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Sortases Make Pili from Three Ingredients

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Sortases Make Pili from Three Ingredients COMMENTARY Sortases make pili from three ingredients So-Young Oh, Jonathan M. Budzik, and Olaf Schneewind* Department of Microbiology, University of Chicago, 920 East 58th Street, Chicago, IL 60637 any bacterial pathogens have evolved hair-like ex- tensions that project from their surfaces and enable Madhesion to host tissues, formation of biofilms, motility, and even transfer of genetic material (1). Gram-positive bac- teria use sortases, membrane-anchored transpeptidases, to cleave precursor pro- teins at cognate recognition motifs within C-terminal sorting signals and assemble pili in their cell wall envelope (2, 3). The transient product of this re- action is an acyl enzyme that, depending on the sortase involved, is relieved by a specific nucleophile (4). Currently stud- ied sortases accept only one nucleophile to complete their transpeptidation reac- tion (5–8). For example, sortase A of Staphylococcus aureus forms an acyl en- zyme with surface proteins bearing LPXTG motif sorting signals, which are subsequently relieved by the nucleophilic attack of the amino group of lipid II, the precursor of peptidoglycan synthesis Fig. 1. Pilus assembly in Corynebacterium diphtheriae. Pilin-specific sortase cleaves the sorting signal of in bacteria (9, 10). In this issue of SpaA and SpaB at the threonine residue (T) of the LPXTG motif, generating an acyl-enzyme intermediate. PNAS, Mandlik et al. (11) provide new Nucleophilic attack by the side chain of lysine (K) in the YPKN motif of SpaA forms an amide bond between insight into sortases and their compli- SpaA or SpaB and provides for pilus polymerization. SpaB is cleaved by the housekeeping sortase. cated recipes for pilus assembly. Nucleophilic attack by lysine 139 (K139) of SpaB connects the minor subunit to the pilus. Nucleophilic attack of lipid II at the acyl intermediate of the housekeeping sortase with SpaB transfers the pilus to the Starting with simple recipes, some cell wall envelope. sortases make pili from two ingredients (12, 13). Bacillus cereus sortase D cleaves the sorting signal of pilin precur- In chemical terms, anchoring of pili to SpaB into SpaA pili. Substitution experi- sors and accepts the side chain ␧-amino the cell wall envelope occurs by a mech- ments identify lysine 139 (K139) as ab- group of lysine (K) within the YPKN anism where another chef, sortase A, solutely required and, similar to the motif of the major pilin protein as its competes with sortase D for the same YPKN motif (3), suggest that the side- nucleophile (14). Products of this reac- ingredient (BcpA substrate). chain amino group of this residue func- tion are covalently polymerized pilin Pili made from three subunits thus far tions as the SpaB nucleophile (11). This discovery provides fresh and important subunits, glued together by an amide have presented an enigma to the pilus bond between the C-terminal carboxyl views on pilus assembly in Gram- sortase hypothesis (3). The pilin-specific positive bacteria, because most sortase group of one subunit and the side-chain sortase of Corynebacterium diphtheriae amino group of the YPKN motif of an- chefs use three ingredients to make pili (sortase A) is responsible for assembly other (14). Conservation of sortase D, and these pili are used for vaccine de- of pili that harbor SpaC at the tip joined as well as the YPKN motif and cell wall velopment (1, 17). If one assumed that to a heteropolymer made of SpaA, the sorting signal of major pilin proteins in C. diphtheriae sortase A cuts three major pilin subunit with YPKN motif, many different species, suggests that this subunits (SpaA, SpaB, and SpaC), the resulting acyl intermediates could be pilus assembly reaction is universal in and SpaB (3) (Fig. 1). Immune electron microscopy studies suggest an occasional relieved by nucleophilic attack of two Gram-positive bacteria (14, 15). A sim- different substrates, SpaA or SpaB, ple recipe for pili therefore requires incorporation of SpaB, a minor subunit that harbors a C-terminal sorting signal which would be a true novelty of sortase only one chef, sortase D, and two ingre- chemistry. This recipe could get even dients, BcpA and BcpB (12). Sortase D but no YPKN motif (3). Mandlik et al. (11) report that deletion of spaB does more complicated because some pili in- acyl intermediates are relieved by the volve two pilin-specific sortases for as- not abrogate pilus assembly; instead, the nucleophilic attack of the BcpA YPKN sembly, leaving open the possibility of motif, thereby generating a pilus with SpaC-SpaAn polymer is released into BcpB at the tip joined to a BcpA ho- the extracellular medium, a phenotype mopolymer (14). Pilus assembly is termi- similar to that of srtF mutants (the C. Author contributions: S.