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 transpeptidation 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., 1999). The genomes of Gram-positive between the carboxyl-group of threonine and the amino- organisms encode at least two, sometimes even more group of the pentaglycine cell wall cross-bridge Schnee- than two, sortase genes Pallen et al., 2001). The wind et al., 1995; Ton-That et al., 1997; Navarre et al., identification of two sortase genes in Methanobacterium 1998; 1999). Anchoring occurs mostly to cross-linked thermoautotrophicum represents a curiosity, as tetrapeptide murein subunits [MurNac- L-Ala-D-iGln-L- this organism synthesizes pseudopeptidoglycan, i.e. N-

Lys surface protein-Gly5)-D-Ala)-GlcNac]n but also to free acetylglucosamine- b1±3)-N-acetyltalosaminurate poly- unsubstituted pentapeptide subunits [MurNac- L-Ala-D- mer, instead of peptidoglycan Kandler and KoÈnig, 1998; iGln-L-Lys surface protein-Gly5)-D-Ala-D-Ala)-GlcNac] Pallen et al., 2001). It is conceivable that sortase may link Ton-That et al., 1997; Navarre et al., 1998). The cross- proteins to the amino-group of lysine, as the side-chain of linking of cell wall anchor peptides is extensive: as many this residue is engaged in cross-linking with g-glutamyl as 11 cell wall peptide subunits and five glycan of neighbouring peptide subunits Kandler and KoÈnig, disaccharide) subunits were found linked to surface 1998). However, no genes encoding a surface protein proteins Navarre et al., 1998; 1999). Using a similar with a C-terminal Cws have been identified thus far in strategy, the cell wall anchor structure of surface proteins Methanobacterium Pallen et al., 2001). in Listeria monocytogenes was determined: the C- The ability of srtA mutant staphylococci to cause an terminal threonine is amide linked to the side-chain infection was assessed using the murine renal abscess amino-group of m-diaminopimelic acid within cell wall model. When compared with the human clinical isolate peptides Dhar et al., 2000). Although several aspects of S. aureus Newman, isogenic srtA knock-out mutants peptidoglycan structure in Gram-positive bacteria are displayed a 2- to 3-log reduction in the number of viable variable Schleifer and Kandler, 1972), the principles of staphylococci recovered from renal abscesses Mazma- surface protein anchoring appear to be conserved nian et al., 2000). As a model for acute infection, the Navarre and Schneewind, 1999). number of sortase mutant staphylococci required to produce a lethal infection after intraperitoneal injection of mice was measured. Compared withthewild-type human Sortase +SrtA) isolate Newman, the LD50 of srtA mutant staphylococci is A screen for S. aureus mutants that fail to cleave the reduced by 1.5 logs Mazmanian et al., 2000). protein A Cws identified srtA, for surface protein sorting A The role of srtA in anchoring surface proteins to the cell Mazmanian et al., 1999). srtA encodes sortase, an wall envelope appears to be conserved in Gram-positive enzyme of 206 amino acids witha putative N-terminal pathogens. Knock-out mutations of the srtA gene in membrane-spanning domain and a C-terminal catalytic Streptococcus gordonii also interfere withthecell wall domain that is presumably translocated across the anchoring and display of surface proteins, which is cytoplasmic membrane S. K. Mazmanian and O. accompanied by a loss of bacterial adhesive properties Schneewind, unpublished). Knock-out mutations of the Bolken et al., 2001). Actinomyces species elaborate srtA gene do not seem to affect growthof staphylococci fimbriae that are composed of Cws-bearing subunit on laboratory media Mazmanian et al., 2000). Sortase proteins Yeung and Cisar, 1990). Knock-out mutations mutants cannot cleave protein A at the LPXTG motif, and of the Actinomyces srtA homologue abolish C-terminal the precursor accumulates in the cytoplasm, membrane processing of fimbrial proteins at the LPXTG motif Yeung and cell wall fractions missorted phenotype) Mazmanian et al., 1998). It is not yet clear whether the fimbrial et al., 2000). Cws of fibronectin-binding proteins FnbA proteins are linked to the cell wall peptidoglycan and/or and FnbB) and clumping factor ClfA) also remain polymerized to form a proteinaceous multisubunit filament uncleaved, suggesting that the srtA mutation causes a Yeung et al., 1998; Pallen et al., 2001). defect in the anchoring of at least some of the other surface proteins Mazmanian et al., 2000). Consistent witha more Sortase-catalysed cleavage at the LPXTG motif general defect in cell wall sorting is the finding that srtA mutant strains cannot display Ig, fibronectin and fibrino- Treatment of staphylococci with the strong nucleophile gen adhesins on the staphylococcal surface Mazmanian hydroxylamine releases surface protein with C-terminal et al., 2000). Genes that are homologous to S. aureus threonine hydroxamate into the extracellular medium srtA are found in all Gram-positive bacterial genomes Ton-That et al., 1999). This observation suggests that sequenced to date, including Actinomyces naeslundii, sortase captures cleaved surface protein as acyl-enzyme Bacillus anthracis, Bacillus subtilis, Clostridium acetabu- intermediates. A model was proposed in which cysteine

