A functional genomics catalogue of activated transcription factors during pathogenesis of pneumococcal disease

Background: Association analysis is an alternative to conventional family-based methods to detect the location of gene(s) or quantitative trait loci (QTL) and provides relatively high resolution in terms of defining the genome position of a gene or QTL. Seed protein and oil concentration are quantitative traits which are determined by the interaction among many genes with small to moderate genetic effects and their interaction with the environment. In this study, a genome-wide association study (GWAS) was performed to identify quantitative trait loci (QTL)controlling seed protein and oil concentration in 298 soybean germplasm accessions exhibiting a wide range of seed protein and oil content. Results: A total of 55,159 single nucleotide polymorphisms (SNPs) were genotyped using various methods including illumina Infinium and GoldenGate assays and 31,954 markers with minor allele frequency >0.10 were used to estimate linkage disequilibrium (LD) in heterochromatic and euchromatic regions. In euchromatic regions, the mean LD (r2) rapidly declined to 0.2 within 360 Kbp, whereas the mean LD declined to 0.2 at 9,600 Kbp in heterochromatic regions. The GWAS results identified 40 SNPs in 17 different genomic regions significantly associated with seed protein. Of these, the five SNPs with the highest associations and seven adjacent SNPs were located in the 27.6-30.0 Mbp region of Gm20. A major seed protein QTL has been previously mapped to the same location and potential candidate genes have recently been identified in this region. The GWAS results also detected 25 SNPs in 13 different genomic regions associated with seed oil. Of these markers, seven SNPs had a significant association with both protein and oil. Conclusions: This research indicated that GWAS not only identified most of the previously reported QTL controlling seed protein and oil, but also resulted in narrower genomic regions than the regions reported as containing these QTL. The narrower GWAS-defined genome regions will allow more precise marker-assisted allele selection and will expedite positional cloning of the causal gene(s)

The pneumococcal alpha-glycerophosphate oxidase enhances nasopharyngeal colonization through binding to host glycoconjugates

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen, causing a broad spectrum of diseases including otitis media, pneumonia, bacteraemia and meningitis. Here we examined the role of a potential pneumococcal meningitis vaccine antigen, alpha-glycerophosphate oxidase (SpGlpO), in nasopharyngeal colonization. We found that serotype 4 and serotype 6A strains deficient in SpGlpO have significantly reduced capacity to colonize the nasopharynx of mice, and were significantly defective in adherence to human nasopharyngeal carcinoma cells in vitro. We also demonstrate that intranasal immunization with recombinant SpGlpO significantly protects mice against subsequent nasal colonization by wild type serotype 4 and serotype 6A strains. Furthermore, we show that SpGlpO binds strongly to lacto/neolacto/ganglio host glycan structures containing the GlcNAcβ1-3Galβ disaccharide, suggesting that SpGlpO enhances colonization of the nasopharynx through its binding to host glycoconjugates. We propose that SpGlpO is a promising vaccine candidate against pneumococcal carriage, and warrants inclusion in a multi-component protein vaccine formulation that can provide robust, serotype-independent protection against all forms of pneumococcal disease

Identification of genes that contribute to the pathogenesis of invasive Pneumococcal Disease by In Vivo transcriptomic analysis

Streptococcus pneumoniae (the pneumococcus) continues to be responsible for a high level of global morbidity and mortality resulting from pneumonia, bacteremia, meningitis, and otitis media. Here we have used a novel technique involving niche-specific, genome-wide in vivo transcriptomic analyses to identify genes upregulated in distinct niches during pathogenesis after intranasal infection of mice with serotype 4 or 6A pneumococci. The analyses yielded 28 common, significantly upregulated genes in the lungs relative to those in the nasopharynx and 25 significantly upregulated genes in the blood relative to those in the lungs in both strains, some of which were previously unrecognized. The role of five upregulated genes from either the lungs or the blood in pneumococcal pathogenesis and virulence was then evaluated by targeted mutagenesis. One of the mutants (ΔmalX) was significantly attenuated for virulence in the lungs, two (ΔaliA and ΔilvH) were significantly attenuated for virulence in the blood relative to the wild type, and two others (ΔcbiO and ΔpiuA) were completely avirulent in a mouse intranasal challenge model. We also show that the products of aliA, malX, and piuA are promising candidates for incorporation into multicomponent protein-based pneumococcal vaccines currently under development. Importantly, we suggest that this new approach is a viable complement to existing strategies for the discovery of genes critical to the distinct stages of invasive pneumococcal disease and potentially has broad application for novel protein antigen discovery in other pathogens such as S. pyogenes, Haemophilus influenzae type b, and Neisseria meningitidis

A transcription factor contributes to pathogenesis and virulence in streptococcus pneumoniae

