Organic solutes in the deepest phylogenetic branches of the Bacteria: identification of α(1-6)glucosyl-α(1-2)glucosylglycerate in Persephonella marina.

The accumulation of organic solutes was investigated in the thermophilic bacteria Persephonella marina and Marinitoga piezophila, two representatives of the deepest lineages in the domain Bacteria. These organisms grow optimally at around 70 °C in medium containing 3 % NaCl. A new disaccharide, accumulating in Persephonella marina, was identified as α(1-6)glucosyl-α(1-2)glucosylglycerate (GGG), by nuclear magnetic resonance. This identification was validated by comparison with the spectra of the compound obtained by chemical synthesis. Besides GGG, the solute pool of Persephonella marina comprised ß-glutamate, di-myo-inositol-1,3'-phosphate and 2-O-α-glucosylglycerate. In contrast, amino acids such as α-glutamate, proline and alanine were the dominant components of the solute pool of Marinitoga piezophila and sugar derivatives were absent. The ability of GGG to protect protein structure against heat denaturation was assessed using model proteins. A genomic search for the biosynthetic pathways of known ionic solutes in Aquificales and Thermotogales shows the inability of this analysis to predict the nature of compatible solutes and underlines the need for efficient cultivation techniques.

Bioassay studies support the potential for iatrogenic transmission of variant Creutzfeldt Jakob Disease through dental procedures.

BACKGROUND: Evidence is required to quantify the potential risks of transmission of variant Creutzfeldt Jakob (vCJD) through dental procedures. Studies, using animal models relevant to vCJD, were performed to address two questions. Firstly, whether oral tissues could become infectious following dietary exposure to BSE? Secondly, would a vCJD-contaminated dental instrument be able to transmit disease to another patient? METHODS: BSE-301V was used as a clinically relevant model for vCJD. VM-mice were challenged by injection of infected brain homogenate into the small intestine (Q1) or by five minute contact between a deliberately-contaminated dental file and the gingival margin (Q2). Ten tissues were collected from groups of challenged mice at three or four weekly intervals, respectively. Each tissue was pooled, homogenised and bioassayed in indicator mice. FINDINGS: Challenge via the small intestine gave a transmission rate of 100% (mean incubation 157±17 days). Infectivity was found in both dental pulp and the gingival margin within 3 weeks of challenge and was observed in all tissues tested within the oral cavity before the appearance of clinical symptoms. Following exposure to deliberately contaminated dental files, 97% of mice developed clinical disease (mean incubation 234±33 days). INTERPRETATION: Infectivity was higher than expected, in a wider range of oral tissues, than was allowed for in previous risk assessments. Disease was transmitted following transient exposure of the gingiva to a contaminated dental file. These observations provide evidence that dental procedures could be a route of cross-infection for vCJD and support the enforcement of single-use for certain dental instruments.

A role for His155 in binding of human prion peptide144-167 to immobilised prion protein.

The interactions and conformational changes that lead to the conversion of the normal prion protein (PrP(c)) to its pathogenic form, PrP(sc), are still being elucidated. Using Surface Plasma Resonance (SPR), we provide evidence that a synthetic peptide (PrP(144-167)) corresponding to residues comprising the alpha helix 1-beta strand 2 domain of PrP(c) is able to interact and bind to immobilised recombinant human PrP (rHuPrP) in a dose-dependent manner. The interaction is pH dependent with an increase in binding observed as the pH is lowered, particularly between pH 6.5 and pH 5.5 suggesting a specific role for His(155) in the interaction, confirmed by covalent modification of this residue in the peptide with diethylpyrocarbonate (DEPC). Circular dichroism analysis of PrP(144-167) revealed no secondary structure motifs across the pH range investigated. Possible pH related structural changes of immobilised rHuPrP are also discussed with regard to the increased affinity for PrP(144-167).

Occurrence of 1-glyceryl-1-myo-inosityl phosphate in hyperthermophiles.

