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Genomic Analyses of Pneumococci Reveal a Wide Diversity of Bacteriocins

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Genomic Analyses of Pneumococci Reveal a Wide Diversity of Bacteriocins Bogaardt et al. BMC Genomics (2015) 16:554 DOI 10.1186/s12864-015-1729-4 RESEARCHARTICLE Open Access Genomic analyses of pneumococci reveal a wide diversity of bacteriocins – including pneumocyclicin, a novel circular bacteriocin Carlijn Bogaardt, Andries J van Tonder and Angela B Brueggemann* Abstract Background: One of the most important global pathogens infecting all age groups is Streptococcus pneumoniae (the ‘pneumococcus’). Pneumococci reside in the paediatric nasopharynx, where they compete for space and resources, and one competition strategy is to produce a bacteriocin (antimicrobial peptide or protein) to attack other bacteria and an immunity protein to protect against self-destruction. We analysed a collection of 336 diverse pneumococcal genomes dating from 1916 onwards, identified bacteriocin cassettes, detailed their genetic composition and sequence diversity, and evaluated the data in the context of the pneumococcal population structure. Results: We found that all genomes maintained a blp bacteriocin cassette and we identified several novel blp cassettes and genes. The composition of the ‘bacteriocin/immunity region’ of the blp cassette was highly variable: one cassette possessed six bacteriocin genes and eight putative immunity genes, whereas another cassette had only one of each. Both widely-distributed and highly clonal blp cassettes were identified. Most surprisingly, one-third of pneumococcal genomes also possessed a cassette encoding a novel circular bacteriocin that we called pneumocyclicin, which shared a similar genetic organisation to well-characterised circular bacteriocin cassettes in other bacterial species. Pneumocyclicin cassettes were mainly of one genetic cluster and largely found among seven major pneumococcal clonal complexes. Conclusions: These detailed genomic analyses revealed a novel pneumocyclicin cassette and a wide variety of blp bacteriocin cassettes, suggesting that competition in the nasopharynx is a complex biological phenomenon. Background capsule or ‘serotype’: over 90 different serotypes have been The pneumococcus is among the most important patho- characterised and new ones continue to be discovered gens worldwide: in 2000, ~14.5 million estimated cases [4, 5]. Consequently, after PCV is introduced, vaccine- of life-threatening pneumococcal diseases like pneumo- serotype disease decreases and nonvaccine-serotype dis- nia, bacteraemia and meningitis occurred and ~826,000 ease often increases [6]. The prevalence of commensal children died [1]. Pneumococcal disease can be treated pneumococci in the paediatric nasopharynx, its ecological with antibiotics, but antibiotic-resistant pneumococci are niche, generally remains the same post-PCV, but reorders found worldwide, e.g. 60 % of pneumococci recovered in in favour of nonvaccine serotypes [7]. Vaccine escape is Asia are multidrug-resistant [2]. Pneumococcal conjugate also possible and new variants can spread rapidly [8–10]. vaccines (PCVs) are administered to children in many de- Nasopharyngeal colonisation of one or more pneumo- veloped countries and some resource-poor countries, coccal serotypes is ubiquitous among children and usu- which has significantly reduced morbidity and mortality ally asymptomatic [11]. The composition of colonising [3]; however, current PCVs only protect against 10 or 13 pneumococci fluctuates over time, indicating the import- pneumococcal types, depending on the vaccine. Pneumo- ance of intraspecies competition in pneumococcal ecology coccal types are defined by an antigenic polysaccharide [12, 13]. Understanding the dynamics of competition is important in the context of understanding how perturba- * Correspondence: [email protected] tions such as vaccine introduction affect the pneumococ- Nuffield Department of Medicine, Peter Medawar Building for Pathogen cal population structure and result in changes in the Research, University of Oxford, Oxford, United Kingdom © 2015 Bogaardt et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bogaardt et al. BMC Genomics (2015) 16:554 Page 2 of 16 pneumococci competing for space and nutrients in the cassettes based on gene content and nucleotide se- nasopharynx. quence, and revealed several novel cassette types. Sur- Bacteriocins are small, ribosomally-synthesised anti- prisingly, we also discovered evidence for a circular microbial peptides or proteins produced by bacteria to bacteriocin cassette, which has not previously been de- inhibit other