Sortase-mediated assembly and surface topology of adhesive pneumococcal pili

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.


Bacterial strains, media, and growth conditions
Pneumococcal strains used, including isogenic mutant derivatives, are described in Supporting Table S1. The insertion-deletion mutagenesis used for most strains as well as the strategy to achieve complementation in trans are described elsewhere (Barocchi et al., 2006;Lau et al., 2002). The primers used for each strain are noted in Table S1, and all primers are described in Supporting Table S2. Resulting strains were checked by PCR, sequencing, and immunogenicity, thereby also demonstrating a lack of polar effects of deletion of one subunit gene on expression of others.
Unless otherwise noted, bacteria were streaked from frozen stocks onto blood plates with appropriate selection for overnight growth, inoculated briefly into pre-warmed DS medium (Dextrose-Serum medium, OXOID Manual, 1990), and then DS was inoculated into prewarmed C+Y medium to achieve an O.D. 620 =0.05. Cultures were permitted to grow to midlog (O.D. 620 =0.4) at 37 o without agitation before collection for experimentation. Bacterial medium was produced by the Karolinska Microbiology laboratory.  Figure S1. Study of pilus expression in T4 and D39∇ populations by quantitative flow cytometry. Cultures of T4 and D39∇ were grown under identical conditions and prepared for measurement of RrgB expression by flow cytometry using a protocol modified from immunofluorescence studies. 10 5 cells from each culture were counted and RrgB expression, determined by Cy3-fluorescence, was plotted on a histogram. In addition, the T4 culture was also prepared in the absence of the anti-RrgB primary antibody, serving as a negative control ("negative"), confined entirely the first peak with low fluorescent intensity. The second peak,

SUPPORTING FIGURE LEGENDS
where the FL3 channel is scored 10 1 or greater (marked by a horizontal line above the peak), contains information about cells successfully stained for RrgB and detected. Note that more D39∇ cells were detected expressing RrgB than T4 cells. This finding suggests that a larger fraction of cells in a D39∇ culture express pili than the fraction of cells in a T4 culture.
Genetic analysis of pilus polymer formation in D39∇ yielded identical results to those seen in T4, so D39∇ was used for ultrastructural analyses.     immunostaining was colored green, and the nucleoid was stained with DAPI and colored blue.
Note that both RrgB and RrgA were found in highly discrete areas, and furthermore, that the two antigens likely overlap. A similar example of RrgB distribution in BHN100 is shown in Fig 3A, and examples of RrgB (Figs 3-5) and RrgA (Fig 3C, S5) topology in T4 are provided.
(B) Genetic determinants of RrgB topology in the 19F strain BHN100 were studied by conventional microscopy. Wild-type 19F BHN100 exhibits discrete RrgB 'banding', similar to that observed by confocal microscopy (A). In contrast, genetic disruption of either srtB ("19FΔsrtB") or srtD ("19FΔsrtD") results in a loss of discrete, organized foci. Instead, many smaller randomly distributed RrgB foci are observed, similar to the phenotype of T4ΔsrtD ( Fig 5C). (C) Genetic determinants of RrgA topology in 19F BHN100 were studied by conventional microscopy. Wild-type 19F BHN100 exhibits discrete RrgA 'banding', as shown by confocal microscopy in panel (A), similar to RrgA distribution in T4 (Fig 3C).