The contribution of these enzymes to virulence is not clearly understood, although lipases may be important for the colonization and persistence of organisms on the skin (17Gribbon E.M. Cunliffe W.J. Holland K.T. J. Gen. Microbiol. 1993; 139: 1745-1751Crossref PubMed Scopus (113) Google Scholar). Another class of proteins that function as collagen binding adhesion receptors are the mammalian integrins. These proteins mediate the attachment of eukaryotic cells to the extracellular matrix. The integrins are transmembrane αβ heterodimeric proteins that mediate cell-cell and cell-matrix interactions of mammalian cells. In this extensive family of proteins, α1β1 and α2β1 are the primary collagen binding integrins. Within the α subunit of the collagen binding integrins, the ligand binding region is called I domain (33Lu C. Shimaoka M. Ferzly M. Oxvig C. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 98: 2387-2392Crossref Scopus (119) Google Scholar). Our data predict that mature GehD may adopt a structure that resembles that of the integrin α1 I-domain. The data described here show that the GehD lipase binds to collagens and may promote S. epidermidis attachment to immobilized collagens. Our data indicate that the GehD lipase may be a bifunctional molecule, acting as a glycerol ester hydrolase and a collagen adhesin. S. epidermidis strains 146, 9491, and from the S. epidermidis 9, and M. (12Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Microbiology. 2000; 146: 1419-1427Crossref PubMed Scopus (59) Google Scholar). S. aureus was of strains in heart the monoclonal antibody and in phosphate-buffered saline and to a cell of using a the A to the cell cells in a The cells for all into and the was it and in attachment A S. epidermidis expression library was as S. epidermidis was with and the to and The to the and with The was into using the The library was and screened using the of from the using the to the as The was to the E. The the for and of the cloned A encoding the mature domain of the GehD lipase was from S. epidermidis The and to the as mature generated cloned into the using the E. as a host and the E. as the expression expression and of recombinant proteins as described using (10Davis S.L. Gurusiddappa S. McCrea K.W. Perkins S. Höök M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). from the as on a The of the proteins was using the of F. L. G. Sci. PubMed Scopus Google Scholar). collagen I was labeled with ester to the To recombinant proteins with of was in of and with of recombinant protein in The was in an for against and collagen and F. S. Höök M. P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) to the S. epidermidis expression The library was using to the protein binding on the with a bovine serum albumin in 20 digoxigenin-labeled collagen in was with the The digoxigenin-labeled collagen was with to in in as antibodies in to as secondary collagen-binding proteins identified the with and To the of the generated against bacterial surface proteins, microtiter coated with of human in of to provide a surface for bacterial The with and 20 and protein binding with a of in of cells of S. epidermidis S. aureus cells and for with to cells. of of monoclonal antibody in of in for with and detected with a of The was with as a and the was using a To microtiter plates coated with of type I collagen in of with and with bovine serum albumin in for the of of the recombinant protein. for with the with 20 was with and to the for the with of was to the was using a of in and with protein to was as and from binding to collagen. as the from a The of antibodies as of proteins binding to collagen was as described that proteins with antibodies and to the The secondary structural of recombinant proteins was data using a with a acid using a of and for in 20 in for the contribution from the was and of secondary structural was of and of and D. These and are from of known protein structures. using the as described S. R.L. A. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The Cna protein was in The α1 I domain was in and mature GehD was in and from the of the for and of the and mature GehD was in to for in and of from and serum using protein plates coated with of type I collagen in of with and with bovine serum albumin in for the of S. epidermidis of and the plates for cells with of and for The plates with and on an To of collagen binding S. epidermidis with of in for The cell to plates coated with of and ability to attach to collagen was as described PubMed Scopus Google Scholar), S. epidermidis with cells E. cells in for and to a for The proteins with ligand cell proteins from the onto a in a binding on the in in for in The was with of digoxigenin-labeled for and with in for The was and collagen-binding proteins with of and of in The S. epidermidis was as a in for MSCRAMMs. We ability to adhere to immobilized bovine collagen type I, human fibrinogen, and human protein was immobilized in microtiter and the to the detected using The results in show that S. has the ability to attach to collagen, fibrinogen and have shown that S. epidermidisattachment to human fibrinogen is proteins such as and M. Frykberg L. Flock J.I. Lindberg M. Guss B. Infect. Immun. 