Synthesis of Mosaic Peptidoglycan Cross-bridges by Hybrid Peptidoglycan Assembly Pathways in Gram-positive Bacteria

Staphylococcus aureus sortase A cleaves surface protein precursors bearing C-terminal LPXTG motif sorting signals between the threonine and glycine residues. Using lipid II precursor as cosubstrate, sortase A catalyzes the amide linkage between the carboxyl group of threonine and the amino group of pentaglycine cross-bridges, thereby tethering C-terminal ends of surface proteins to the bacterial cell wall envelope. Staphylococcal sortase B also anchors its only known substrate, the IsdC precursor with a C-terminal NPQTN motif sorting signal, to the cell wall envelope. Herein, we determined the cell wall anchor structure of IsdC. The sorting signal of IsdC is cleaved between threonine and asparagine of the NPQTN motif, and the carboxyl group of threonine is amide-linked to the amino group of pentaglycine crossbridges. In contrast to sortase A substrates, the anchor structure of IsdC displays shorter glycan strands and significantly less cell wall cross-linking. A model is proposed whereby sortases A and B recognize unique features of sorting signals and peptidoglycan substrates to deposit proteins with distinct topologies in the cell wall envelope.

Section: Methodsmentioning

confidence: 99%

Anchor Structure of Staphylococcal Surface Proteins

Marraffini

Schneewind²

2005

“…The disaccharide-tetrapeptide containing meso-diaminopimelic acid (mesoDAP 3 ) was purified from E. coli strain ATCC 25113. The procedures used for peptidoglycan preparation, digestion with muramidases, and reduction of MurNAc to muramitol with sodium borohydride have been previously described for enterococci (24) and E. coli (25). The resulting muropeptides were separated by RP-HPLC in acetonitrile gradients containing trifluoroacetic acid (24) and identified by mass spectrometry (MS).…”

Section: Methodsmentioning

confidence: 99%

“…The procedures used for peptidoglycan preparation, digestion with muramidases, and reduction of MurNAc to muramitol with sodium borohydride have been previously described for enterococci (24) and E. coli (25). The resulting muropeptides were separated by RP-HPLC in acetonitrile gradients containing trifluoroacetic acid (24) and identified by mass spectrometry (MS). The concentration of the muropeptides was estimated by amino acid analysis after acidic hydrolysis with a Hitachi autoanalyzer (26).…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, dimers were generated in vitro as described above except that the disaccharide-peptides used as substrates were not reduced. The reaction mixture was treated with ammonium hydroxide, desalted using a micro column (ZipTipC 18 , Millipore), and the resulting lactoyl-peptides were analyzed by nanoelectrospray MS/MS using N 2 as the collision gas (24).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Specificity of L,D-Transpeptidases from Gram-positive Bacteria Producing Different Peptidoglycan Chemotypes

Magnet

Arbeloa

Mainardi

et al. 2007

Self Cite

The bacterial cell wall peptidoglycan is a net-like macromolecule that surrounds the cytoplasmic membrane (1). The polymer is essential because it supplies the cell with mechanical protection against the osmotic pressure of the cytoplasm. The peptidoglycan subunit contains ␤-1,4-linked N-acetylglucosamine (GlcNAc) 2 and N-acetylmuramic acid (MurNAc) substituted by a peptide stem ( The assembly pathway of peptidoglycan precursors and its mode of polymerization are generally highly conserved in eubacteria. Variations in the structure of peptidoglycan subunits involve mainly the fifth (C-terminal) and third positions of the pentapeptide stem (5). The specificity of peptidoglycan cross-linking enzymes for the donor is the bottleneck that limits emergence of resistance to glycopeptides because the modifications of the precursors that prevent drug binding should be * This work was supported by the program "ACI Microbiologie 2003" from the Fonds National de la Science (Grant ACIM-4-9), the European Community (COBRA, contract LSHM-CT-2003-503335, 6th PCRD), NIAID, National Institutes of Health (Grant R01 AI45626), and the Fondation pour la Recherche Mé dicale. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

“…3A). Following affinity purification by Ni-NTA, BcpA MH6 was cleaved with CNBr and amino sugars of C-terminal anchor peptides were reduced with sodium borohydride (32). Glycopeptides were subjected to RP-HPLC with a C 18 column, and anchor peptides were found to elute at 35% CH 3 CN-0.1% formic acid.…”

Section: Substrate Properties Control the Fate Of Pilin Precursors-mentioning

confidence: 99%

Cell Wall Anchor Structure of BcpA Pili in Bacillus anthracis

Budzik¹,

Schneewind

2008

Assembly of pili in Gram-positive bacteria and their attachment to the cell wall envelope are mediated by sortases. In Bacillus cereus and its close relative Bacillus anthracis, the major pilin protein BcpA is cleaved between the threonine and the glycine of its C-terminal LPXTG motif sorting signal by the pilin-specific sortase D. The resulting acyl enzyme intermediate is relieved by the nucleophilic attack of the side-chain amino group of lysine within the YPKN motif of another BcpA subunit. Cell wall anchoring of assembled BcpA pili requires sortase A, which also cleaves the LPXTG sorting signal of BcpA between its threonine and glycine residues. We show here that sortases A and D require only the C-terminal sorting signal of BcpA for substrate cleavage. Unlike sortase D, which accepts the YPKN motif as a nucleophile, sortase A forms an amide bond between the BcpA C-terminal carboxyl group of threonine and the sidechain amino group of diaminopimelic acid within the cell wall peptidoglycan of bacilli. These results represent the first demonstration of a cell wall anchor structure for pili, which are deposited by sortase A into the envelope of many different microbes.Many Gram-positive bacteria use pili, protein fibers that project from microbial surfaces, to adhere to and invade host tissues or form biofilms (1). Gram-positive pili are assembled from pilin subunits that are synthesized as precursors with N-terminal signal peptides and C-terminal sorting signals in the bacterial cytoplasm (P1) (2). The signal peptide is cleaved and precursors translocated through the bacterial membrane by the Sec machinery, thereby generating the P2 precursor (3, 4). Assembly of pili involves the polymerization of the major pilin protein, a reaction that is catalyzed by pilinspecific sortase (2, 5), designated sortase D in pathogenic Bacillus sp. (6). Pilin-specific sortases perform a transpeptidation reaction, whereby the C-terminal LPXTG motif sorting signal of the major pilin protein, BcpA in bacilli, is cleaved between the threonine and the glycine residue (4). Intermediary product of this reaction is a thioester-linked acyl-enzyme between the active site cysteine residue of sortase and the carboxyl group of threonine (7,8). Nucleophilic attack of the ⑀-amino group of lysine within the YPKN pilin motif of another BcpA subunit resolves the sortase D intermediate and generates the amide bond between the C-terminal carboxyl group of threonine and the side-chain amino group of lysine of adjacent BcpA subunits (4). Conservation of pilin-specific sortase, the YPKN motif, and C-terminal sorting signal in the major subunit of pilin proteins suggests that pilus assembly occurs by a universal mechanism in Gram-positive bacteria (2).Pili are anchored to the cell wall envelope of Gram-positive bacteria by a mechanism that requires sortase A (6, 9 -11). Sortase A is otherwise known to cut the C-terminal LPXTG sorting signal of surface proteins and immobilize anchored products in the cell wall envelope of 13). In Staphylococcus aureus, s...