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 GlcNAcß1-3Galß 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.

GlpC gene is responsible for biofilm formation and defense against phagocytes and imparts tolerance to pH and organic solvents in Proteus vulgaris.

Biofilm-forming bacteria are highly resistant to antibiotics, host immune defenses, and other external conditions. The formation of biofilms plays a key role in colonization and infection. To explore the mechanism of biofilm formation, mutant strains of Proteus vulgaris XC 2 were generated by Tn5 random transposon insertion. Only one biofilm defective bacterial species was identified from among 500 mutants. Inactivation of the glpC gene coding an anaerobic glycerol-3-phosphate dehydrogenase subunit C was identified by sequence analysis of the biofilm defective strain. Differences were detected in the growth phenotypes of the wild-type and mutant strains under pH, antibiotic, and organic solvent stress conditions. Furthermore, we observed an increase in the phagocytosis of the biofilm defective strain by the mouse macrophage RAW264.7 cell line compared to the wild-type strain. This study shows that the glpC gene plays an important role in biofilm formation, in addition to imparting pH, organic solvent, and antibiotic tolerance, and defense against phagocytosis to Proteus sp. The results further clarified the mechanism of biofilm formation at the genomic level, and indicated the importance of the glpC gene in this process. This data may provide innovative therapeutic measures against P. vulgaris infections; furthermore, as an important crocodile pathogen, this study also has important significance in the protection of Chinese alligators.

Cattle immunized against the pathogenic L-α-glycerol-3-phosphate oxidase of Mycoplasma mycoides subs. mycoides fail to generate neutralizing antibodies and succumb to disease on challenge.

The membrane-associated enzyme L-α-glycerol-3-phosphate oxidase (GlpO) of Mycoplasma mycoides subs. mycoides (Mmm), the causal agent of contagious bovine pleuropneumonia (CBPP) has been identified as a virulence factor responsible for the release of toxic by-products such as H2O2 that mediate host cell injury. Since CBPP pathogenesis is based on host inflammatory reactions, we have determined the capacity of recombinant GlpO to generate in vivo protective responses against challenge in immunized cattle. We also investigated whether sera raised against recombinant GlpO in cattle and mice inhibit production of H2O2 by Mmm. Immunization of cattle with recombinant GlpO did not protect against challenge with a virulent strain of Mmm. Further, although both murine and bovine antisera raised against recombinant GlpO detected recombinant and native forms of GlpO in immunoblot assays with similar titres, only murine antibodies could neutralize GlpO enzymatic function. The data raise the possibility that Mmm has adapted to evade potential detrimental antibody responses in its definitive host.

Identification of a novel pneumococcal vaccine antigen preferentially expressed during meningitis in mice.

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.

Glycerol-3-phosphate and systemic immunity.

Glycerol-3-phosphate (G3P), a conserved three-carbon sugar, is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis. G3P can be derived via the glycerol kinase-mediated phosphorylation of glycerol or G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate. Previously, we showed G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Inoculation of Arabidopsis with C. higginsianum correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in GLY1 encoded G3Pdh accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Recently, we showed that G3P is also a potent inducer of systemic acquired resistance (SAR) in plants. SAR is initiated after a localized infection and confers whole-plant immunity to secondary infections. SAR involves generation of a signal at the site of primary infection, which travels throughout the plants and alerts the un-infected distal portions of the plant against secondary infections. Plants unable to synthesize G3P are defective in SAR and exogenous G3P complements this defect. Exogenous G3P also induces SAR in the absence of a primary pathogen. Radioactive tracer experiments show that a G3P derivative is translocated to distal tissues and this requires the lipid transfer protein, DIR1. Conversely, G3P is required for the translocation of DIR1 to distal tissues. Together, these observations suggest that the cooperative interaction of DIR1 and G3P mediates the induction of SAR in plants.

Glycerol-3-phosphate is a critical mobile inducer of systemic immunity in plants.

Glycerol-3-phosphate (G3P) is an important metabolite that contributes to the growth and disease-related physiologies of prokaryotes, plants, animals and humans alike. Here we show that G3P serves as the inducer of an important form of broad-spectrum immunity in plants, termed systemic acquired resistance (SAR). SAR is induced upon primary infection and protects distal tissues from secondary infections. Genetic mutants defective in G3P biosynthesis cannot induce SAR but can be rescued when G3P is supplied exogenously. Radioactive tracer experiments show that a G3P derivative is translocated to distal tissues, and this requires the lipid transfer protein, DIR1. Conversely, G3P is required for the translocation of DIR1 to distal tissues, which occurs through the symplast. These observations, along with the fact that dir1 plants accumulate reduced levels of G3P in their petiole exudates, suggest that the cooperative interaction of DIR1 and G3P orchestrates the induction of SAR in plants.

[Morphodensitometric evaluation of lymphocytes enzymatic status in pathogenesis of chronic alcoholism]. / Morfodensitometricheskaia otsenka fermentativnogo statusa limfotsitov pri issledovanii patogeneza khronicheskogo alkogolizma.

