Cell Wall Anchor Structure of BcpA Pili in Bacillus anthracis

Abstract

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...

Sortase D Forms the Covalent Bond That Links BcpB to the Tip of Bacillus cereus Pili

“…BcpB H6 -BcpA was purified by a second round of Ni-NTA affinity chromatography under denaturing conditions and separated by reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection using a C18 column with a linear gradient from 1 to 99% acetonitrile (CH 3 CN) in 0.1% formic acid in 100 min, as previously reported for BcpA pilin peptides (16) and the BcpA anchor structure (19).…”

“…For immunoblots with ␣-BcpA and ␣-BcpB antisera, 3 ml of cell culture was digested with 100 units of mutanolysin in 0.05 M Tris-HCl, pH 6.3, 1.5 mM MgCl 2 , and 1 mM phenylmethanesulfonyl chloride. For purification of BcpB MH6 -BcpA from B. anthracis strains harboring pJB213, 6 liters of cell culture were digested with 20,000 units of mutanolysin, and cells were processed as described previously (19). Reactions were incubated at 37°C for 16 h, and the solubilized cell wall species was subjected to immunoblotting and purified by Ni-NTA affinity chromatography.…”

“…IsdX1-BcpA SS -GST is a translational hybrid between IsdX1, the sorting signal of BcpA (BcpA SS ) and E. coli glutathione S-transferase (Fig. 3A) (19). Cleavage of the hybrid precursor (P1/P2) into the mature form (M) was monitored by immunoblotting with ␣-GST serum (Fig.…”

“…Sortase D cleaves the BcpA LPXTG motif sorting signal between the threonine and glycine residues to form an amide bond to the ⑀-amino group of the lysine within the YPKN motif of adjacent BcpA subunits (16). However, sortase A also cleaves BcpA precursors, which are subsequently linked to the side chain amino group of meso-diaminopimelic acid within lipid II (19). The latter reaction serves to terminate fiber elongation, immobilizing BcpA pili in the cell wall envelope (19).…”

“…However, sortase A also cleaves BcpA precursors, which are subsequently linked to the side chain amino group of meso-diaminopimelic acid within lipid II (19). The latter reaction serves to terminate fiber elongation, immobilizing BcpA pili in the cell wall envelope (19).…”