The accumulation of compatible solutes was studied in the hyperthermophilic bacterium Aquifex pyrophilus as a function of the temperature and the NaCl concentration of the growth medium. Nuclear magnetic resonance analysis of cell extracts revealed the presence of alpha- and beta-glutamate, di-mannosyl-di-myo-inositol phosphate, di-myo-inositol phosphate, and an additional compound here identified as 1-glyceryl-1-myo-inosityl phosphate. All solutes accumulated by A. pyrophilus are negatively charged at physiological pH. The intracellular levels of di-myo-inositol phosphate increased in response to supraoptimal growth temperature, while alpha- and beta-glutamate accumulated in response to osmotic stress, especially at growth temperatures below the optimum. The newly discovered compound, 1-glyceryl-1-myo-inosityl phosphate, appears to play a double role in osmo- and thermoprotection, since its intracellular pool increased primarily in response to a combination of osmotic and heat stresses. This work also uncovered the nature of the unknown compound, previously detected in Archaeoglobus fulgidus (L. O. Martins et al., Appl. Environ. Microbiol. 63:896-902, 1997). The curious structural relationship between diglycerol phosphate (found only in Archaeoglobus species), di-myo-inositol phosphate (a canonical solute of hyperthermophiles), and the newly identified solute is highlighted. This is the first report on the occurrence of 1-glyceryl-1-myo-inosityl phosphate in living systems.

Methods to minimize the risks of Creutzfeldt-Jakob disease transmission by surgical procedures: where to set the standard?

New prion-related disorders have emerged over the past 20 years, of which the most notable in the human context is variant Creutzfeldt-Jakob disease (CJD). This disorder is a challenge to medical and public health professionals seeking early detection and diagnosis, provision of therapy, and support for persons affected and a better understanding of transmission risks. The risk of iatrogenic transmission of the disease remains a significant threat, given the well documented cases of CJD transmission via surgery, organ transplantation, and blood transfusion. This review discusses our current understanding of the prevalence of variant CJD, the distribution of tissue infectivity, and new methods for the decontamination of surgical instruments. A comparison of emerging technologies is provided on the basis of our current perception of surgical risk to identify methods that are likely to provide sufficient safety margins and to stimulate debate about the standards needed to protect against variant CJD and CJD transmission.

Proteolytic inactivation of the bovine spongiform encephalopathy agent.

Thermostable proteases have been investigated for their ability to provide a novel biological solution to decontamination of prion agents responsible for transmissible spongiform encephalopathies (TSEs). Proteases were identified that digested total mouse brain homogenate (MBH) protein from uninfected mice. These proteases were then evaluated for digestion of BSE (301V) infectious MBH over a range of pH and temperatures, screened for loss of anti-prion antibody 6H4 immunoreactivity and protease-treated infectious MBH assessed in mouse bioassay using VM mice. Despite a number of proteases eliminating all 6H4-immunoreactive material, only the subtilisin-enzyme Properase showed a significant extension in incubation period in mouse bioassays following a 30-min incubation at 60 degrees C and pH 12. These results demonstrate the potential of the method to provide a practical solution to the problems of TSE contamination of surgical instruments and highlight the inadequacy of using Western blot for assessment of decontamination/inactivation of TSE agents.

Comparative study of the thermostabilizing properties of mannosylglycerate and other compatible solutes on model enzymes.

The protection of mannosylglycerate, at 0.5 M concentration, against heat inactivation of the model enzyme lactate dehydrogenase (LDH) was compared to that exerted by other compatible solutes, namely, trehalose, ectoine, hydroxyectoine, di- myo-inositol phosphate, diglycerol phosphate, and mannosylglyceramide. Mannosylglycerate and hydroxyectoine were the best stabilizers of the enzyme and showed comparable protective effects. Diglycerol phosphate, trehalose, and mannosylglyceramide protected the enzyme to a lower extent. Ectoine conferred no protection, and di- myo-inositol phosphate had a strong destabilizing effect. The superior ability of mannosylglycerate to prevent LDH inactivation was accompanied by a higher efficiency in preventing LDH aggregation induced by heat stress. Moreover, mannosylglycerate induced an increase of 4.5 degrees C in the melting temperature of LDH, whereas the same molar concentration of trehalose caused an increase of only 2.2 degrees C. The effectiveness of mannosylglycerate in protecting LDH was also compared to that of other chemically related compounds: mannose, methyl-mannoside, potassium glycerate, glucosylglycerol, glycerol, and glucose. Mannosylglycerate conferred the highest protection, but glucosylglycerol and potassium glycerate were very efficient; glucose exerted a low degree of protection, glycerol and methyl-mannoside had no significant effect, and mannose caused destabilization. Mannosylglycerate was also a good thermoprotectant of glucose oxidase from Aspergillus niger, an enzyme with a net charge opposite to that of LDH under the working conditions. Given the superior performance of mannosylglycerate as a thermoprotectant of enzyme activity in vitro, it is conceivable that it also fulfills a protein thermoprotective function in vivo.