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Sortase-Catalysed Anchoring of Surface Proteins to the Cell Wall of Staphylococcus Aureus

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Sortase-Catalysed Anchoring of Surface Proteins to the Cell Wall of Staphylococcus Aureus Molecular Microbiology 2001) 40 5), 1049±1057 MicroReview Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus Sarkis K. Mazmanian, Hung Ton-That and properties that may explain why these microbes can cause Olaf Schneewind* abscesses at many anatomical sites Flock, 1999). In all Department of Microbiology and Immunology, cases examined, the adhesive and serum-binding proper- UCLA School of Medicine, 10833 Le Conte Avenue, ties of staphylococci have been traced back to the Los Angeles, CA 90095, USA. expression of surface proteins Foster and HoÈoÈk, 1998). Many of these surface proteins are anchored to the cell wall envelope by a mechanism that requires a C-terminal cell wall Summary sorting signal Cws) Navarre and Schneewind, 1999). Many surface proteins of Gram-positive bacteria are anchored to the cell wall envelope by a transpeptida- tion mechanism, requiring a C-terminal sorting signal Protein A ± a cell wall-anchored surface protein with a conserved LPXTG motif. Sortase, a membrane protein of Staphylococcus aureus, cleaves polypep- Protein A, the first identified surface protein of S. aureus, tides between the threonine and the glycine of the binds to the Fc portion of antibodies and causes precipitation LPXTG motif and catalyses the formation of an of immunoglobulin Ig) Jensen, 1958; SjoÈdahl, 1977). The amide bond between the carboxyl-group of threonine N-terminal region of protein A, including five Ig-binding and the amino-group of peptidoglycan cross-bridges. domains, is displayed on the bacterial surface, whereas S. aureus mutants lacking the srtA gene fail to anchor the C-terminal region X is buried in the cell wall envelope and display some surface proteins and are impaired SjoÈquist et al., 1972a; Guss et al., 1984). SjoÈquist and in the ability to cause animal infections. Sortase acts colleagues showed that cleavage of the staphylococcal on surface proteins that are initiated into the secre- peptidoglycan withlysostaphin,a glycyl-glycine endopep- tion +Sec) pathway and have their signal peptide tidase that cuts cell wall cross-bridges, solubilizes protein removed by signal peptidase. The S. aureus genome A as a species withuniform mass SjoÈquist et al., 1972b). encodes two sets of sortase and secretion genes. It is Digestion of the glycan strands with muramidase releases conceivable that S. aureus has evolved more than protein A as a spectrum of polypeptide fragments with one pathway for the transport of 20 surface proteins linked cell wall SjoÈquist et al., 1972a, b). Molecular to the cell wall envelope. cloning and DNA sequencing of the spa gene revealed that protein A is synthesized as a precursor, containing an N-terminal signal peptide that directs the polypeptide into Introduction the secretory Sec) pathway LoÈfdahl et al., 1983; UhleÂn et al., 1984). The predicted C-terminus of protein A Staphylococcus aureus is an important human pathogen harbours the 35-residue Cws that is conserved in surface that causes abscesses in many organ tissues, septicaemia proteins of Gram-positive bacteria Fischetti et al., 1990; and several other diseases Lowy, 1998). Molecular Schneewind et al., 1992). An LPXTG motif is followed by biologists have examined staphylococci for many years in a hydrophobic domain and a tail of mostly positively an effort to explain the extraordinary pathogenic potential of charged residues Fischetti et al., 1990). Although Cws this microorganism Novick, 1991). S. aureus is capable of resemble known membrane anchor peptides, the hydro- binding several human serum factors Foster and HoÈoÈk, phobic domain of protein A and other Cws cannot insert 1998). Multiple pathogenic strategies have been suspected, fused reporter proteins into the lipid bilayer Schneewind and staphylococcal resistance to phagocytic killing seems to et al., 1992). Truncation of the Cws charged tail causes be the underlying goal in binding serum proteins. Staphylo- staphylococci to secrete mutant protein A into the extra- cocci also adhere to several different human tissues, cellular medium Schneewind et al., 1992). In contrast, Accepted 15 February, 2001. *For correspondence. E-mail olafs@ deletion of the LPXTG motif prevents cleavage and cell ucla.edu; Tel. 11) 310 206 0997; Fax 11) 310 267 0173. wall anchoring of protein A; mutant polypeptides fractionate Q 2001 Blackwell Science Ltd 1050 S. K. Mazmanian, H. Ton-That and O. Schneewind withthecell wall and cytoplasmic membrane compart- subsequently identified Cws. This procedure identified ments Schneewind et al., 1992). When tethered to the C- 19 surface protein genes Table 1). Nine genes encode terminus of polypeptides