Pneumococcal pili are composed of protofilaments exposing adhesive clusters of Rrg A

Abstract

Pili have been identified on the cell surface of Streptococcus pneumoniae, a major cause of morbidity and mortality worldwide. In contrast to Gram-negative bacteria, little is known about the structure of native pili in Gram-positive species and their role in pathogenicity. Triple immunoelectron microscopy of the elongated structure showed that purified pili contained RrgB as the major compound, followed by clustered RrgA and individual RrgC molecules on the pilus surface. The arrangement of gold particles displayed a uniform distribution of anti-RrgB antibodies along the whole pilus, forming a backbone structure. Antibodies against RrgA were found along the filament as particulate aggregates of 2-3 units, often co-localised with single RrgC subunits. Structural analysis using cryo electron microscopy and data obtained from freeze drying/metal shadowing technique showed that pili are oligomeric appendages formed by at least two protofilaments arranged in a coiled-coil, compact superstructure of various diameters. Using extracellular matrix proteins in an enzyme-linked immunosorbent assay, ancillary RrgA was identified as the major adhesin of the pilus. Combining the structural and functional data, a model emerges where the pilus RrgB backbone serves as a carrier for surface located adhesive clusters of RrgA that facilitates the interaction with the host.

Figure 1. Micrograph of Negative Stained Whole Cell Streptococcus pneumoniae TIGR4.

Sample stained with 1% buffered phosphotungstic acid (PTA). Open arrows indicate an individual single pilus; arrow indicates bundles of individual pili. Scale bar, 200 nm. (Philips TEM CM200 FEG microscope at 50000× magnification, working at low-dose conditions).

Figure 2. Multi-Step Purification of Native Pneumococcal Pili.

SN of mutanolysin treated T4 bacteria was applied to a 25–56% sucrose gradient (A). Pili containing fractions (no. 5–8) were further purified using size exclusion chromatography: HMW pili (peak A*) were separated from lower molecular weight material (peak B and C) (B). Summary of the purification procedure is shown in C (silver stained SDS-PAGE, and respective western analysis with α-RrgB antibodies). Lanes: 1: HMW marker, 2: T4 whole cell lysate, 3: SN mutanolysin digestion, 4: sucrose gradient pool (fractions no. 5–8), 5: HMW pili – size exclusion chromatography pool peak A (*).

Figure 3. Triple Immunoelectron-Microscopy (IEM) Analysis of the Pilus Subunits of Streptococcus pneumoniae.

Isolated pili material (A) was incubated with antisera raised against His-tagged RrgA, RrgB and RrgC and conjugated respectively to 15 nm, 5 nm and 10 nm gold particles. The image shows the pilus backbone stained with gold-labelled antibodies raised against the main pneumococcal pilus component (RrgB). Clusters of RrgA ancillary proteins (open arrows) are present along the entire pilus. Single copies of the ancillary protein RrgC (arrows) were found alone or co-localized with the RrgA clusters. Scale bar, 100 nm. The same protocol for triple immunogold EM has been applied to bacteria preparation of Streptococcus pneumoniae T4 (B), showing a similar pattern of gold distribution (scale bar, 100 nm).

Figure 4. Cryo Electron Microscopy (Cryo-EM) Image of Isolated Single Pili.

The image shows the presence of different sized individual pili (open arrow and arrow) distributed on the EM grid where they form a net of elongated structures. Image of the vitrified sample has been taken by cryo-EM low-dose conditions in a TEM CM200FEG microscope at 50000× magnification. Scale bar, 100 nm.

Figure 5. Freeze Drying/Metal Shadowing and Immunogold EM Images of Purified Pili Preparation.

Gallery of shadowed (A, B and C) and immunogold labelled (D, E and F) pili showing the diversity of pili morphology and sizes. The protofilament is visible in (A and D), pili are visible in (B, C, E and F). For IEM isolated pili material was incubated with antisera raised against His-tagged RrgB followed by secondary gold antibodies. The metal shadow underlines the increased complexity when going from protofilaments to pili. Scale bar, 100 nm.

Figure 6. Density Profile of Thin Individual Pili.

(A) Six short boxed thin pili extracted from micrographs containing purified single pili. Scale bar, 10 nm. (B) Averaged thin pili after translational and rotational alignment of the boxed regions. Scale bar, 10 nm. (C) Density profiles across the pili axis of the averaged thin pili projected onto the short axis. Calculated average diameter (double-headed arrow) for thin pili is 9.6±0.3 nm.

Figure 7. Images of Gaussian Filtered Thin Individual Pili Show Their Structural Composition.

(A) Images of original pilus taken at low dose conditions. (B) Gaussian filtered thin individual pili show their structural composition. Inset reveals an enlarged view of the thin pilus corresponding to the pilus crossover. Crossover position (arrow) for thin pilus is indicated. The protofilament diameter was measured as 3.5 nm. Crossover diameters (arrows) were calculated as 6.8 nm for thin pili (9.5 nm diameter). Scale bars, 10 nm.

Figure 8. Dose Dependent Binding of RrgA to Selected Extracellular Matrix (ECM) Components.

Shown are the results of binding increasing concentrations of purified T4 pilus proteins HisTag-RrgA, -RrgB and -RrgC (A) and HMW pilus preparations (B) to fibronectin, collagen I and laminin. BSA and delta pilus mock preparation served as negative controls. Binding was quantified by ELISA at an absorbance of 405 nm. Points represent the means (error bars, standard errors of the means) of measurements made in triplicate.

Figure 9. Model of a Pneumococcal Pilus.

Model showing T4 pneumococcal pilus composed of at least two RrgB protofilaments (green) arranged in a coiled-coil superstructure with surface located ancillary proteins (RrgA and RrgC) is based on cryo-EM, freeze drying/metal shadowing EM, IEM and biochemical data. (R) and (L) illustrate a possible right and left handed orientation of the thin pilus respectively. Outlines are not drawn to scale.