1998; 66: 2666-2673Crossref PubMed Google Scholar, S.L. Gurusiddappa S. McCrea K.W. Perkins S. Höök M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), the bacterial components that mediate attachment to collagen to this not A of monoclonal antibodies was generated against theS. aureus MSCRAMM F. S. Höök M. P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We the that of these with collagen-binding proteins on S. epidermidis a of strains epidermidis 146, 9491, and and with cells of all the S. epidermidis strains of these antibodies against the ligand binding region of these antibodies shown to inhibit collagen binding to Cna and recognize in the ligand binding of F. S. Höök M. P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). all of the bind aureus cells. The S. epidermidisstrains are recognized only two These results that S. epidermidis on surface proteins that epitopes similar to those present on Cna and that these proteins are recognized and We an expression library from S. epidermidis to Using and as well as digoxigenin-labeled collagen, we screened We that with and labeled collagen. of the that of the and the an of sequence that the cloned of the sequence from the is to the identified S. epidermidis second lipase (12Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Microbiology. 2000; 146: 1419-1427Crossref PubMed Scopus (59) Google Scholar) of GehD and lipases have shown that they are and as acid that are processed post-translationally to extracellular mature lipases of amino with a of (12Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Microbiology. 2000; 146: 1419-1427Crossref PubMed Scopus (59) Google Scholar). To the native protein in the mature we the to recombinant mature GehD The encoding mature GehD was cloned into the expression The protein was expressed as a C-terminal and GehD appears as a acid sequence not any the amino acid of Cna and mature the spectra of mature GehD shown in A is different from that of Cna of the mature GehD data using the and that the predicted secondary structure of mature GehD of and secondary structure differs from that of the structure of and D. B. Infect. Immun. 2001; PubMed Scopus Google Scholar). In the mature GehD spectra strongly resembles that of a mammalian cell-surface collagen binding known as the α1 integrin I domain The secondary structure of this domain is and which is with that of mature The collagen binding of the mature GehD was ligand protein was to a and with digoxigenin-labeled collagen, and In this the mature GehD binds collagen and be that a second is detected in and is a that is recognized the secondary antibody to and The collagen binding of the biotin-labeled mature GehD was also a GehD in a saturable to collagens I, II, and IV coated on microtiter the binding to coated with albumin was the assay we that binding mature In addition, we the ability of mature GehD to inhibit the binding of mature GehD to immobilized collagen. GehD inhibit the binding of the labeled protein to immobilized collagen, a fibrinogen binding recombinant protein from S. epidermidis not that biotin-labeled and mature GehD bind with similar to immobilized collagen. We also to the binding of mature GehD to collagen surface In this recombinant mature GehD is a coated with type I collagen. Using a we a for the Using a we data and a of and binding for mature GehD collagen However, not only was the of mature GehD with collagen on the but the collagen binding as the mature GehD was for of these are of the mature GehD binding to collagen that we not the and be with We a microtiter well attachment assay to the adherence of S. epidermidis to collagen. of S. epidermidis a of the gene show a ability to attach to immobilized collagen with S. epidermidis However, the has a collagen A similar a in the gene has a ability to attach to collagen with These data that there may be cell surface cell attachment to collagen. The of recombinant mature GehD on bacterial adherence in in which microtiter with of recombinant mature GehD for S. epidermidis mature GehD the attachment of S. epidermidis to collagen in a but it does not the attachment of a We generated antibodies against the mature GehD protein and ability to with the binding of GehD to collagen. GehD inhibit the binding of biotin-labeled mature GehD to immobilized collagen, A similar was monoclonal antibodies The monoclonal generated against Cna the binding of mature GehD, an not inhibit In addition, we the of these using E. and S. epidermidis cell extracts. The GehD recognize a of in cell of E. gene S. is not present in the cell These data show that anti-GehD are specific for mature We these antibodies in a microtiter well attachment assay to ability to inhibit the attachment of S. epidermidis cells to immobilized collagen. S. epidermidis cells with of GehD antibodies the cell to microtiter cells detected using GehD antibodies inhibit the attachment of S. epidermidis to collagen. The not to the attachment of the A similar was the monoclonal was to the bacterial cells These data that surface-associated mature GehD may as a collagen and mediate the attachment of S. epidermidis to surfaces. In to S. aureus, the adherence of S. epidermidis to extracellular matrix proteins has not been well is known that S. epidermidis can adhere to fibrinogen, fibronectin, (7Herrmann M. Vaudaux P., D. Pittet R.A. Lew P.D. Schumacher- Perdreau F. Peters G. Waldvogel F.A. J. Infect. Dis. 1988; 158: 693-701Crossref PubMed Scopus (429) Google Scholar), and vitronectin (11Heilmann C. Hussain M. Peters G. Gotz F. Mol. Microbiol. 1997; 24: 1013-1024Crossref PubMed Scopus (574) Google Scholar). The adherence to fibrinogen is protein adhesins such as M. Frykberg L. Flock J.I. Lindberg M. Guss B. Infect. Immun. 