To date, the role of transcription factors (TFs) in the progression of disease for many pathogens is yet to be studied in detail. This is probably due to transient, and generally low expression levels of TFs, which are the central components controlling the expression of many genes during the course of infection. However, a small change in the expression or specificity of a TF can radically alter gene expression. In this study, we combined a number of quality-based selection strategies including structural prediction of modulated genes, gene ontology and network analysis, to predict the regulatory mechanisms underlying pathogenesis of Streptococcus pneumoniae (the pneumococcus). We have identified two TFs (SP_0676 and SP_0927 [SmrC]) that might control tissue-specific gene expression during pneumococcal translocation from the nasopharynx to lungs, to blood and then to brain of mice. Targeted mutagenesis and mouse models of infection confirmed the role of SP_0927 in pathogenesis and virulence, and suggests that SP_0676 might be essential to pneumococcal viability. These findings provide fundamental new insights into virulence gene expression and regulation during pathogenesis.Layla K. Mahdi, Esmaeil Ebrahimie, David L. Adelson, James C. Paton, Abiodun D. Ogunniy

Comparative GO: a web application for comparative Gene Ontology and Gene Ontology-based gene selection in bacteria

Extent: 8p.The primary means of classifying new functions for genes and proteins relies on Gene Ontology (GO), which defines genes/proteins using a controlled vocabulary in terms of their Molecular Function, Biological Process and Cellular Component. The challenge is to present this information to researchers to compare and discover patterns in multiple datasets using visually comprehensible and user-friendly statistical reports. Importantly, while there are many GO resources available for eukaryotes, there are none suitable for simultaneous, graphical and statistical comparison between multiple datasets. In addition, none of them supports comprehensive resources for bacteria. By using Streptococcus pneumoniae as a model, we identified and collected GO resources including genes, proteins, taxonomy and GO relationships from NCBI, UniProt and GO organisations. Then, we designed database tables in PostgreSQL database server and developed a Java application to extract data from source files and loaded into database automatically. We developed a PHP web application based on Model-View-Control architecture, used a specific data structure as well as current and novel algorithms to estimate GO graphs parameters. We designed different navigation and visualization methods on the graphs and integrated these into graphical reports. This tool is particularly significant when comparing GO groups between multiple samples (including those of pathogenic bacteria) from different sources simultaneously. Comparing GO protein distribution among up- or down-regulated genes from different samples can improve understanding of biological pathways, and mechanism(s) of infection. It can also aid in the discovery of genes associated with specific function(s) for investigation as a novel vaccine or therapeutic targets.Mario Fruzangohar, Esmaeil Ebrahimie, Abiodun D. Ogunniyi, Layla K. Mahdi, James C. Paton, David L. Adelso

The effects of methionine acquisition and synthesis on Streptococcus pneumoniae growth and virulence

Extent: 14 p.Bacterial pathogens need to acquire nutrients from the host, but for many nutrients their importance during infection remain poorly understood. We have investigated the importance of methionine acquisition and synthesis for Streptococcus pneumoniae growth and virulence using strains with gene deletions affecting a putative methionine ABC transporter lipoprotein (Sp_0149, metQ) and/or methionine biosynthesis enzymes (Sp_0585 - Sp_0586, metE and metF). Immunoblot analysis confirmed MetQ was a lipoprotein and present in all S. pneumoniae strains investigated. However, vaccination with MetQ did not prevent fatal S. pneumoniae infection in mice despite stimulating a strong specific IgG response. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry demonstrated that MetQ has both a high affinity and specificity for L-methionine with a KD of ~ 25 nM, and a DmetQ strain had reduced uptake of C14-methionine. Growth of the ΔmetQ/ΔmetEF strain was greatly impaired in chemically defined medium containing low concentrations of methionine and in blood but was partially restored by addition of high concentrations of exogenous methionine. Mixed infection models showed no attenuation of the ΔmetQ, ΔmetEF and ΔmetQ/DmetEF strains in their ability to colonise the mouse nasopharnyx. In a mouse model of systemic infection although significant infection was established in all mice, there were reduced spleen bacterial CFU after infection with the ΔmetQ/ΔmetEF strain compared to the wild-type strain. These data demonstrate that Sp_0149 encodes a high affinity methionine ABC transporter lipoprotein and that Sp_0585 – Sp_0586 are likely to be required for methionine synthesis. Although Sp_0149 and Sp_0585-Sp_0586 make a contribution towards full virulence, neither was essential for S. pneumoniae survival during infection.Shilpa Basavanna, Suneeta Chimalapati, Abbas Maqbool, Bruna Rubbo, Jose Yuste, Robert J. Wilson, Arthur Hosie, Abiodun D. Ogunniyi, James C. Paton, Gavin Thomas and Jeremy S. Brow

