Ficolin-2 does not bind to other <i>wciG</i>-encoding serotypes.

Strains of the indicated serotype were incubated with 10% normal human serum for 1 hour and stained with either an anti-ficolin-2 antibody (ficolin-2) or an isotype matched monoclonal antibody specific for the serotype 11A capsule (hyp11AG1) prior to analysis by FACS. Serotypes 11A and 11E served as positive and negative controls, respectively, for both antibodies. Data are presented as mean ± standard deviation of two technical replicates.</p

Pneumococcal strains used in this study.

Pneumococcal strains used in this study.</p

Ficolin-2 binds the serotype 35B capsular polysaccharide in a manner dependent on WciG-mediated O-acetylation.

A, Serotype 35B strain 3009–06 was incubated with 10% normal human serum in the presence of increasing amounts of purified serotype 35B capsular polysaccharide (“35B PS”) or C-polysaccharide (“CWPS”) for 1 hour at 4°C. After washing, bacteria were incubated with a ficolin-2-specific monoclonal antibody, washed, and incubated with a phycoerythrin-conjugated secondary antibody prior to washing and analysis via flow cytometry. B, Serotype 35B strain 3009–06 (“35B”) and serotype 35D strain 3431–06 (“35D”), along with their derivatives KAG1014 (“35BΔwciG”); KAG1019 (“35BΔwciG; wciG3431-06”); KAG1023 (“35BΔwciG; wciG+); and KAG1022 (“35D; wciG+); were incubated with 10% serum at 4°C for 1 hour prior to detection of ficolin-2 as above (“10% Serum”). In half of wells, the anti-ficolin-2 antibody was omitted (“2° Only”). Data are presented as mean ± standard deviation for two technical replicates and representative of at least three independent experiments.</p

Frequencies of invasive pneumococcal disease by serotype.

Frequencies of invasive pneumococcal disease by serotype.</p

Studies of ficolin-2 binding and ficolin-2-mediated complement deposition.

A, ficolin-2 (FCN2) binds to representative serotype 11A, 35B, and 31 strains but not their isogenic O-acetyl transferase-deficient derivatives (11E, 35D, and 31X1). Bacteria were incubated with buffer (light gray fill), 10% heat-inactivated normal human serum (dark gray line), or 10% normal human serum (black line) for 1 hour at 4°C. After incubation, cells were stained with a ficolin-2-specific antibody and analyzed by flow cytometry. B, ficolin-2 stimulates complement deposition on serotypes 11A and 35B but not serotype 31. Bacteria were pre-incubated with buffer (light gray shading), recombinant ficolin-2 (solid black line), or heat-inactivated recombinant ficolin-2 (dashed black line). After washing, bacteria were incubated with 10% C1q-depleted serum (C1q-dpl). Complement fragment C4 degradation products (C4b and C4c) were detected with antibodies and assayed by flow cytometry. Ficolin-2 alone (not shown) did not elicit complement deposition.</p

The isolate 7236-07 <i>cps</i> locus does not contain capsular biosynthetic or novel surface protein genes.

<p>Schematic of the <i>cps</i> locus between primers 5419 and 3419 from isolate 7236-07 (deposited to GenBank, accession no. KJ363164). Genes and spacing are to scale. <i>dexB</i> and <i>aliA</i> are truncated at their 5′ and 3′ ends, respectively, in the sequence coverage. Annotations of <i>tnp</i> pseudogenes were assigned corresponding to the serotype 5 <i>cps</i> locus (GenBank no. CR931637).</p

Mutations in PCR-serotype 8 Isolates.

a<p>Sequence numbers correspond to GenBank accession No. AJ239004.</p>b<p>Polymorphisms present in an alternative serotype 8 sequence (accession No. CR931644) were ignored.</p>c<p>Residue numbers correspond to amino acid sequence deduced from <i>cap8C</i> (Wzd), <i>cap8D</i> (Wze), or <i>cap8E</i> (WchA) in GenBank accession No. AJ239004.</p>d<p>FS, frameshift;</p><p>*, stop.</p>e<p>Residue previously identified as a site of inactivating mutation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097825#pone.0097825-Xayarath1" target="_blank">[29]</a>.</p

Population-Based Analysis of Invasive Nontypeable Pneumococci Reveals That Most Have Defective Capsule Synthesis Genes

<div><p>Since nasopharyngeal carriage of pneumococcus precedes invasive pneumococcal disease, characteristics of carriage isolates could be incorrectly assumed to reflect those of invasive isolates. While most pneumococci express a capsular polysaccharide, nontypeable pneumococci are sometimes isolated. Carriage nontypeables tend to encode novel surface proteins in place of a capsular polysaccharide synthetic locus, the <i>cps</i> locus. In contrast, capsular polysaccharide is believed to be indispensable for invasive pneumococcal disease, and nontypeables from population-based invasive pneumococcal disease surveillance have not been extensively characterized. We received 14,328 invasive pneumococcal isolates through the Active Bacterial Core surveillance program during 2006–2009. Isolates that were nontypeable by Quellung serotyping were characterized by PCR serotyping, sequence analyses of the <i>cps</i> locus, and multilocus sequence typing. Eighty-eight isolates were Quellung-nontypeable (0.61%). Of these, 79 (89.8%) contained <i>cps</i> loci. Twenty-two nontypeables exhibited serotype 8 <i>cps</i> loci with defects, primarily within <i>wchA</i>. Six of the remaining nine isolates contained previously-described <i>aliB</i> homologs in place of <i>cps</i> loci. Multilocus sequence typing revealed that most nontypeables that lacked capsular biosynthetic genes were related to established non-encapsulated lineages. Thus, invasive pneumococcal disease caused by nontypeable pneumococcus remains rare in the United States, and while carriage nontypeables lacking <i>cps</i> loci are frequently isolated, such nontypeable are extremely rare in invasive pneumococcal disease. Most invasive nontypeable pneumococci possess defective <i>cps</i> locus genes, with an over-representation of defective serotype 8 <i>cps</i> variants.</p></div

Multi-locus sequence types of Group II Nontypeable Isolates, 2006–2009.

1<p>Data obtained from MLST.net, accessed January 26, 2014.</p

PCR-8 nontypeable isolates contain inactivating mutations in <i>wchA</i>.

<p>A. Schematic of the serotype 8 <i>cps</i> locus (GenBank accession No. AJ239004). Alternative nomenclature is shown for regulatory genes. B. Molecular model of WchA as predicted by TMPRED (<a href="http://www.ch.embnet.org/software/TMPRED_form.html" target="_blank">http://www.ch.embnet.org/software/TMPRED_form.html</a>). Black arrows indicate nonsense (*) and frameshift (**) mutations; gray arrows indicate missense mutations. Residue numbers correspond to the predicted primary amino acid sequence for WchA.</p