Molecular architecture of Streptococcus pneumoniae TIGR4 pili

Abstract

Although the pili of Gram-positive bacteria are putative virulence factors, little is known about their structure. Here we describe the molecular architecture of pilus-1 of Streptococcus pneumoniae, which is a major cause of morbidity and mortality worldwide. One major (RrgB) and two minor components (RrgA and RrgC) assemble into the pilus. Results from TEM and scanning transmission EM show that the native pili are approximately 6 nm wide, flexible filaments that can be over 1 microm long. They are formed by a single string of RrgB monomers and have a polarity defined by nose-like protrusions. These protrusions correlate to the shape of monomeric RrgB-His, which like RrgA-His and RrgC-His has an elongated, multi-domain structure. RrgA and RrgC are only present at the opposite ends of the pilus shaft, compatible with their putative roles as adhesin and anchor to the cell wall surface, respectively. Our structural analyses provide the first direct experimental evidence that the native S. pneumoniae pilus shaft is composed exclusively of covalently linked monomeric RrgB subunits oriented head-to-tail.

Figure 1

The pili of S. pneumoniae TIGR4 bacteria imaged by negative-stain TEM. (A) Fine pili protruding from the bacterial surface (arrowheads). (B) Second example illustrating the variability in pilus number. Note that the individual pili (arrowhead) appear wider close the cell surface (arrow) due to different negative staining. (C) Third example illustrating the pilus bundles (#) and tangles (^*) that can form. Some individual pili can also be detected (arrowhead). Pili can be followed back to the bacterial surface. Negative stain: 2% phosphotungstic acid (PTA). A high-pass filter was applied to increase pilus visibility. Scale bar: 100 nm.

Figure 2

Constituents of isolated TIGR4 pili and their structural significance. (A) TEM image of TIGR4 pili isolated in Tris buffer and negatively stained with 2% UAc. A beaded structure is evident in an enlarged view (inset). (B) Western blot analysis of the HMW pilus fractions. RrgB forms a ladder of HMW polymers (lane 1, anti-RrgB-His antibody). Loading equal volumes showed the HMW pilus material to contain different amounts of RrgA (lane 2), RrgB (lane 3) and RrgC (lane 4) as measured by Western blotting with anti-RrgA–His, anti-RrgB–His and anti-RrgC–His antibodies, respectively. (C) TEM images of TIGR4 pili after incubation with antibodies to RrgB–His. Single antibodies link the pili laterally to form ladder-like structures. (D) TEM image of TIGR4 pili after incubation with antibodies to RrgA–His. A cluster of antibodies (white arrow) links the pili together at their ends; the individual antibodies are difficult to distinguish. Such v-shaped pilus assemblies were typical. Inset: various orientations of individual antibodies and traces indicating their outer contours. (E) TEM image of a TIGR4 pilus after incubation with antibodies to RrgC-His. A single antibody is attached to the end of the pilus (white arrow). Note the absence of antibodies along the pilus shafts in panels D and E. Scale bars: panel A, 100 nm, inset 20 nm; panels C–E and inset in panel D, 20 nm.

Figure 3

EM of the purified pilus constituents. Representative negative stain (2% UAc) TEM averages (left gallery) and contrast reversed STEM single-shot, dark-field images (right gallery) are shown. (A) RrgA–His. The averages were calculated by sorting 592 single projections into 19 classes. (B) RrgB–His. The averages were calculated by sorting 706 single projections into 14 classes. (C) RrgC–His. The averages were calculated by sorting 256 single projections into 12 classes. Scale bar: 10 nm.

Figure 4

STEM analysis of isolated TIGR4 pili. (A) STEM dark-field image of unstained, freeze-dried TIGR4 pili; the regions selected for mass-per-length measurement are indicated. (B) The mass-per-length histogram obtained from this sample. The peak at 6.4 (±1.4)kDa/nm (n=395) shows the pilus filament to be a single string of RrgB monomers and predicts the presence of one monomer every 10.2 (±0.5)nm. (C, D) Contrast-reversed STEM dark-field images recorded at a magnification of × 10⁶ from negatively stained TIGR4 (2% PTA). A nose-like protrusion is present at irregular intervals and gives the filaments a defined polarity. Indeed, individual pilus subunits can be distinguished and have the same shape as RrgB–His monomers; compare with Figure 3B. Subunit boundaries are indicated by white lines in panel C (analysed in Supplementary Figure S9) and for panel D by the model in panel E. (E) Highly contoured TEM images of RrgB–His monomers (average length 12.2 (±0.5)nm) matched, without straightening, to the subunits of pilus (D), illustrating that its RrgB subunits overlap; the RrgB monomers are shown in alternating shades of grey and their ends are marked by dotted lines to facilitate visualisation. (F) Outline of the outer contours of panel E superimposed on panel D. Scale bar: panel A, 30 nm and panels C–F, 10 nm.

Figure 5

Localisation of the ancillary proteins, RrgA and RrgC. TEM of negatively stained (2% UAc) immunolabelled TIGR4 pili, enlarged to show pilus polarity. (A) Pili labelled with anti-RrgA-His; the noses point away from the antibody cluster. The antibody cluster is indicated by a white arrow. (B) Pilus labelled with anti-RrgC–His; the noses point towards the antibody. The antibody is indicated by a white arrow. As RrgC anchors the pilus to the cell wall, as suggested by the release of pili in the absence of RrgC (Supplementary Figure S11), the black arrows in panels A and B point away from the bacterium. Scale bar: panels A and B, 20 nm.

Figure 6

Model of S. pneumoniae pilus. (A) The TIGR4 pilus consists of a shaft composed of RrgB, with RrgA at its distal and RrgC at its proximal end. Sortase SrtC-1 mediates the polymerisation of RrgB (red) via the LPXTG motif (^*, IPQTG) and pilin motif (#) (Manzano et al, 2008) into a single string of monomers, covalently linked head-to-tail. The nose-like feature of RrgB gives the pilus a clear polarity. C-type sortases also control the addition of the ancillary proteins RrgA and RrgC, and anchor RrgC to the peptidoglycan cell wall, depending on the recognition of the respective LPXTG motifs YPRTG (RrgA) and VPDTG (RrgC; LeMieux et al, 2008; Neiers et al, 2009). RrgA is located at the distal end of the pilus, while RrgC is proximal to the bacterium. This is in agreement with the proposed role of RrgA as adhesin (Nelson et al, 2007; Hilleringmann et al, 2008) and the release of pili into the supernatant when RrgC is not present. RrgA and RrgC do not localise together in these single pili and are neither incorporated into the pilus shaft nor present along its length as proposed earlier (LeMieux et al, 2006; Fälker et al, 2008; Hilleringmann et al, 2008). (B) The ability of RrgA and RrgC to form a heterodimer (LeMieux et al, 2008) suggests a site in RrgC that can be covalently linked to an LPXTG motif from either RrgA or RrgB. The inability of RrgA and/or RrgC to form polymers may indicate that (i) RrgA has no site to interact with an LPXTG motif and (ii) RrgC has an LPXTG motif that activates only the C-type sortase, which links it to the cell wall.