Engineering the Substrate Specificity of Staphylococcus aureus Sortase A

The Staphylococcus aureus transpeptidase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine and the pentaglycine cross-bridge of cell wall peptidoglycan. Genetic and biochemical studies strongly suggest that SrtA and SrtB exhibit exquisite specificity for their recognition motifs. To better understand the origins of substrate specificity within these two isoforms, we used sequence and structural analysis to predict residues and domains likely to be involved in conferring substrate specificity. Mutational analyses and domain swapping experiments were conducted to test their function in substrate recognition and specificity. Marked changes in the specificity profile of SrtA were obtained by replacing the β6/β7 loop in SrtA with the corresponding domain from SrtB. The chimeric β6/β7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a kcat/Kmapp of 0.0062 ± 0.003 m-1 s-1. This enzyme was unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpeptidation to occur. The overall catalytic specificity profile (kcat/Kmapp(NPQTN)/kcat/Kmapp(LPETG)) of SrtLS was altered 700,000-fold from SrtA. These results indicate that the β6/β7 loop is an important site for substrate recognition in sortases. The Staphylococcus aureus transpeptidase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine and the pentaglycine cross-bridge of cell wall peptidoglycan. Genetic and biochemical studies strongly suggest that SrtA and SrtB exhibit exquisite specificity for their recognition motifs. To better understand the origins of substrate specificity within these two isoforms, we used sequence and structural analysis to predict residues and domains likely to be involved in conferring substrate specificity. Mutational analyses and domain swapping experiments were conducted to test their function in substrate recognition and specificity. Marked changes in the specificity profile of SrtA were obtained by replacing the β6/β7 loop in SrtA with the corresponding domain from SrtB. The chimeric β6/β7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a kcat/Kmapp of 0.0062 ± 0.003 m-1 s-1. This enzyme was unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpeptidation to occur. The overall catalytic specificity profile (kcat/Kmapp(NPQTN)/kcat/Kmapp(LPETG)) of SrtLS was altered 700,000-fold from SrtA. These results indicate that the β6/β7 loop is an important site for substrate recognition in sortases. Recently, much attention has been focused on the role of surface protein virulence factors in the pathogenesis of Gram-positive bacteria (1Paterson G.K. Mitchell T.J. Trends Microbiol. 2004; 12: 89-95Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 2Sabet C. Lecuit M. Cabanes D. Cossart P. Bierne H. Infect. Immun. 2005; 73: 6912-6922Crossref PubMed Scopus (134) Google Scholar, 3Tendolkar P.M. Baghdayan A.S. Shankar N. J. Bacteriol. 2006; 188: 2063-2072Crossref PubMed Scopus (94) Google Scholar). These covalently anchored proteins mediate such diverse processes as adherence to and invasion of host endothelial tissues, immune system evasion, and iron acquisition (4Marraffini L.A. Dedent A.C. Schneewind O. Microbiol. Mol. Biol. Rev. 2006; 70: 192-221Crossref PubMed Scopus (532) Google Scholar, 5Maresso A.W. Schneewind O. Biometals. 2006; 19: 193-203Crossref PubMed Scopus (99) Google Scholar). Inhibition of surface protein anchoring has thus emerged as a possible new therapeutic strategy for treatment of Gram-positive infections (6Cazzola M. Page C.P. Matera M.G. Curr. Opin. Pulm. Med. 2004; 10: 204-210Crossref PubMed Scopus (9) Google Scholar). Sortases are a family of transpeptidases that catalyze the covalent attachment of these surface proteins to the peptidoglycan layer of the Gram-positive cell wall (4Marraffini L.A. Dedent A.C. Schneewind O. Microbiol. Mol. Biol. Rev. 2006; 70: 192-221Crossref PubMed Scopus (532) Google Scholar). Several lines of evidence support a role for sortases in the pathogenicity of infections. Strains of Staphylococcus aureus in which srtA was genetically deleted are unable to anchor surface proteins in vivo and show a reduced ability to form renal abscesses in mice (7Mazmanian S.K. Liu G. Jensen E.R. Lenoy E. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5510-5515Crossref PubMed Scopus (385) Google Scholar). The srtA knock-out in Listeria monocytogenes has been shown to be less invasive in vitro, fails to anchor internalin to the cell wall in vivo, and is reduced in its ability to colonize the liver and spleen in a mouse model of infection (8Garandeau C. Reglier-Poupet H. Dubail I. Beretti J.L. Berche P. Charbit A. Infect. Immun. 