Pneumococcal Gene Complex Involved in Resistance to Extracellular Oxidative Stress

Streptococcus pneumoniae is a Gram-positive bacterium which is a member of the normal human nasopharyngeal flora but can also cause serious disease such as pneumonia, bacteremia, and meningitis. Throughout its life cycle, S. pneumoniae is exposed to significant oxidative stress derived from endogenously produced hydrogen peroxide (H2O2) and from the host through the oxidative burst. How S. pneumoniae, an aerotolerant anaerobic bacterium that lacks catalase, protects itself against hydrogen peroxide stress is still unclear. Bioinformatic analysis of its genome identified a hypothetical open reading frame belonging to the thiol-specific antioxidant (TlpA/TSA) family, located in an operon consisting of three open reading frames. For all four strains tested, deletion of the gene resulted in an approximately 10-fold reduction in survival when strains were exposed to external peroxide stress. However, no role for this gene in survival of internal superoxide stress was observed. Mutagenesis and complementation analysis demonstrated that all three genes are necessary and sufficient for protection against oxidative stress. Interestingly, in a competitive index mouse pneumonia model, deletion of the operon had no impact shortly after infection but was detrimental during the later stages of disease. Thus, we have identified a gene complex involved in the protection of S. pneumoniae against external oxidative stress, which plays an important role during invasive disease.

Cations and oxidative stress response in Streptococcus pneumoniae

Streptococcus pneumoniae is a bacterium, which colonizes the human nasopharynx and can cause serious disease, such as pneumonia, otitis media, meningitis, and bacteremia. Generally, groups at risk for invasive pneumococcal disease are young children, elderly and immuno-compromised patients, both in developed and developing countries. Throughout the host, S. pneumoniae encounters various oxygen radicals, which can kill or damage the bacterium. The ability to withstand the damaging effects of oxygen radicals is crucial for the bacterial ability to cause disease. Our understanding of the pneumococcal defenses against oxidative stress is far from complete. In the present thesis, a novel protein complex is described, which protects the bacteria from the hazardous effects of oxygen radicals, most likely by repairing damaged proteins. Pneumococcal mutants lacking this complex were also less virulent in mouse models mimicking disease. Moreover, the presented results show for the first time that oxidative stress resistance is vital for the bacteria to survive on dry surfaces. This desiccation tolerance is thought to aid the transmission of the bacteria. Two key components necessary for the survival of desiccation stress are described in this thesis. Altogether, the present studies have provided further insights into (1) how S. pneumoniae copes with oxygen radicals and (2) the significance of oxidative stress resistance in infection and pathogenesis

The effect of phenylbutazone injection on serumic levels of thyroid hormones in the horse

This study was conducted to evaluate the effect of phenylbutazone injection on serumic levels of thyroid hormones in the Arabian horse. Twelve Arabian horses were allocated to two groups of control and treatment each consisting of six animals. In the treatment group, 3 mg/kg phenylbutazone was injected intravenously for 6 days while in the control group equal values of 0.9% NaCl solution was used for the injection. Blood samples of all animals were collected from the jugular vein at days 0(before injection), 1, 2, 3, 4, 5 and 6, their sera separated by centrifuging and the levels of T3 and T4 were measured using the ELISA technique. There was a significant difference (

Streptococcus pneumoniae and reactive oxygen species:an unusual approach to living with radicals

Streptococcus pneumoniae, an aerotolerant anaerobe, is an important human pathogen that regularly encounters toxic oxygen radicals from the atmosphere and from the host metabolism and immune system. Additionally, S. pneumoniae produces large amounts of H2O2 as a byproduct of its metabolism, which contributes to its virulence but also has adverse effects on its biology. Understanding how S. pneumoniae defends against oxidative stress is far from complete, but it is apparent that it does not follow the current paradigm of having canonical enzymes to detoxify oxygen radicals or homologues of typical oxidative stress responsive global regulators. We will give an overview of how S. pneumoniae copes with oxygen radicals. Furthermore, we draw parallels with other pathogenic streptococcal species and provide future research perspectives

Effect of <i>spxB</i> mutation and addition of catalase on <i>cps</i> transcription.