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184 C184) of sortase performs a nucleophilic attack on peptidoglycan precursor UDP-MurNac-L-Ala-D-iGln-L-Lys-D- the carbonyl carbon at the scissile peptide bond between Ala-D-Ala; Park's nucleotide) is linked to undecaprenol- threonine and glycine, followed by the formation of a pyrophosphate, generating lipid I Matsuhashi, 1994). The thioester with the carboxyl-group of threonine Ton-That membrane-bound intermediate is modified further by the et al., 1999). During this reaction, the amino-group of the addition of GlcNac and pentaglycine [undecaprenolpyr- cleaved LPXTG is released, and the Cws is subsequently ophosphate-MurNac -L-Ala-D-iGln- NH2-Gly5)L-Lys-D-Ala- degraded. This model is supported by several observa- D-Ala)- b1±4)-GlcNac; lipid II] and translocated across the tions. i) Methyl-methanethiosulphonates, reagents that cytoplasmic membrane Matsuhashi et al., 1965). Lipid II form disulphide with sulphydryl, act as inhibitors of sortase serves as a substrate for the transglycosylation reaction, Ton-That and Schneewind, 1999). The inhibited enzyme polymerizing the glycan strands of the bacterial cell wall is regenerated by treatment withthereducing reagent to yield the repeating disaccharide MurNac-GlcNac)n dithiothreitol DTT) Ton-That et al., 1999). ii) Mutational Nakagawa et al., 1984). Cell wall pentapeptides L-Ala-D- replacement of C184 with alanine abolishes the enzy- iGln- NH2-Gly5)L-Lys-D-Ala-D-Ala) of nascent peptidogly- matic activity of sortase Ton-That et al., 1999). iii) C184 can strands are cross-linked via the transpeptidation is conserved within sortase genes Mazmanian et al., reaction, thereby generating a three-dimensional cell wall 1999). iv) Thioester-linked acyl-enzyme intermediates network Tipper and Strominger, 1965). Penicillin is an are sensitive to hydroxylaminolysis Ton-That et al., inhibitor of the transpeptidation reaction, but does not 2000). v) In the three-dimensional structure of sortase, interfere withthetransglycosylation reaction Strominger C184 and several other conserved residues cluster into et al., 1967). Vancomycin binds to the D-Ala-D-Ala moiety a surface-exposed pocket that seems to represent the of lipid II Bugg et al., 1991) and prevents both enzyme active site Ilangovan et al., 2001). transglycosylase and transpeptidase reactions Tipper To obtain a soluble sortase and examine its properties, and Strominger, 1965), whereas moenomycin is an the N-terminal membrane anchor segment of SrtA inhibitor of transglycosylation alone van Heijenoort et al., residues 2±25) was replaced witha six-histidinetag 1987). The addition of vancomycin leads to a steady