bearing N-terminal signal pep- the well-characterized surface proteins Spa, FnbA, FnbB tides, the Cws promotes anchoring of hybrid polypeptides fibrinonectin-binding proteins), ClfA, ClfB fibrinogen- to the cell wall envelope Pozzi et al., 1992; Schneewind binding clumping factors), SdrC, SdrD, SdrE and Pls et al., 1993; Medaglini et al., 1995; Navarre and Schnee- [containing serine S) aspartate D) repeat regions wind, 1996; Strauss and GoÈtz, 1996; Piard et al., 1997). upstream of the Cws] UhleÂn et al., 1984; Flock et al., 1987; JoÈnsson et al., 1991; McDevitt et al., 1994; Josefsson et al., 1998a; NõÂ Eidhin et al., 1998). The gene Sorting signals and surface proteins encoding S. aureus collagen adhesin Cna), a Cws-bearing Ten Cws of surface proteins from different Gram-positive adhesin for bone tissue Patti et al., 1992), is not present in bacteria have been fused to the C-terminus of either the four S. aureus genome sequences. Cna is found in truncated protein A lacking its own Cws or staphylococcal staphylococcal isolates from bone and connective tissue enterotoxin B Seb) Schneewind et al., 1993). Five Cws infections Patti et al., 1994). Ten genes encode unknown were functional in staphylococci and caused cell wall surface proteins, herein referred to as sas S. aureus anchoring of hybrid proteins. Some of the non-functional surface protein). All surface proteins appear to be exported Cws were altered by mutation and examined again for by an N-terminal signal peptide. The Cws is invariably anchoring. In all cases examined, the mutant Cws could located at the C-terminal end and contains LPXTG gain function, which required an alteration in the residue sequences withvariable residues at theX and T positions. spacing between the LPXTG motif and the positively One Cws has a replacement of alanine A) for threonine T). charged tail Schneewind et al., 1993). Mutational The X position can be occupied by acidic [glutamate E), analysis of the protein A Cws revealed that two arginines aspartate D)], uncharged [alanine A), glutamine Q), R), positioned 31 and 32 residues downstream of the asparagine N)] and basic residues [lysine K)]. LPXTG motif, function as a signal to retain the polypeptide within the secretory pathway. It appears that positive Cell wall anchor structure charge is a signal for retention, as lysine, but not histidine, can substitute for arginines Schneewind et al., 1993). During cell wall anchoring, the Cws of protein A is cleaved The staphylococcal databases were searched for between the threonine and the glycine of the LPXTG motif surface protein genes using the protein A Cws as a Navarre and Schneewind, 1994). The cell wall anchor BLAST search query followed by other searches with structure of surface proteins released from peptidoglycan Table 1. S. aureus surface protein genes sas). sas Codons Ligand Predicted sorting signal Reference spa 508 IgG/vWBF LPETGEENPFIGTTVFGGLSLALGAALLAGRRREL UhleÂn et al. 1984) fnbA 1018 Fibronectin/ LPETGGEESTNKGMLFGGLFSILGLALLRRNKKNHKA SignaÈs et al. 1989) fibrinogen fnbB 914 Fibronectin/ LPETGGEESTNNGMLFGGLFSILGLALLRRNKKNHKA JoÈnsson et al. 1991) fibrinogen clfA 933 Fibrinogen LPDTGSEDEANTSLIWGLLASIGSLLLFRRKKENKDKK McDevitt et al. 1994) clfB 913 Fibrinogen LPETGDKSENTNATLFGAMMALLGSLLLFRKRKQDHKEKA NõÂ Eidhin et al. 1998) cna 1183 Collagen LPKTGMKIITSWITWVLGLYLILRKRFNS Patti et al. 1992) sdrC 947 Unknown LPETGSENNNSNNGTLFGGLFAALGSLLSFGRRKKQNK Josefsson et al. 1998a) sdrD 1315 Calcium LPETGNENSGSNNATLFGGLFAALGSLLLFGRRKKQNK Josefsson et al. 1998a, b) sdrEa 1166 Unknown LPETGSENNGSNNATLFGGLFAALGSLLLFGRRKKQNK Josefsson et al. 1998a) pls 1637 Unknown LPDTGNDAQNNGTLFGSLFAALGGLFLVGRRRKNKNNEEK AF115379b sasA 2261 Unknown LPDTGDSIKQNGLLGGVMTLLVGLGLMKRKKKKDENDQDDSQA sasB 937 Unknown LPDTGMSHNDDLPYAELALGAGMAFLIRRFTKKDQQTEE de Wu and Lencastre 1999); Komatsuzawa et al. 2000) sasC 2186 Unknown LPNTGSEGMDLPLKEFALITGAALLARRRTKN sasD 241 Unknown LPAAGESMTSSILTASIAALLLVSGLFLAFRRRSTNK sasE 354 Unknown LPKTGLTSVDNFISTVAFATLALLGSLSLLLFKRKESK AB042826b sasF 637 Unknown LPKAGETIKEHWLPISVIVGAMGVLMIWLSRRNKLKNKA sasG 1117 Unknown LPKTGLESTQKGLIFSSIIGIAGLMLLARRRKN sasH 308 Unknown LPKTGTNQSSSPEAMFVLLAGIGLIATVRRRKAS sasI 895 Unknown LPKTGETTSSQSWWGLYALLGMLALFIPKFRKESK sasJ 645 Unknown LPQTGEESNKDMTLPLMALLALSSIVAFVLPRKRKN a. A variant of SdrE binds bone sialoprotein Tung et al., 2000). b. GenBank accession number, Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1049±1057 Surface protein anchoring by sortase 1051 via N-acetylmuramidase, N-acetylglucosaminidase, N- tylicum, Corynebacterium diphtheriae, Enterococcus acetylmuramyl-L-Ala amidase, D-Ala-Gly endopeptidase faecalis, Listeria monocytogenes, Streptococcus mutans, F11 enzyme) and lysostaphin showed that the surface Streptococcus pneumoniae and Streptococcus pyogenes proteins of S. aureus are linked via an amide bond Mazmanian et al.,
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