<p>Transcription of the <i>cps</i> promoter was estimated by measuring the β-galactosidase activities of strains D39 (bars with white background), D39<i>spxB</i> (bars with grey background) and D39<i>spxB</i> and its parent in the presence of 200 U/ml bovine liver catalase (spotted bars) harbouring pORI P<i>cps</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068277#tab1" target="_blank">Table 1)</a> in mid-exponential (A, left panel and B) and late-exponential (A, right panel) phases of growth. Cultures were grown in CDM containing 1% (wt/vol) Glc (A) or in BHI (B) at 37^°C, and under semi-aerobic conditions. All the determinations were done at least in triplicate and the values are means ± SD. a.u. = arbitrary units.</p

Growth profiles of strains D39, D39<i>spxB</i> and D39<i>spxB</i>^<i>+</i> under semi-aerobic conditions.

<p>Cultures were grown in A) M17 without pH control (initial pH 6.5) or B) CDM with pH control (pH 6.5), with 1% (wt/vol) Glc at 37^°C, in both cases under semi-aerobic conditions (for details see Materials and Methods). The plotted growth curves are from a representative experiment. For each condition at least three independent experiments were performed, except for the complemented strain (D39<i>spxB</i>^<i>+</i>) which was performed twice, and the error was below 15%. Symbols: (triangles), D39<i>spxB</i>; (squares), D39; (diamonds), D39<i>spxB</i>^<i>+</i>. Arrows in the CDM grown cultures indicate sampling time for capsule determination and intracellular metabolite analysis (Figures 2 and 5).</p

Inactivation of <i>spxB</i> led to an increase in capsule production.

<p>(A) Phenotype on Glc-M17 agar plate of D39 (D39), D39 containing the pGh9 T7 plasmid (D39pGh9 T7) and D39 in which the plasmid is excised leaving the ISS1 element in <i>spxB</i> (D39<i>spxB</i>). (B) Phenotype on Glc-M17 agar plate of TIGR4 transparent variant (T4 transparent), TIGR4 opaque variant (T4 opaque) and TIGR4 in which the pGh9 T7 plasmid is inserted into the genome (T4 pGh9 T7). (C–F) TEM pictures of <i>S. pneumoniae</i> grown in broth to exponential phase and stained with LRR. (C) D39, 9700 times magnified; (D) D39<i>spxB</i> 9700 times magnified; (E) D39, 135.000 times magnified; (F) D39<i>spxB</i> 135.000 times magnified.</p

Model of the <i>spxB</i> deletion effect on glucose metabolism and capsule production in <i>S. pneumoniae</i>.

<p>Glucose is converted by the conventional glycolytic pathway to pyruvate. Fermentation products result from the action of different competing enzymes (lactate dehydrogenase, pyruvate formate-lyase, pyruvate oxidase and hypothetically the pyruvate dehydrogenase complex). Overall, our data show that SpxB represses carbohydrate specific pathways (ketogluconate and hyaluronic acid utilization), capsule production, and stimulates formation of acetyl-P and acetate at the expense of pyruvate and lactate. Pyruvate and end-products are represented by three-dimensional bars and the height of the bar represents the relative amount; grey slashed arrows, function not experimentally verified; boxed metabolites indicate higher metabolite accumulation. Thick pointed-line, repression; Thick black arrow, activation. KDG, ketogluconate; HA, hyaluronic acid; GlcNAc, N-acetylglucosamine, GlcUA, glucuronic acid; Ac–P, acetyl-phosphate; α-G1P, α-glucose 1-phosphate; G6P, glucose 6-phosphate; FBP, fructose 1,6-bisphosphate; PEP, phosphoenolpyruvate.</p