2002; 70: 1382-1390Crossref PubMed Scopus (107) Google Scholar, 9Bierne H. Mazmanian S.K. Trost M. Pucciarelli M.G. Liu G. Dehoux P. Jansch L. Garcia-del Portillo F. Schneewind O. Cossart P. Mol. Microbiol. 2002; 43: 869-881Crossref PubMed Scopus (194) Google Scholar). In Streptococcus gordonii, srtA inactivation leads to decreased surface protein display, reduced fibronectin binding in vitro, and a reduced ability to colonize the oral mucosa of mice (10Bolken T.C. Franke C.A. Jones K.F. Zeller G.O. Jones C.H. Dutton E.K. Hruby D.E. Infect. Immun. 2001; 69: 75-80Crossref PubMed Scopus (96) Google Scholar). Likewise, srtA knockouts from Streptococcus family members S. mutans (11Lee S.F. Boran T.L. Infect. Immun. 2003; 71: 676-681Crossref PubMed Scopus (103) Google Scholar), S. pneumonia (12Paterson G.K. Mitchell T.J. Microbes Infect. 2006; 8: 145-153Crossref PubMed Scopus (65) Google Scholar), S. pyogenes (13Barnett T.C. Scott J.R. J. Bacteriol. 2002; 184: 2181-2191Crossref PubMed Scopus (103) Google Scholar), S. suis (14Osaki M. Takamatsu D. Shimoji Y. Sekizaki T. J. Bacteriol. 2002; 184: 971-982Crossref PubMed Scopus (80) Google Scholar), S. sanguinis (15Yamaguchi M. Terao Y. Ogawa T. Takahashi T. Hamada S. Kawabata S. Microbes Infect. 2006; 8: 2791-2796Crossref PubMed Scopus (63) Google Scholar), and S. agalactiae (16Lalioui L. Pellegrini E. Dramsi S. Baptista M. Bourgeois N. Doucet-Populaire F. Rusniok C. Zouine M. Glaser P. Kunst F. Poyart C. Trieu-Cuot P. Infect. Immun. 2005; 73: 3342-3350Crossref PubMed Scopus (95) Google Scholar) have been shown to have attenuated pathogenicity. Most Gram-positive bacterial species whose genomes have been sequenced to date contain at least one sortase homolog, and many species contain multiple sortase genes (17Pallen M.J. Lam A.C. Antonio M. Dunbar K. Trends Microbiol. 2001; 9: 97-102Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In many cases, putative sortase genes are located within operons that contain genes encoding potential sortase substrates (proteins with C-terminal LPXTG motifs). This raises the possibility that many of these additional sortase genes are co-expressed along with their substrates, suggesting that they might play discrete functional roles by anchoring different subsets of surface proteins (17Pallen M.J. Lam A.C. Antonio M. Dunbar K. Trends Microbiol. 2001; 9: 97-102Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). the of S. are two sortase isoforms, Sortase A (SrtA) and Sortase B SrtA is for anchoring proteins a C-terminal which of an LPXTG followed by a less conserved domain and a Schneewind O. Mol. Microbiol. PubMed Scopus Google Scholar, O. P. 70: Full Text PDF PubMed Scopus Google Scholar). the domain and the to the putative surface protein in the to SrtA the LPXTG sequence between the threonine and residues by of a site H. Mazmanian S.K. L. Schneewind O. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is which is by from an on the cross-bridge of This leads to covalent attachment of the of the surface protein to from which is the bacterial cell wall H. Mazmanian S.K. Schneewind O. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). SrtB to SrtA with two SrtB has a specificity profile different from that of SrtA. selectively anchors the NPQTN-containing protein and anchor proteins S.K. H. K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). the anchor for SrtB is peptidoglycan is the cross-bridge of peptidoglycan from the cell wall L.A. Schneewind O. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In the for SrtB is located along with its substrate in an which is of iron S.K. H. K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). the acquisition of iron is an for bacterial of host Rev. Infect. PubMed Scopus Google Scholar), is likely that SrtB an function for virulence by anchoring a protein with a role in iron acquisition to a anchor in the cell wall S.K. M. P. J. A. Schneewind O. 2003; PubMed Scopus Google Scholar). SrtA and SrtB are for their recognition to date is these between their we the of domain and analysis to of SrtA and SrtB. were from used multiple for and were and were used with were from and used was on an protein system with a and a were used to the was an system with an and a a was on a was on an and of was in the M. P. 2004; 43: PubMed Scopus Google Scholar). were in at to an of was to of and were by after were in and with an was by and to a The was with for was a of and were were and was to was a m-1 was by of was used as a for of by the The was the and was and were by and of was from the of S. aureus the and The was with and and with the which been with and to the This was and with a and the sequence was by and were used to the from the and was a to the This was and were in with were to