SrtADN) Ton-That et al., 1999). In the absence of the accumulation of surface protein precursor withuncleaved peptidoglycan substrate, SrtADN catalyses peptide bond Cws, indicating that this compound causes a reduction in hydrolysis and cleaves LPETG between the threonine and the sorting reaction Ton-That and Schneewind, 1999). A the glycine Ton-That et al., 2000). Glycine, NH2-Gly2 and similar, albeit weaker, effect is observed when moeno-

NH2-Gly3 can be used as substitutes for peptidoglycan mycin is added to staphylococcal cultures Ton-That and substrate and increase the rate of sortase-mediated Schneewind, 1999). In contrast, penicillin G does not alter cleavage at LPETG peptides Ton-That et al., 2000). In the rate of cell wall sorting. These results suggests that the presence of NH2-Gly3, sortase catalyses only the sortase may use lipid II as a substrate for surface protein transpeptidation reaction of cell wall sorting, forming anchoring. This is corroborated by the finding that

LPET-Gly3 and NH2-G, but there is no hydrolysis at staphylococcal protoplasts, in which the mature LPETG peptides Ton-That et al., 2000). assembled cell wall has been removed by digestion with Nuclear magnetic resonance NMR) spectroscopy muralytic enzyme, catalyse cleavage of surface proteins revealed the three-dimensional structure of the catalyti- at the LPXTG motif similar to bacteria with an intact cell cally active SrtAD2259 as a b-barrel fold witheight b- wall Ton-That and Schneewind, 1999). strands aligned in a unique antiparallel and parallel Does staphylococcal sortase recognize mutationally manner Ilangovan et al., 2001). The surface of the barrel altered cell wall cross-bridges? The FemA, FemB factor is decorated with two short helices and two large loops, essential for methicillin resistance) and FmhB proteins which define the enzyme active site. The active-site Fem homologue) are thought to catalyse the addition of pocket is open at two sides, forming an elongated tunnel Gly to the 1-amino of L-Lys within lipid I Berger-BaÈchi, with a hydrophobic surface that can accommodate an 1994; Rohrer et al., 1999). Mutant staphylococci lacking unfolded polypeptide chain Ilangovan et al. 2001). This is the FemB protein synthesize NH2-Gly3 but no NH2-Gly5 in agreement withthepresumed scanning function of cross-bridges, whereas femA and femX fmbH) mutant sortase, examining newly translocated polypeptides for strains synthesize NH2-Gly1 or NH2-Gly1/NH2-L-Lys LPXTG motif sequences. Once the C-terminal Cws cross-bridges respectively Kopp et al., 1996). The arrests polypeptide translocation, sortase cuts at the femA, femB, femAB and femAX femA, fmbH) mutant LPXTG motif and transfers surface protein to the cell wall. staphylococci display a reduction in the rate of surface

protein anchoring, consistent with the notion that NH2- Gly is the preferred substrate of the sorting reaction Peptidoglycan substrate of the sorting reaction 5 Ton-That et al., 1998). Nonetheless, surface protein was

During bacterial cell wall synthesis, a soluble cytoplasmic found linked to NH2-Gly3,NH2-Gly1 and NH2-Gly4Ser1