bacteria, and the producer strain has a ded- scribed among pneumococci. icated immunity system that protects it from its own bacteriocin. Bacteriocins are a diverse group of com- Results pounds in terms of size, mode of action and immunity Presence, nomenclature and classification of blp cassettes mechanisms, and are produced by both Gram-positive All 336 of the pneumococcal genomes investigated and Gram-negative bacteria. Given that the producer (Additional file 1) had a blp bacteriocin cassette. In the and target strains are often of the same bacterial species, literature there are two sets of gene names associated the bacteriocins are largely believed to be involved in with these cassettes in pneumococci and we have identi- competition for limited resources within an ecological fied additional novel genes in this study, adding to the niche, but they may also be contributing to the mainten- confusion around nomenclature. Therefore, the gene ance of microbial diversity at a population level [14–16]. names used in this study are presented in Table 1. Unre- Pneumococci have been shown to mediate intraspecies solvable ambiguities were encountered in seven cassette competition by the production of bacteriocins encoded assemblies and these were excluded from further analyses. by the highly variable blp (also known as spi/pnc)cas- Sequences for the 329 remaining blp cassettes were sepa- sette [17–22]. This cassette typically contains several rated by gene content into 33 Categories and by sequence bacteriocin-like peptide genes, clustered together with similarity into 79 Groups. 79 Group prototype cassettes genes for putative membrane proteins that may have were analysed further. functions in immunity, in a region labelled the ‘bacteri- ocin/immunity region’ (BIR). The predicted pneumococcal Description of the blp cassettes at the level of gene bacteriocin peptides show homology to type II bacteriocin organisation precursor peptides, and consist of a conserved N-terminal The length of the blp bacteriocin gene cassettes ranged leader sequence ending in a double glycine motif and an from 9.1 to 17.5 kb (Fig. 1). All Categories had a number alanine/glycine-rich mature peptide [17]. Previous studies of genes in common at the start and end of the cassette, have demonstrated the existence of genes for at least 10 including the regulatory, ABC transporter and CAAX such peptides within the blp cassette [17, 20–22]. protease genes (possibly related to bacteriocin self- The blp locus is regulated by quorum sensing, in a immunity [25]), and a membrane protein gene putatively manner reminiscent of the regulation of competence by associated with immunity (Tables 1 and 2). One excep- ComCDE [17, 18]. This is typically effected by a three- tion to this was that blpY and blpZ were missing in Cat- component system consisting of sensor histidine kinase egory 4, having been replaced by the remnants of an BlpH (SpiH), response regulator BlpR (SpiR2), and pep- insertion sequence (IS) element. tide pheromone BlpC (SpiP). At high extracellular con- The demarcation of the ABC transporter genes (blpA) centrations of BlpC the pheromone binds to BlpH, in and transport accessory protein gene (blpB) was highly turn activating BlpR [23]. BlpR then binds to conserved variable even within Categories, and the frequent div- motifs in promoters, inducing expression of all genes in ision of blpA into multiple ORFs was consistent with the locus, resulting in the production of more BlpC and findings from previous studies [21, 22]. Son et al. bacteriocins. Similar to the bacteriocin-like peptides, BlpC analysed the sequences of the transporter genes and has an N-terminal leader sequence [17, 18]; upon recogni- linked the frequent presence of frameshift-causing re- tion by the dedicated ABC transporter BlpAB (SpiABCD), peatsanddeletionsinblpA to ‘cheater’ (immunity only, the signal peptide is cleaved off, and the mature peptide is non-inhibitory) phenotypes. transported out of the cell [24]. Sequence analyses and Located between the common genes, the composition induction experiments showed that different BlpC se- of the BIR was highly variable among Categories (Table 2). quences correspond to separate pherotypes, with no cross- The number of BIR genes ranged from 1–15 and included induction of the blp genes [17, 18]. structural bacteriocin genes, putative immunity genes, a The aims of our study were to: i) provide a detailed CAAX protease and genes with products of unknown characterisation and comparison of the blp bacteriocin function. cassettes from a large and diverse set of historical and Overall, most cassettes had one copy of each gene,
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