an of and was to of were at and by were in the and protein was in the as for was to was the m-1 was by and of Sortase of S. aureus SrtA and SrtB were used as for a S.F. T.L. J. PubMed Scopus Google Scholar) were on to of to whereas two of with to SrtB. these and proteins with of analysis reduced to that with SrtA SrtB. T.J. PubMed Scopus Google Scholar) was used to a multiple sequence of these C. J. J. Mol. Biol. 2000; PubMed Scopus Google Scholar) was used to an of a of were T.L. C. Proc. Mol. Biol. Google Scholar). analysis was on a of SrtA SrtB were a multiple sequence Scholar) and the of SrtA and SrtB In of sortase studies M. P. 2004; 43: PubMed Scopus Google Scholar), analysis putative sequence and residues likely to be involved in substrate and of a β6/β7 the sequence of the of by was used PubMed Scopus Google Scholar). The M. P. 2004; 43: PubMed Scopus Google Scholar) and were used as for of were to which the sequence of from the to the β6/β7 the sequence of for the and the sequence of from the loop to the of the by was to the of PubMed Scopus Google Scholar). were and used as for the of The were used are from The was with and that been with and and of the and of M. P. 2004; 43: PubMed Scopus Google Scholar) was in which were in with were to an of with and at were by at for in and with an The was by and to a The was with and was a of were and a with were and to was the m-1 of and and the were the by the and used for of SrtA. were by of and were at and were in the protein were of the were obtained m-1 m-1 m-1 was to a with a for were on an model were at from to in with a of were for and were and for The was to the in is the protein is the in and is the of residues in the protein for for and for of and were by the strategy on on a an were from the with a for The were the of the was in a and to A was used to the to was used to the of the for Sortase of P. 2004; PubMed Scopus Google Scholar) for SrtA was to SrtB with the were with of enzyme and the substrates and were in a in at and the enzyme to was of the were by the of the were for and were by the of of of the was a and a from to corresponding to of the substrate and were at the corresponding to of the substrate and the transpeptidation were by with at and at of the of the substrate and at for of the of substrate to of SrtB was by formation as a function of the of the of enzyme was used in a these were to of the The substrate was from to whereas was at was used to the to the of the and for of and SrtLS with were conducted a of enzyme and were for and and the of was as for SrtB. of the kcat/Kmapp were obtained the is the is the of and is the of for enzyme with were and of the of from the were with a and a were in of in and an were in and corresponding to putative were and by SrtA and SrtB have different specificity SrtA anchors whereas SrtB is for NPQTN-containing proteins S.K. H. K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, M. P. 2004; 43: PubMed Scopus Google Scholar). In to better understand the for we used sequence analysis to at SrtA and SrtB from Gram-positive for sequence that were conserved SrtA and SrtB were analysis of the of S. aureus SrtA and SrtB to domains that between the two Y. H. Schneewind O. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Mazmanian S.K. Schneewind O. 2004; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). and were used to the of on SrtA specificity. and of Sortase A and B multiple sequence of of Sortase A and B from different Gram-positive species was the of the of conserved residues and The site in was conserved sortase as were the and residues and in These conserved residues are located within two conserved domains along the and which the sortase analysis of a of the of conserved of these were to both SrtA and SrtB these were the of SrtA and SrtB from S. they to structural and to the and which the of the sortase the and of both of the The of both proteins is and sequence to a conserved in SrtA and SrtB was used to for conserved in of SrtA and SrtB from Gram-positive as to both SrtA and SrtB were and of these conserved structural and and which the for the sortase and contain the catalytic and was SrtB and a structural in SrtA. and are to SrtB and the structural β6/β7 of on β6/β7 β6/β7 loop in a new In to these sortase was a in SrtA and two that were to SrtB was in SrtB the of was to to the a structural in SrtA and were to SrtB and they to the structural the and and the β6/β7 This was the in analysis in SrtA and SrtB to an structural the of of these within SrtA and SrtB and their between isoforms, that is of the their and their are in multiple sequence and analysis are for at between related these between isoforms, the much of SrtA the To between