1998; 66: 2666-2673Crossref PubMed Google Scholar) (10Davis S.L. Gurusiddappa S. McCrea K.W. Perkins S. Höök M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and attachment to vitronectin to be the autolysin However, the proteins for the interactions with collagen and have not been to for we a expression library from the S. epidermidis To we of a of that against the collagen binding MSCRAMM from S. of these and to epitopes present on the surface of S. epidermidis cells. we and labeled collagen to expression library and a collagen binding the that to and collagen expressed an N-terminal of the GehD S. lipase has the organization as the lipases and F. Chem. 1998; PubMed Scopus Google Scholar). These lipases to be as of major and mature lipase. The is for and it is during of the protein. The domain has been found to be important for and during F. Mol. Gen. 1986; PubMed Scopus Google Scholar). data (12Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Microbiology. 2000; 146: 1419-1427Crossref PubMed Scopus (59) Google Scholar) that GehD is similarly translated as a and post-translationally processed into mature lipase. The of this extracellular lipase is The mature of GehD can be found associated to cells and in lysostaphin from the cell G. the LPXTG motif associated with the proteins found in most bacterial surface proteins is not present in the of the GehD protein. cell-surface adhesins that not a LPXTG motif have been These the binding adhesins A.R. K.W. M. S. Mol. Microbiol. 2001; PubMed Scopus Google Scholar) from Infect. Immun. PubMed Google Scholar) from and the binding S. M. S. Mol. Microbiol. 2001; PubMed Scopus Google Scholar) from S. A possible for cell surface of these adhesins has been described for S. M. S. Mol. Microbiol. 2001; PubMed Scopus Google Scholar) from S. a is an and can with receptors on is it binds to and the of into the host cells. the of the GehD and the S. epidermidis cell surface is not understood, it is to similarly to it associated to the bacterial surface proteins with adhesive on the surface LPXTG J. Infect. Dis. 2000; PubMed Scopus Google Scholar), a surface of S. J. 1997; PubMed Scopus Google Scholar), a surface of A and theS. epidermidis autolysin AtlE, which specifically binds to vitronectin (11Heilmann C. Hussain M. Peters G. Gotz F. Mol. Microbiol. 1997; 24: 1013-1024Crossref PubMed Scopus (574) Google Scholar). proteins with have also been described D. B. Infect. Immun. 2001; PubMed Scopus Google Scholar, B. A. J. 2001; Scopus Google Scholar). the of adhesins identified in in the but it is not these proteins are virulence The lipases have been virulence factors in localized infections such as absesses (13Hedström S.A. Acta Pathol. Microbiol. Scand. Sect. B Microbiol. 1975; 83: 285-292Google Scholar, 14Hedström S.A. Nilsson-Ehle P. Acta Pathol. Microbiol. Scand. Sect. B Microbiol. 1983; 91: 169-173Google Scholar, 15Rollof J. Hedström S.A. Nilsson-Ehle P. Acta Pathol. Mocrobiol. Immunol. Scand. Sect. B Microbiol. 1987; 95: 109-113PubMed Google Scholar), and in (16Lowe A.M. Beattie D.T. Deresiewicz R.L. Mol. Microbiol. 1998; 27: 967-976Crossref PubMed Scopus (164) Google Scholar) that lipase gene expression is during infection in a murine The contribution of these enzymes to virulence is not clearly understood, although lipases may be important for the colonization and persistence of organisms on the acid sequence has shown that and GehD are to is to lipase from S. aureus GehD has to the S. aureus and the lipase of Staphylococcus (12Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Microbiology. 2000; 146: 1419-1427Crossref PubMed Scopus (59) Google Scholar). the lipases are a of the predicted secondary conserved be of to any of these lipases have adhesive in to The ability of this to be bi-functional may be of to the S. epidermidis colonization and on skin and artificial surfaces. of S. epidermidis in GehD to the of GehD in bacterial interactions with collagen. GehD can mediate bacterial attachment to immobilized collagen. was mature In addition, two monoclonal antibodies against Cna and antibodies against the mature GehD lipase inhibit the attachment ofS. epidermidis to collagen. the show a attachment to collagen, which the that also with collagen. is to that we not in library for collagen are two that this have a binding for collagen, a to the the sequence have been in a different to that of the The ability of recombinant to bind to collagen has not been but it is of GehD was identified as a collagen binding using against However, amino acid sequence not any the amino acid of Cna and mature the spectra of mature GehD is different from that of Cna. of the mature GehD data that the predicted secondary structure of mature GehD of and secondary structure differs from that of the structure of and J. M. K. M. D. L. Höök M. Biol. 1997; Scopus Google Scholar). These data that these proteins may have radically different structures. In the mature GehD spectra strongly resembles that of a mammalian cell surface collagen binding known as the α1 integrin I The secondary structure of this domain is and which is with that of mature We common mature GehD and the integrin α1 I bind to have similar and of and bind for and have Gotz F. 2000; PubMed Scopus Google Scholar, C. Shimaoka M. Ferzly M. Oxvig C. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 98: 2387-2392Crossref Scopus (119) Google Scholar, F. Chem. 1998; PubMed Scopus Google Scholar, C. M. A. D. M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of these common it is to that GehD and the integrin α1 I domain may bind to collagen, similar these highly may provide of the collagen binding behavior of GehD, they also the for a lipase The data described in this predict that GehD and Cna have radically different secondary structures. However, and recognize has been shown that recognizes epitopes in the of and it has been that it ligand binding with collagen the binding F. S. Höök M. P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the that the collagen binding epitopes present on recognized and may also be found in they to recognize a collagen binding these can be as to collagen-binding proteins in