Activities of succinate dehydrogenase (SDH), alpha-glycerophosphate dehydrogenase (alpha-GPDH), and nonspecific esterase (NE) were studied in blood lymphocytes of healthy men and chronic alcoholics. SDH activity was notably decreased, while the levels of alpha-GPDH and NE were increased in alcoholics. The detected metabolic changes suggest lymphocyte dysfunction and hence, impairment of neuroimmune relationships, which may be the central component in the pathogenesis of chronic alcoholism.

Enzyme-linked immunosorbent assay of autoantibodies against mitochondrial glycerophosphate dehydrogenase in insulin-dependent and non-insulin-dependent diabetic subjects.

The mitochondrial enzyme FAD-linked glycerophosphate dehydrogenase (mGDH) plays a key role in the recognition of glucose as a stimulus for insulin release from the pancreatic islet B-cell. In the present study, an ELISA procedure was used for the measurement of mGDH antibodies in both insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetic patients. Positive readings, exceeding the upper limit of the normal range, were recorded in 7 out of 12 IDDM patients, as distinct (P < 0.01) from 2 out of 12 nondiabetic subjects of comparable age. The study conducted in 41 NIDDM patients and 15 control subjects of similar age indicated that the incidence of mGDH-positive cases was not significantly different in the diabetic (4/41) and control (1/15) groups, the measurement of optical density in the positive cases barely exceeding the upper limit of the normal range. These findings indicate that the mitochondrial enzyme mGDH often acts as an antigenic determinant in IDDM, but not in NIDDM, patients.

Autoantibodies against mitochondrial glycerophosphate dehydrogenase in patients with IDDM.

The mitochondrial enzyme FAD-linked glycerophosphate dehydrogenase (mGDH) plays a key role in the recognition of D-glucose as a stimulus for insulin release from the pancreatic islet B-cell. This study reveals that autoantibodies against this enzyme are not uncommonly found in patients with insulin-dependent diabetes mellitus (IDDM) examined at the onset of the disease. Antibodies reacting with a recombinant mGDH fragment product were observed in the serum of four out of 15 type-1 diabetics, but in none of 15 control subjects. The serum of patients positive for the recombinant mGDH fragment also recognized native mGDH in a rat testis extract, provided that the enzymatic protein was first exposed to an anti-mGDH rabbit serum. Antibodies against mGDH were also found in four out 12 patients with autoimmune thyroiditis. These findings reveal that a mitochondrial enzyme, that represents an essential component of the islet B-cell glucose-sensing device, may act as an antigenic determinant in patients with IDDM or other autoimmune diseases.

Evidence for glucocorticoid target cells in the rat optic nerve. Hormone binding and glycerolphosphate dehydrogenase induction.

Biochemical evidence suggests that neuroglia are responsive to glucocorticoids, yet previous studies of glucocorticoid localization have typically failed to demonstrate significant uptake by neuroglial cells. To further investigate this problem, we measured glycerol-3-phosphate dehydrogenase (GPDH) activity and glucocorticoid receptor binding capacity in normal rat optic nerves and in those undergoing Wallerian (axonal) degeneration. Binding studies were also performed on hippocampus and anterior pituitary for comparison purposes. Normal optic nerve preparations possessed a high level of GPDH activity that was glucocorticoid-inducible and that increased further following axonal degeneration. Antibody inactivation experiments demonstrated the presence of more enzyme molecules in the degenerating nerve preparations. correlative immunocytochemical studies found GPDH-positive reaction product only in morphologically identified oligodendrocytes, a result that is consistent with the previously reported localization of this enzyme in rat brain. Optic nerve cytosol fractions displayed substantial high-affinity binding of both dexamethasone (DEX) and corticosterone (CORT) that, like GPDH, was elevated approximately two fold in degenerating nerves. Finally, in vivo accumulation of [3H]DEX and [3H]CORT by optic nerve and other myelinated tracts was examined using nuclear isolation and autoradiographic methods. Although neither steroid was found to be heavily concentrated by these tissues in vivo, a small preference for DEX was observed in the nuclear uptake experiments. These results are discussed in terms of the hypothesis that glial cells are targets for glucocorticoid hormones.

Microcomplement fixation studies on the evolution of alpha-glycerophosphate dehydrogenase within the genus Drosophila.

Antisera were prepared against purified alpha-glycerophosphate dehydrogenase (EC 1.1.1.8) (alphaGPDH) from Drosophila melanogaster, D. virilis, and D. busckii. The immunological distances between the enzymes from the 3 species and those from 31 additional drosophilid species agree in general with the accepted phylogeny of the genus. These data permit an estimate that the subgenus Sophophora diverged 52 million years ago from the line leading to the subgenus Drosophila. The antiserum against melanogaster alphaGPDH was capable of distinguishing allelic variants of alphaGPDH. On the basis of presumed single amino acid substitutions, no drosophilid alphaGPDH tested differed from the melanogaster enzyme by more than eight or nine substitutions. The study was extended to include representatives of six other dipteran families. The immunological distances between alphaGPDH from Drosophila and alphaGPDH from these dipterans were reasonably consistent with a phylogeny of the order Diptera established by more conventional means. The unit evolutionary period of this enzyme was estimated to be 18 million years.