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1049±1057 Surface protein anchoring by sortase 1053 cross-bridges, but not to the 1-amino group of L-Lys Ton- translocation pore Economou and Wickner, 1994). Once That et al., 1998). It appears that sortase recognizes polypeptides have been translocated, signal peptidase some, but not all, cross-bridges as substrates for the lepB ± leader peptidase in E. coli) removes the signal sorting reaction. This notion is also corroborated by the peptide from the precursor, and mature protein is released observation that S. carnosus cells, engineered to synthe- into the periplasmic space Dalbey and Wickner, 1985). size Gly3/Ser2 cross-bridges, are capable of anchoring A second mechanism of protein secretion involves surface protein to the cell wall peptidoglycan Strauss the co-translational translocation of membrane proteins et al., 1998). Ulbrandt et al., 1997). The signal recognition particle SRP), Ffhand 4.5S RNA ffs)inE. coli, binds to signal peptide bearing nascent polypeptides, an interaction that A second sortase gene +srtB) is thought to stall ribosomal translation Poritz et al., A second sortase gene, srtB, was identified by BLAST 1990). Once the SRP complex is bound to its receptor searches using the srtA gene as a query Pallen et al., FtsY in E. coli) and the ribosome has docked on the 2001). All S. aureus strains examined thus far harbour translocation pore, translation resumes and presumably both srtA and srtB genes. What can be the role of two provides the force to translocate polypeptides across the sortase enzymes in staphylococci? Replacement of the membrane Miller et al., 1994). Three membrane proteins, srtB gene of S. aureus Newman withthe ermC marker SecD, SecF and YajC, associate withtheSecYEG pore does not cause a defect in the cell wall anchoring of and appear to regulate SecA-dependent translocation protein A, FnbA, FnbB or ClfA S. K. Mazmanian and O. activity Duong and Wickner, 1997). Schneewind, unpublished). Nevertheless, it is possible We have searched staphylococcal genome databases that srtB mutant staphylococci display a sorting defect for for the presence of secretion genes secABDEFGY, ffh, other surface proteins. Clearly, SrtA and SrtB cannot fulfil ftsY and lepB) Table 2). The S. aureus genome encodes redundant functions, but it is conceivable that the two secAYEG and yajC similar to E. coli and B. subtilis sortases catalyse similar reactions using different surface Blattner et al., 1997; Kunst et al., 1997). A secB gene protein substrates. could not be found; however, a second set of secretion genes, secA-2 and secY-2, was identified. As observed for B. subtilis,theS. aureus genome encodes for a secDF Which genes act on the sorting pathway? fusion gene but not for single secD and secF genes The staphylococcal cell wall is composed of peptidogly- Bolhuis et al., 1998). Two signal peptidase genes spsA can, a covalently linked macromolecule spanning 40 nm and spsB) are present in the S. aureus chromosome in diameter Giesbrecht et al., 1998). This envelope Cregg et al., 1996), whereas ffh and ftsY are present in structure is impermeable for large and small $ 10 kDa) single copy only. The duplicate secretion genes secA2 polypeptides Schneewind et al., 1992). We presume that and secY2) are located in a single large operon inserted the secretion of proteins into the extracellular medium into the genome between sasA and sasB. What can be requires genes whose sole function may be to promote the function of additional secretion genes in the staphy- polypeptide translocation across the cell wall. Such lococcal genome? One possibilty is the assembly of two cell wall translocation would obviously have to be co- secretion pathways that transport polypeptides to distinct ordinated with the translocation of proteins across the locations, perhaps membrane and extracellular medium? plasma membrane. Protein secretion has not been Many other models can of course be proposed. If one studied in S. aureus or other Gram-positive pathogens; assumes that two secretion pathways do indeed exist in however, these organisms harbour several genes S. aureus, additional questions come to mind. Do surface implicated in protein secretion in Escherichia coli. The protein translocation pore of E. coli is composed of Table 2. Secretion genes in S. aureus, B. subtilis and E. coli. three membrane proteins, SecYEG, which can accept Gene S. aureus B. subtilis E. coli substrates in two ways Duong et al., 1997; PohlschroÈder et al., 1997). Signal peptide-bearing precursor proteins secA secA-1, secA-2 secA secA are maintained in a secretion-competent state by binding secB ± ± secB secD secDF secDF secD to chaperones, for example SecB Randall, 1992). B. secE secE secE secE subtilis makes do without SecB, and other chaperones secF ± ± secF presumably function as substitutes Kunst et al., 1997). secG secG secG secG secY secY-1, secY-2 secY secY Translocation substrate is transferred to SecA, an yajC yajC yajC yajC ATPase that undergoes conformational rearrangements ffh ffh ffh ffh upon interacting with the secretion machinery, a process ftsY ftsY ftsY ftsY lepB spsA, spsB sipSTUVW lepB that is thought to push precursor proteins through the