SrtA and SrtB isoforms, we a of a SrtA and SrtB the conserved site we for residues that between SrtA and SrtB residues in The residues to the catalytic in S. aureus in S. aureus is conserved as an in of the SrtA as a in the SrtB This an site of for SrtA for SrtB. the residues to the catalytic in S. aureus in S. aureus is an in SrtA and in SrtB. the of sortases are of these residues residues from the catalytic be to have their to the catalytic residues and be to and the The of the in as with the in might to the binding of the NPQTN sequence the LPXTG of SrtA In the and residues in the SrtB site are both of which might better the substrate for by To test these we the residues and from SrtA for of SrtA the SrtA and substrate we the SrtA and as as the SrtA that the and were an NPQTN The SrtA was both and NPQTN the of the was to SrtA and to substrate of the β6/β7 as a on the specificity of we to domains that between SrtA and SrtB and thus might be to be important for specificity. A of the of SrtA and SrtB was to domains that in between the two sequence have shown that S. aureus SrtA and SrtB have sequence residues G. Mazmanian S.K. P. Schneewind O. A. 2004; 12: Full Text Full Text PDF PubMed Scopus Google their show overall The overall with a of within the the two proteins are with a of of the structural between SrtA and SrtB in and in the Most these structural are the of an domain in SrtB and the different and of the β6/β7 in the two In is a loop whereas in is and of a an and In the β6/β7 loop conserved such as and which are by residues in such as and the and of the β6/β7 are different in SrtA and they structural with the of of the the in both at the in at the The loop in SrtA and the loop of the domain in SrtB are both at the of the site and to be located to the residues of the recognition in SrtA substrates, in SrtB binding is to be required for SrtA U. H. J. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). analyses by N. U. J. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) that the of the β6/β7 loop in SrtA that the loop is in the of In the the loop between a in which the loop is the and an in which the loop is from the The form of SrtA a binding of residues and loop and a conserved on the β6/β7 binding thus the of the and β6/β7 the β6/β7 loop the N. U. J. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). with SrtB a putative site on the loop and and a conserved on the β6/β7 To a analysis of the of SrtB has been the between the and of SrtA and to that SrtB to the of its β6/β7 for substrate binding and of a β6/β7 on the lines of we that the β6/β7 might be an important role in substrate and the different of SrtA and SrtB. In to test we a loop swap enzyme we the β6/β7 loop of SrtA with the corresponding loop from SrtB. This chimeric protein the sequence of SrtA for residues and the sequence of SrtB for the residues in in and the sequence of SrtA for the in in the β6/β7 loop is in SrtB in SrtA by the C-terminal residues in SrtLS to be from their corresponding residues in SrtA from SrtA in of SrtB with as a for we SrtB an to one by to the of SrtA P. 2004; PubMed Scopus Google Scholar). In of the SrtA substrate we used the NPQTN-containing SrtB is less and of enzyme SrtB and were required to of of the were with SrtB a of ± and a for of ± studies have a of ± for and a of ± for with D.E. 2005; PubMed Scopus Google Scholar). These show that SrtB has a for substrate that is within the of that for SrtA a that is of and changes to the sequence of a protein changes in the and In to the of the loop swap on the of we the of and SrtLS is a of SrtA and was that the of SrtLS a of the of SrtA and SrtB. for and SrtLS are shown in The for SrtA a of and with the of the enzyme Y. H. Schneewind O. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, U. H. J. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). SrtA is the used for and is to be SrtB a with an of and which is in with the for protein G. Mazmanian S.K. P. Schneewind O. A. 2004; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). SrtLS has a that is a of the of the of SrtA and SrtB has the as the SrtA has in the The domain from SrtB an that is in SrtA. the of domain in SrtLS be to to the which is was on these that SrtLS structural to both the of SrtA and in a SrtLS for and altered substrate specificity. of Sortase the role of the β6/β7 loop swap on substrate we of and SrtLS with LPXTG and NPQTN substrates to and the SrtLS and SrtLS as as the SrtLS were on their catalytic their of and substrate were used be the the of of of the was to of substrate in to of of obtained for SrtA and SrtB were to obtained by of and and was to be for SrtA with its substrate with a kcat/Kmapp of ± m-1 in with P. 2004; PubMed Scopus Google Scholar, D.E. 2005; PubMed Scopus Google Scholar). SrtB the after at and an enzyme of This is with studies in which was for SrtB in S.K. H. K. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). SrtLS was to the substrate with a reduced kcat/Kmapp of ± m-1 This to a in LPXTG cleavage by the loop swap enzyme suggesting that of the β6/β7 loop was to recognition and of the for sortase were at to the for of kcat/Kmapp as to the of of of the β6/β7 loop from SrtA with the loop from SrtB an enzyme (SrtLS) with a kcat/Kmapp for NPQTN SrtB and a specificity 700,000-fold from ± ± ± ± ± ± ± ± ± ± in a new with SrtA a of the such of NPQTN cleavage in SrtA. the of cleavage was with kcat/Kmapp to ± m-1 s-1. with its cleavage an for NPQTN-containing recognition by SrtA. SrtB the NPQTN as with a kcat/Kmapp of ± m-1 in with from the of SrtB The loop swap enzyme SrtLS the NPQTN with an kcat/Kmapp of ± m-1 a of SrtLS for NPQTN to the kcat/Kmapp of SrtLS is that of SrtB. the swapping of a surface loop was to the specificity profile for NPQTN for of SrtA by a of the results of these strongly that the β6/β7 loop is an important site in SrtA and SrtB. of SrtLS to the of the conserved and residues by we of and as as the of these was and and for and NPQTN The SrtLS was to the at a kcat/Kmapp of ± m-1 of the of SrtLS was with a kcat/Kmapp of ± of the of SrtLS The SrtLS was for to the of in of the substrate the of the and on the of SrtLS these the NPQTN cleavage of they the ability of SrtLS to the to an overall in the specificity of SrtLS The of these suggest that the residues and play an important role in recognition by whereas the corresponding residues from SrtB to be important for NPQTN of the of we the from the SrtB and SrtLS with by The of both the SrtB and SrtLS at a of analysis of of the of an at corresponding to the cleavage analysis of the of the the of the SrtB at a of analysis of to contain an with corresponding to the transpeptidation of in the was an with which to the analysis of both of these their the of the SrtLS at was the at was analysis of its as the corresponding to the transpeptidation were that whereas SrtLS is of the NPQTN is unable to perform transpeptidation of the the the is by from The that is to the transpeptidation stage of the in in that are in the binding for the cell wall peptidoglycan substrates of SrtA and SrtB. A by and Schneewind L.A. Schneewind O. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus (63) Google Scholar) evidence for a anchor for the protein SrtB in suggest that surface proteins are to peptidoglycan that has and is the for This that are different peptidoglycan substrates for SrtA and SrtB. is the be in the of SrtB that to its less peptidoglycan In pentaglycine was to as an anchor for NPQTN by SrtB by was to as an anchor for that been by SrtA is possible that the of the loop in SrtLS pentaglycine from the site and thus is the of the of possible is that be additional domains on the surface of SrtB that are in SrtLS and that the of these domains is for transpeptidation of the to occur. domain for is the conserved domain in analysis which to a between and on SrtB. This is in the of SrtA and as a recognition site for the peptidoglycan substrate of SrtB. are in to swap loop to its for transpeptidation of enzyme for altered specificity has been a to recognition and In the of a was to a with a of H. T. PubMed Scopus Google Scholar). for many of changes are required to specificity A. Sci. PubMed Scopus Google Scholar, M. J. Mol. Biol. PubMed Scopus (96) Google Scholar). In of these cases, domain swapping has been as a for the specificity of related for which are H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, L. L. PubMed Scopus Google Scholar). and H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) used loop as a to In their on the specificity of of surface with the corresponding from with a in of in their on the of to L. L. PubMed Scopus Google Scholar) that was to swap two of the surface in to the specificity of the A in of was swapping of these a for SrtA that was one the in of SrtA and SrtB on the of for SrtA and for is a of the β6/β7 loop in SrtA was to to an with an of cleavage SrtB. and domain swapping have been used as to specificity and recognition in S. aureus sortase from analysis of SrtA and SrtB the specificity profile of suggesting that these residues play a role in the of the SrtA of the β6/β7 loop in SrtA with the corresponding loop from SrtB was to the specificity profile of SrtA by were unable to the of these results support the that the β6/β7 loop is a substrate recognition site in sortase studies are to additional that are involved in the recognition and of sortase substrates and that