title: Sortase-mediated assembly and surface topology of adhesive pneumococcal pili.
abstract: The rlrA genetic islet encodes an extracellular pilus in the Gram-positive pathogen Streptococcus pneumoniae. Of the three genes for structural subunits, rrgB encodes the major pilin, while rrgA and rrgC encode ancillary pilin subunits decorating the pilus shaft and tip. Deletion of all three pilus-associated sortase genes, srtB, srtC and srtD, completely prevents pilus biogenesis. Expression of srtB alone is sufficient to covalently associate RrgB subunits to one another as well as linking the RrgA adhesin and the RrgC subunit into the polymer. The active-site cysteine residue of SrtB (Cys 177) is crucial for incorporating RrgC, even when the two other sortase genes are expressed. SrtC is redundant to SrtB in permitting RrgB polymerization, and in linking RrgA to the RrgB filament, but SrtC is insufficient to incorporate RrgC. In contrast, expression of srtD alone fails to mediate RrgB polymerization, and a srtD mutant assembles heterotrimeric pilus indistinguishable from wild type. Topological studies demonstrate that pilus antigens are localized to symmetric foci at the cell surface in the presence of all three sortases. This symmetric focal presentation is abrogated in the absence of either srtB or srtD, while deletion of srtC had no effect. In addition, strains expressing srtB alone or srtC alone also displayed disrupted antigen localization, despite polymerizing subunits. Our data suggest that both SrtB and SrtC act as pilus subunit polymerases, with SrtB processing all three pilus subunit proteins, while SrtC only RrgB and RrgA. In contrast, SrtD does not act as a pilus subunit polymerase, but instead is required for wild-type focal presentation of the pilus at the cell surface.
authors.affiliation: Department of Bacteriology, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden.
authors.forename: Stefan
authors.initials: S
authors.lastname: Fälker
authors.forename: Aaron L
authors.initials: AL
authors.lastname: Nelson
authors.forename: Eva
authors.initials: E
authors.lastname: Morfeldt
authors.forename: Kristina
authors.initials: K
authors.lastname: Jonas
authors.forename: Kjell
authors.initials: K
authors.lastname: Hultenby
authors.forename: Johannes
authors.initials: J
authors.lastname: Ries
authors.forename: Ojar
authors.initials: O
authors.lastname: Melefors
authors.forename: Staffan
authors.initials: S
authors.lastname: Normark
authors.forename: Birgitta
authors.initials: B
authors.lastname: Henriques-Normark