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

<p>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.</p

Sequence Analysis of 96 Genomic Regions Identifies Distinct Evolutionary Lineages within CC156, the Largest <i>Streptococcus pneumoniae</i> Clonal Complex in the MLST Database

<div><p>Multi-Locus Sequence Typing (MLST) of <i>Streptococcus pneumoniae</i> is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize <i>S. pneumoniae</i> strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.</p> </div

CC156 strain panel used in this study.

<p>For each strain name, ST, serotype/serogroup, country of isolation, number of MLST alleles in common with ST156 and ST4945, data source, strain source and lineage (as identified by 96-MLST hierarchical clustering, <i>see</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061003#pone-0061003-g002" target="_blank">Figure 2</a>) are indicated.</p

Triple Immunoelectron-Microscopy (IEM) Analysis of the Pilus Subunits of <i>Streptococcus pneumoniae</i>.

<p>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 <i>of Streptococcus pneumoniae</i> T4 (B), showing a similar pattern of gold distribution (scale bar, 100 nm).</p

Minimum Spanning Tree analysis based on 96-MLST allelic profiles identifies seven distinct lineages by imposing a maximum threshold of 75 different loci.

<p>The Minimum Spanning Tree analysis was performed by using PHYLOVIZ on the 96-MLST alleles of the 66 strains considered in this study. The lineages identified by applying the threshold of 75/96 different loci are highlighted with shadowed shapes and named according to the lineage identification of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061003#pone-0061003-g002" target="_blank">Figure 2</a>.</p

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

<p>(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.</p

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

<p>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.</p

Model of a Pneumococcal Pilus.

<p>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.</p

Micrograph of Negative Stained Whole Cell <i>Streptococcus pneumoniae</i> TIGR4.

<p>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).</p

Multi-Step Purification of Native Pneumococcal Pili.

<p>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 (*).</p