-Y.O., J.M.B., and O.S. wrote the nated when sortase A cuts BcpA and diphtheriae homolog of sortase A) (16). paper. accepts its nucleophile, lipid II. Because What is the nucleophilic attribute that The authors declare no conflict of interest. BcpA can still be joined to the pilus via makes SpaB an ingredient of pili? See companion article on page 14147. its YPKN motif, incorporation of lipid Mandlik et al. use a molecular genetic *To whom correspondence should be addressed. E-mail: II at the C-terminal end causes pili to approach and identify the smallest do- [email protected]. be immobilized in the cell wall envelope. main that allows for incorporation of © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807334105 PNAS ͉ September 16, 2008 ͉ vol. 105 ͉ no. 37 ͉ 13703–13704 Downloaded by guest on September 24, 2021 multiple substrate specificities (18, 19). cocci), where deletion of the minor sub- In sum, recipes for pili with three in- Whatever models turn out to be true, unit also results in the release of pili gredients seem complicated and require currently available data suggest that (20). Unlike bacilli, corynebacteria, or sortases that cleave multiple substrates some pilin-specific sortases must accept group B streptococci, pili of Streptococ- and accept multiple nucleophiles. What’s more than one nucleophile to complete cus pneumoniae were found localized to needed now are biochemical approaches their transpeptidation reaction (11). An- the cell wall in a mutant that lacked the that recapitulate these assembly reac- tions in vitro and reveal the molecular other attribute of recipes with three in- housekeeping sortase A, the enzyme details of substrate recognition and sor- gredients is that two chefs (sortases A that accepts lipid II nucleophiles (21). If tase catalysis. Looking deep into chefs’ and F) may compete for the minor pilin, so, should we expect that pilin-specific recipes will not only provide insight into whereas the major subunit is apparently sortases of S. pneumoniae accept two Gram-positive pili, but should also gen- only cut by sortase A (16). Similar nucleophiles from different chemical erate menus of astonishing variety that mechanisms may apply for pili of Strep- groups, pilin precursor (a protein) and can be used in imaginative ways for bio- tococcus agalactiae (group B strepto- lipid II (a glycolipid)? logical investigation (22). 1. Telford JL, Barocchi MA, Margarit I, Rappuoli R, Grandi 8. Marraffini LA, Schneewind O (2007) Sortase C-medi- 15. Kang HJ, Coulibaly F, Clow F, Proft T, Baker EN (2007) G (2006) Pili in gram-positive pathogens. Nat Rev Mi- ated anchoring of BasI to the cell wall envelope of Stabilizing isopeptide bonds revealed in gram-positive crobiol 4:509–519. Bacillus anthracis. J Bacteriol 189:6425–6436. bacterial pilus structure. Science 318:1625–1628. 2. Marraffini LA, Dedent AC, Schneewind O (2006) Sorta- 9. Ton-That H, Mazmanian SK, Faull KF, Schneewind O 16. Swaminathan A, et al. (2007) Housekeeping sortase ses and the art of anchoring proteins to the envelopes (2000) Anchoring of surface proteins to the cell wall of facilitates the cell wall anchoring step of pilus polymers of gram-positive bacteria. Microbiol Mol Biol Rev Staphylococcus aureus sortase catalyzed in vitro in Corynebacterium diphtheriae. Mol Microbiol 70:192–221. transpeptidation reaction using LPXTG peptide and 66:961–974. 3. Ton-That H, Schneewind O (2003) Assembly of pili on NH2-Gly3 substrates. J Biol Chem 275:9876–9881. 17. Maione D, et al. (2005) Identification of a universal the surface of Corynebacterium diphtheriae. Mol Mi- 10. Perry AM, Ton-That H, Mazmanian SK, Schneewind O Group B streptococcus vaccine by multiple genome crobiol 50:1429–1438. (2002) Anchoring of surface proteins to the cell wall of screen. Science 309:148–150. 4. Ton-That H, Liu G, Mazmanian SK, Faull KF, Schnee- Staphylococcus aureus Lipid II is an in vivo peptidogly- 18. Gaspar AH, Ton-That H (2006) Assembly of distinct pilus wind O (1999) Purification and characterization of sor- can substrate for sortase-catalyzed surface protein an- structures on the surface of Corynebacterium diphthe- tase, the transpeptidase that cleaves surface proteins choring. J Biol Chem 277:16241–16248. riae. J Bacteriol 188:1526–1533. of Staphylococcus aureus at the LPXTG motif. Proc Natl 11. Mandlik A, Das A, Ton-That H (2008) The molecular 19. Rosini R, et al. (2006) Identification of novel genomic Acad Sci USA 96:12424–12429. switch that activates the cell wall anchoring step of islands coding for antigenic pilus-like structures in 5. Ton-That H, Faull KF, Schneewind O (1997) Anchor pilus assembly in Gram-positive bacteria. Proc Natl Streptococcus agalactiae. Mol Microbiol 61:126–141. structure of staphylococcal surface proteins. I. A Acad Sci USA 105:14147–14152. branched peptide that links the carboxyl terminus of 12. Budzik JM, Marraffini LA, Schneewind O (2007) Assem- 20. Nobbs AH, et al. (2008) Sortase A utilizes ancillary proteins to the cell wall.
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