A molecular mechanism for bacterial susceptibility to zinc

Transition row metal ions are both essential and toxic to microorganisms. Zinc in excess has significant toxicity to bacteria, and host release of Zn(II) at mucosal surfaces is an important innate defence mechanism. However, the molecular mechanisms by which Zn(II) affords protection have not been defined. We show that in Streptococcus pneumonia extracellular Zn(II) inhibits the acquisition of the essential metal Mn(II) by competing for binding to the solute binding protein PsaA. We show that, although Mn(II) is the high-affinity substrate for PsaA, Zn(II) can still bind, albeit with a difference in affinity of nearly two orders of magnitude. Despite the difference in metal ion affinities, high-resolution structures of PsaA in complex with Mn(II) or Zn(II) showed almost no difference. However, Zn(II)-PsaA is significantly more thermally stable than Mn(II)-PsaA, suggesting that Zn(II) binding may be irreversible. In vitro growth analyses show that extracellular Zn(II) is able to inhibit Mn(II) intracellular accumulation with little effect on intracellular Zn(II). The phenotype of S. pneumoniae grown at high Zn(II):Mn(II) ratios, i.e. induced Mn(II) starvation, closely mimicked a DpsaA mutant, which is unable to accumulate Mn(II). S. pneumoniae infection in vivo elicits massive elevation of the Zn(II):Mn(II) ratio and, in vitro, these Zn(II):Mn(II) ratios inhibited growth due to Mn(II) starvation, resulting in heightened sensitivity to oxidative stress and polymorphonuclear leucocyte killing. These results demonstrate that microbial susceptibility to Zn(II) toxicity is mediated by extracellular cation competition and that this can be harnessed by the innate immune response.Christopher A. McDevitt, Abiodun D. Ogunniyi, Eugene Valkov, Michael C. Lawrence, Bostjan Kobe, Alastair G. McEwan and James C. Pato

A Variable Region within the Genome of Streptococcus pneumoniae Contributes to Strain-Strain Variation in Virulence

The bacterial factors responsible for the variation in invasive potential between different clones and serotypes of Streptococcus pneumoniae are largely unknown. Therefore, the isolation of rare serotype 1 carriage strains in Indigenous Australian communities provided a unique opportunity to compare the genomes of non-invasive and invasive isolates of the same serotype in order to identify such factors. The human virulence status of non-invasive, intermediately virulent and highly virulent serotype 1 isolates was reflected in mice and showed that whilst both human non-invasive and highly virulent isolates were able to colonize the murine nasopharynx equally, only the human highly virulent isolates were able to invade and survive in the murine lungs and blood. Genomic sequencing comparisons between these isolates identified 8 regions >1 kb in size that were specific to only the highly virulent isolates, and included a version of the pneumococcal pathogenicity island 1 variable region (PPI-1v), phage-associated adherence factors, transporters and metabolic enzymes. In particular, a phage-associated endolysin, a putative iron/lead permease and an operon within PPI-1v exhibited niche-specific changes in expression that suggest important roles for these genes in the lungs and blood. Moreover, in vivo competition between pneumococci carrying PPI-1v derivatives representing the two identified versions of the region showed that the version of PPI-1v in the highly virulent isolates was more competitive than the version from the less virulent isolates in the nasopharyngeal tissue, blood and lungs. This study is the first to perform genomic comparisons between serotype 1 isolates with distinct virulence profiles that correlate between mice and humans, and has highlighted the important role that hypervariable genomic loci, such as PPI-1v, play in pneumococcal disease. The findings of this study have important implications for understanding the processes that drive progression from colonization to invasive disease and will help direct the development of novel therapeutic strategies

Extracellular matrix formation enhances the ability of streptococcus pneumoniae to cause invasive disease

Extent: 17p.During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100- fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease.Claudia Trappetti, Abiodun D. Ogunniyi, Marco R. Oggioni and James C. Pato

Contribution of a genomic accessory region encoding a putative cellobiose phosphotransferase system to virulence of Streptococcus Pneumoniae

Streptococcus pneumoniae (the pneumococcus) is a formidable human pathogen, responsible for massive global morbidity and mortality. The ability to utilize carbohydrates in a variety of host niches appears to be integral to pneumococcal pathogenesis. In this study we investigated a genomic island, which includes a ROK family protein, a putative cellobiose phosphotransferase system (PTS) and a putative sulfatase. This accessory region is widespread in the pneumococcus in strains of various serotypes and levels of virulence. We have performed simple bioinformatic analysis of the region and investigated its role in vivo in 2 strains with markedly different virulence profiles (WCH206 of serotype 3, ST180; Menzies5 of serotype 11A, ST662). Deleting and replacing the entire island with an antibiotic resistance cassette caused the virulent serotype 3 strain to become attenuated in a murine pneumonia/sepsis model. Further mutants were constructed and used to show that various components of the island contribute significantly to the fitness of WCH206 in a variety of niches of this model, including the nasopharynx, ears and blood, but especially in the lungs. In addition, the island conferred a competitive advantage in nasopharyngeal colonization for the serotype 11A strain, which was essentially avirulent in the pneumonia/sepsis model. The contribution of this island to both pathogenesis and colonization may explain why this accessory region is widespread in the pneumococcus.Lauren J. McAllister, Abiodun D. Ogunniyi, Uwe H. Stroeher and James C. Pato