Preparation of C-reactive protein latex reagent of human serum

C-reactive protein (CRP) is an acute phase reactant the concentration of which increases significantly following tissue injury or inflammation. It is present in the albumin fraction of the serum of patients and is antigenic upon injection into rabbits. We report here the development of a local latex reagent coated with rabbit anti-CRP. For this purpose CRP was isolated from pooled sera of patients and injected into rabbits. CRP rabbit antiserum was purified by using ammonium sulfate precipitation. Followed by passing on ion exchange chromatography (Diethylaminoethyl 'DEAE' cellulose). Then, latex particles were coated with purified antisera. The prepared CRP latex reagent agglutination was observed upon the addition of patients' sera and compared with that of normal sera. In addition, it was evaluated against a standard CRP latex reagent. The prepared reagent gave positive results (100%) with patients' sera and the positive control of the standard kit. While, the negative control of the standard kit and 30% of the normal sera gave negative results

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 variable region of the pneumococcal pathogenicity island 1 is responsible for the unusually high virulence of a serotype 1 isolate

Streptococcus pneumoniae is the leading infectious cause of death in children in the world. However, the mechanisms that drive the progression from asymptomatic colonization to disease are poorly understood. Two virulence-associated genomic accessory regions (ARs) were deleted in a highly virulent serotype 1 clinical isolate (strain 4496) and examined for their contribution to pathogenesis. Deletion of a prophage encoding a platelet-binding protein (PblB) resulted in reduced adherence, biofilm formation, reduced initial infection within the lungs, and a reduction in the number of circulating platelets in infected mice. However, the region’s overall contribution to the survival of mice was not significant. In contrast, deletion of the variable region of pneumococcal pathogenicity island 1 (vPPI1) was also responsible for a reduction in adherence and biofilm formation but also reduced survival and invasion of the pleural cavity, blood, and lungs. While the 4496�PPI1 strain induced higher expression of the genes encoding interleukin-10 (IL-10) and CD11b in the lungs of challenged mice than the wild-type strain, very few other genes exhibited altered expression. Moreover, while the level of IL-10 protein was increased in the lungs of 4496�PPI1 mutant-infected mice compared to strain 4496-infected mice, the levels of gamma interferon (IFN-�), CXCL10, CCL2, and CCL4 were not different in the two groups. However, the 4496�PPI1 mutant was found to be more susceptible than the wild type to phagocytic killing by a macrophage-like cell line. Therefore, our data suggest that vPPI1 may be a major contributing factor to the heightened virulence of certain serotype 1 strains, possibly by influencing resistance to phagocytic killing

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

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

Investigation into the controversial association of Streptococcus gallolyticus with colorectal cancer and adenoma

Background: The seroprevalence of IgG antibodies of Streptococcus gallolyticus subspecies gallolyticus, CIP 105428, was evaluated to investigate the controversial association of S. gallolyticus with colorectal carcinoma and adenoma in attempt to investigate the nature of such association if any, by exploring the mRNA expression of NF-κB and IL-8. Moreover, the serological behavior of S. gallolyticus IgG antibodies was compared to that of an indicator bacterium of bowel, Bacteroides fragilis. Methods: ELISA was used to measure IgG antibodies of S. gallolyticus and B. fragilis in sera of 50 colorectal cancer, 14 colorectal adenoma patients, 30 age- and sex- matched apparently healthy volunteers (HV) and 30 age- and sex- matched colonoscopically-proven tumor-free control subjects. NF-κB and IL-8 mRNA expression was evaluated in tumorous and non-tumorous tissue sections of carcinoma and adenoma patients in comparison with that of control subjects by using in situ hybridization assay. Results: Colorectal cancer and adenoma patients were associated with higher levels of serum S. Gallolyticus IgG antibodies in comparison with HV and control subjects (P 0.05). ELISA cutoff value for the seropositivity of S. gallolyticus IgG was calculated from tumor-free control group. The expression of NF-κB mRNA was higher in tumorous than non-tumorous tissue sections of adenoma and carcinoma, higher in carcinoma/adenoma sections than in control subjects, higher in tumorous sections of carcinoma than in adenoma patients, and higher in S. gallolyticus IgG seropositive than in seronegative groups in both tumorous and non-tumorous sections (P < 0.05). IL-8 mRNA expression in tumorous sections of adenoma and carcinoma was higher than in non-tumorous sections, higher in carcinoma/adenoma than in control subjects, and higher in S. gallolyticus IgG seropositive than in seronegative groups in tumorous rather than non-tumorous sections (P < 0.05). Conclusion: S. gallolyticus most likely plays an essential role in the oncogenic progression of normal colorectal mucosa to adenoma and to CRC. This promoting/propagating role of S. gallolyticus might take place by utilizing certain inflammatory, anti-apoptotic, and angiogenic factors of transformation including NF-κB and IL-8.Ahmed S Abdulamir, Rand R Hafidh, Layla K Mahdi, Tarik Al-jeboori and Fatimah Abubake

Pneumococcal Virulence Gene Expression and Host Cytokine Profiles during Pathogenesis of Invasive Disease▿ †

Pneumococcal disease continues to account for significant morbidity and mortality worldwide. For the development of novel prophylactic and therapeutic strategies against the disease spectrum, a complete understanding of pneumococcal behavior in vivo is necessary. We evaluated the expression patterns of the proven and putative virulence factor genes adcR, cbpA, cbpD, cbpG, cpsA, nanA, pcpA, piaA, ply, psaA, pspA, and spxB after intranasal infection of CD1 mice with serotype 2, 4, and 6A pneumococci by real-time reverse transcription-PCR. Simultaneous gene expression patterns of selected host immunomodulatory molecules, CCL2, CCL5, CD54, CXCL2, interleukin-6, and tomor necrosis factor alpha, were also investigated. We show that pneumococcal virulence genes are differentially expressed in vivo, with some genes demonstrating niche- and serotype-specific differential expression. The in vivo expression patterns could not be attributed to in vitro differences in expression of the genes in transparent and opaque variants of the three strains. The host molecules were significantly upregulated, especially in the lungs, blood, and brains of mice. The pneumococcal-gene expression patterns support their ascribed roles in pathogenesis, providing insight into which protein combinations might be more appropriate as vaccine antigens against invasive disease. This is the first simultaneous comparison of bacterial- and host gene expression in the same animal during pathogenesis. The strategy provides a platform for prospective evaluation of interaction kinetics between invading pneumococci and human patients in culture-positive cases and should be feasible in other infection models