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EuroPneumo Special Issue / pneumonia 2015 Oct 21;7:I–72 P2.45 Structural analysis of the choline-binding sites in CbpL from Streptococcus pneumoniae Javier Gutiérrez-Fernández 1 , Martín Alcorlo Pagés 1 , Malek Saleh 2 , Sergio G. Bartual 1 , Thomas Pribyl 2 , Sven Hammerschmidt 2 , Juan A. Hermoso 1 1 Spanish National Research Council, Madrid, Spain; 2 Ernst Moritz Arndt University of Greifswald, Greifswald, Germany Four families of surface proteins decorate the cell surface of the human pathogen Streptococcus pneumoniae. Besides lipoproteins and LPXTG proteins, also present in other Gram-positive bacteria, the pneumococcus presents the nonclassical surface proteins and the choline-binding protein family. Choline binding proteins (CBPs) show a modular organisation including, at least, the choline-binding domain and a domain exerting a biological function. The cholinebinding domain interacts with choline molecules from teichoic and lipoteichoic acids, attaching the whole protein to the peptidoglycan layer. Here, we show the three-dimensional structure of the choline-binding domain of CbpL displaying 8 choline-binding sites. Four of them follow the canonical sequence while the other 4 are different. The alternate configuration of canonical and non-canonical sites is a unique property of CbpL among CBP family. Structural analysis and specific features of this module will be provided. P2.46 Assessment of the specific role of the phosphorylcholine esterase Pce in the modification of pneumococcal teichoic acids Franziska Waldow 1 , Thomas Kohler 2 , Dominik Schwudke 1, 3 , Sven Hammerschmidt 2 , Nicolas Gisch 1 1 Division of Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany; 2 Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany; 3 Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Germany Pneumococcal diseases are a global burden and Streptococcus pneumoniae is one of the most important pathogens in bacterial meningitis, otitis media, and sinusitis. Unlike other Gram-positive bacteria, the lipoteichoic acid (LTA) and wall teichoic acid (WTA) of S. pneumoniae possess the same structure within their repeating units. Pneumococci have a unique nutritional requirement for choline for growth to be able to attach phosphorylcholine (P-Cho) residues to the N-acetylgalactosamine moieties of their teichoic acids (TAs). This structural feature is of physiological importance for the anchoring of surface-localised choline-binding proteins (CBPs) to the pneumococcal cell wall. CBPs like the pneumococcal surface protein C (PspC) and the phosphorylcholine esterase (Pce) have been shown to be involved in pneumococcal adhesion to host cells. Interestingly, Pce hydrolyses about 15–30% of the total P-Cho residues attached to pneumococcal TAs. In the study presented here, we investigate which P-Cho residues are specifically removed from the TAs by the Pce. LTAs of different pneumococcal strains were isolated by citric buffer/butan-1-ol extraction and purified by hydrophobic interaction chromatography. These LTA preparations were further treated with anhydrous hydrazine to remove fatty acids and D-alanine residues. This procedure has two major advantages: less complex mass spectrometry data can be obtained and 1 H as well as 31 P NMR spectra show significantly higher resolutions. Importantly, in these spectra of O-deacylated and therefore non-aggregated LTA molecules specific moieties can be quantified. We have shown recently that the terminus of the pneumococcal LTA can vary in the P-Cho substitution pattern in dependency on the strain (D39 vs. TIGR4) and the culturing conditions. Here, the role of Pce in the modification of pneumococcal TAs will be elucidated by detailed structural investigation of LTA isolated from TIGR4∆cps∆pce as well as by treatment of fully P-Cho-decorated pneumococcal LTA with heterologously expressed Pce. pneumonia 2015 Volume 7 47
EuroPneumo Special Issue / pneumonia 2015 Oct 21;7:I–72 Oral abstracts O1.1 Identification of pneumococcal colonisation determinants in the stringent response pathway, facilitated by genomic diversity Yuan Li 1 , Nicholas J. Croucher 1 , Claudette M. Thompson 1 , Krzysztof Trzcinski 2 , William P. Hanage 1 , Marc Lipsitch 1 1 Harvard T.H. Chan School of Public Health, Boston, MA, USA, 2 UMC Utrecht, Utrecht, The Netherlands Understanding genetic determinants of a microbial phenotype generally involves creating and comparing isogenic strains differing at the locus of interest, but the naturally existing genomic and phenotypic diversity of microbial populations has rarely been exploited. Here we report use of a diverse collection of 616 carriage isolates and their genome sequences to help identify a novel determinant of pneumococcal colonisation. A spontaneously arising laboratory variant (SpnYL101) of a capsule-switched TIGR4 strain (TIGR4:19F) showed reduced ability to establish mouse nasal colonisation and lower resistance to non-opsonic neutrophil-mediated killing in vitro, a phenotype correlated with in vivo success. Whole genome sequencing revealed 5 single nucleotide polymorphisms (SNPs) affecting 4 genes in SpnYL101 relative to its ancestor. To evaluate the effect of variation in each gene, we performed an in silico screen of 616 previously published genome sequences to identify pairs of closely related, serotype-matched isolates that differ at the gene of interest, and compared their resistance to neutrophil killing. This method allowed rapid examination of multiple candidate genes and found phenotypic differences apparently associated with variation in SP_1645, a RelA/ SpoT homolog (RSH) involved in the stringent response. To establish causality, the alleles corresponding to SP_1645 were switched between the TIGR4:19F and SpnYL101. The wild-type SP_1645 conferred higher resistance to neutrophil killing and competitiveness in mouse colonisation. Using a similar strategy, variation in another RSH gene (TIGR4 locus tag SP_1097) was found to alter resistance to neutrophil killing. O1.2 Genomics reveals the worldwide distribution of multidrug-resistant serotype 6E pneumococci Andries van Tonder 1 , James Bray 1 , Lucy Roalfe 2 , Rebecca White 2 , Marta Zancoli 2 , Sigríður Quirk 3 , Gunnstein Haraldsson 3 , Keith Jolley 1 , Martin Maiden 1 , Stephen Bentley 5 , Ásgeir Haraldsson 3, 4 , Helga Erlendsdóttir 3, 4 , Karl Kristinsson 3, 4 , David Goldblatt 2 , Angela Brueggemann 1 1 University of Oxford, Oxford, UK, 2 University College London, London, UK, 3 University of Iceland, Reykjavik, UK, 4 Landspitali University Hospital, Reykjavik, UK, 5 Wellcome Trust Sanger Institute, Hinxton, UK The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that there are nearly 100 antigenically distinct serotypes. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and a common coloniser of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analysed a diverse dataset of approximately 1000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analysed the genetic diversity among serogroup 6 pneumococci and investigated whether pneumococcal conjugate vaccine (PCV)-induced serotype 6A and 6B antibodies inhibited serotype 6E pneumococci. We found that 43% of all genomes were serotype 6E and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, 3 of which were multidrug-resistant, described approximately 90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to kill serotype 6E pneumococci. We also revealed 3 major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term due to the emergence of serotypes that can evade vaccine- pneumonia 2015 Volume 7 48
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