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Fig. 1. Surface protein anchoring in Staphylococcus aureus. i) Export. Precursor proteins withan N-terminal signal peptide are initiated into the secretory Sec) pathway, and the signal peptide is removed. ii) Retention. The C-terminal sorting signal retains polypeptides within the secretory pathway. iii) Cleavage. Sortase cleaves between the threonine and the glycine of the LPXTG motif, resulting in the formation of a thioester enzyme intermediate. iv) Linkage. Nucleophilic attack of the free amino group of lipid II at the thioester bond resolves the acyl- enzyme intermediate, synthesizing the amide bond between surface proteins and the pentaglycine cross-bridge and regenerating the active- site sulphydryl. v) Cell wall incorporation. Lipid-linked surface protein is first incorporated into the cell wall via the transglycosylation reaction. The murein pentapeptide subunit with attached surface protein is then cross-linked to other cell wall peptides, generating the mature murein tetrapeptide. proteins travel only one or bothsecretion pathways?Can signal peptidase and sortase genes in the chromosome of any one of the sec genes be removed without a loss of cell S. aureus suggests that this microorganism may have wall sorting? The list of `Gedanken Experimente' could go evolved more than one pathway for surface protein on, but one can say only this with certainty ± very little transport. Some 20 different polypeptides seem to be is known about protein secretion and cell wall sorting anchored by the sortase-catalysed pathway and play pathways in S. aureus. important roles during the pathogenesis of S. aureus infections.

Conclusions and prospects Acknowledgements Based on the evidence described above, cell wall sorting of surface proteins can be divided into five steps Fig. 1). S.K.M. is supported by the Predoctoral Training Program in i) Export. Precursor proteins withan N-terminal signal Genetics GM07104), and H.T.-T. by the Microbial Patho- peptide are initiated into the secretory Sec) pathway, and genesis Training Grant AI07323). Work in O.S. laboratory is supported by a grant from the NIH-NIAID, Infectious Disease the signal peptide is removed. ii) Retention. The C- Branch AI33987. We apologize to the many authors whose terminal Cws retains polypeptides within the secretory work could not be adequately described because of space pathway. iii) Cleavage. Sortase cleaves between the constraints. threonine and the glycine of the LPXTG motif, resulting in the formation of a thioester enzyme intermediate. iv) Linkage. Nucleophilic attack of the free amino-group of References lipid II at the thioester bond resolves the acyl-enzyme Berger-BaÈchi, B. 1994) Expression of resistance to methi- intermediate, generating the amide bond between surface cillin. Trends Microbiol 2: 389±309. protein and the pentaglycine cross-bridge. v) Cell wall Blattner, F.R., Plunkett, G.R., Bloch, C.A., Perna, N.T., incorporation. Lipid-linked surface protein is incorporated Burland, V., Riley, M., et al. 1997) The complete genome into the cell wall via the transglycosylation and transpep- sequence of Escherichia coli K-12. Science 277: 1453± 1474. tidation reactions of bacterial cell wall synthesis. The Bolhuis, A., Broekhuizen, C.P., Sorokin, A., van Roosmalen, sorting pathway appears to be composed of Sec M.L., Venema, G., Bron, S., et al. 1998) SecDF of Bacillus machinery, signal peptidase and sortase, but will probably subtilis, a molecular Siamese twin required for the efficient require several additional genes that promote surface secretion of proteins. J Biol Chem 273: 21217±21224. protein retention and translocation across the cell wall Bolken, T.C., Franke, C.A., Jones, K.F., Zeller, G.O., Jones, envelope. The presence of multiple sets of secretion, C.H., Dutton, E.K., Hruby, D.E. 2001) Inactivation of the

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