The pneumococcal histidine triad (Pht) proteins are a recently recognized family of surface proteins, comprising 4 members: PhtA, PhtB, PhtD, and PhtE. They are being promoted for inclusion in a multicomponent pneumococcal protein vaccine currently under development, but to date, their biological functions and their relative contributions to pathogenesis have not been clarified. In this study, the involvement of these proteins in pneumococcal virulence was investigated in murine models of sepsis and pneumonia by using defined, nonpolar mutants of the respective genes in Streptococcus pneumoniae D39. In either challenge model, mutagenesis of all 4 genes was required to completely abolish virulence relative to the wild-type, suggesting significant functional redundancy among Pht proteins. The in vivo expression of pht genes was significantly up-regulated in the nasopharynx and lungs compared with blood. We provide unequivocal molecular evidence for Zn(2+)-dependent, AdcR-mediated, regulation of pht gene expression by real-time reverse transcriptase-polymerase chain reaction, Western blotting, and electrophoretic mobility-shift assays. We also present the first direct evidence for the biological function of this protein family by demonstrating that Pht proteins are required for inhibition of complement deposition on the pneumococcal surface through the recruitment of complement factor H.

The importance of Mn 2؉ for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn 2؉ is required are yet to be fully elucidated. Here, we analyzed the effect of Mn 2؉ limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn 2؉ transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn 2؉ -dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn 2؉ -SodA and virulence-associated genes pcpA and prtA. We also demonstrate the upregulation of at least one oxidativeand nitrosative-stress-response gene cluster, comprising adhC, nmlR, and czcD, in response to Mn 2؉ stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn 2؉ -replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn 2؉ having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn 2؉ transport by PsaA in pneumococcal competence, physiology, and metabolism and elucidate mechanisms underlying the response to Mn 2؉ stress.

BackgroundThe 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.MethodsELISA 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.ResultsColorectal 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) while no similar association was found with serum IgG antibodies of B. fragilis (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).ConclusionS. 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.

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.Availability http://turing.ersa.edu.au/BacteriaGO.

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 pneumococcalgene 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.

ABSTRACTStreptococcus 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-widein vivotranscriptomic 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 (ΔaliAand ΔilvH) were significantly attenuated for virulence in the blood relative to the wild type, and two others (ΔcbiOand ΔpiuA) were completely avirulent in a mouse intranasal challenge model. We also show that the products ofaliA,malX, andpiuAare 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 asS. pyogenes,Haemophilus influenzaetype b, andNeisseria meningitidis.

Streptococcus pneumoniae is the most common cause of severe bacterial meningitis in children, the elderly, and immunocompromised individuals. To identify virulence factors preferentially expressed during meningitis, we conducted niche-specific genome-wide in vivo transcriptomic analysis after intranasal infection of mice with serotype 4 or 6A pneumococci. The expression of 34 bacterial genes was substantially altered in brain tissue of mice infected with either of the 2 strains. Ten upregulated genes were common to both strains, 7 of which were evaluated for their role in the development of meningitis. One previously uncharacterized protein, α-glycerophosphate oxidase (GlpO), was cytotoxic for human brain microvascular endothelial cells (HBMECs) via generation of H 2 O 2 . A glpO deletion mutant was defective in adherence to HBMECs in vitro as well as in progression from the blood to the brain in vivo. Mutant bacteria also induced markedly reduced meningeal inflammation and brain pathology compared with wild type, despite similar levels of bacteremia. Immunization of mice with GlpO protected against invasive pneumococcal disease and provided additive protection when formulated with pneumolysin toxoid. Our results provide the basis of a strategy that can be adapted to identify genes that contribute to the development of meningitis caused by other pathogens.

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