sodium-dodecyl-sulfate and Pneumococcal-Infections

Choline-binding modules (CBMs) have a ββ-solenoid structure composed of choline-binding repeats (CBR), which consist of a β-hairpin followed by a short linker. To find minimal peptides that are able to maintain the CBR native structure and to evaluate their remaining choline-binding ability, we have analysed the third β-hairpin of the CBM from the pneumococcal LytA autolysin. Circular dichroism and NMR data reveal that this peptide forms a highly stable native-like β-hairpin both in aqueous solution and in the presence of trifluoroethanol, but, strikingly, the peptide structure is a stable amphipathic α-helix in both zwitterionic (dodecylphosphocholine) and anionic (sodium dodecylsulfate) detergent micelles, as well as in small unilamellar vesicles. This β-hairpin to α-helix conversion is reversible. Given that the β-hairpin and α-helix differ greatly in the distribution of hydrophobic and hydrophilic side chains, we propose that the amphipathicity is a requirement for a peptide structure to interact and to be stable in micelles or lipid vesicles. To our knowledge, this "chameleonic" behaviour is the only described case of a micelle-induced structural transition between two ordered peptide structures.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Choline; Circular Dichroism; Humans; Micelles; Models, Molecular; N-Acetylmuramoyl-L-alanine Amidase; Nuclear Magnetic Resonance, Biomolecular; Peptides; Phosphorylcholine; Pneumococcal Infections; Protein Structure, Secondary; Sodium Dodecyl Sulfate; Streptococcus pneumoniae; Surface-Active Agents; Trifluoroethanol

The mechanisms that control Streptococcus pneumoniae's ability to colonize the nasopharynx or to invade the middle ear and cause acute otitis media are not understood. Focused study of these mechanisms requires efficient methods for the extraction of microbial RNA from minute clinical samples. Several lysis/extraction methods were tested and compared to determine the optimal conditions for isolating intact total RNA from pneumococcal cells. The sensitivity and efficiency of the extractions were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Compared to other methods, mechanical homogenization in TRIZOL was the most efficient for releasing microbial RNA, and addition of polyinosinic acid (Poly I) as an RNA carrier increased the assay sensitivity to 10(2) colony forming units when detected by RT-PCR amplification of 16S ribosomal RNA or messenger RNA for penicillin binding protein 2b. Quantitative results were confirmed using a ribonuclease protection assay. Penicillin binding protein 2b was also detected in rat middle ear mucosa recovered 5 weeks after middle ear challenge with S. pneumoniae. This study describes a useful core methodology for use in identifying pneumococcal virulence genes from small titer samples and has promising applications in clinical studies of pneumococcal nasopharyngeal colonization and otitis media pathogenesis.

Topics: Animals; Endopeptidase K; Endopeptidases; Guanidines; Humans; Otitis Media; Phenols; Pneumococcal Infections; Poly I; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sodium Dodecyl Sulfate; Streptococcus pneumoniae

There has been considerable recent research into protein based Streptococcus pneumoniae vaccines as alternatives to the existing capsular antigen vaccines. PiuA and PiaA (formerly Pit1A and Pit2A) are recently identified lipoprotein components of S. pneumoniae iron uptake ABC transporters which are required for full virulence and are likely to be expressed on the surface of the bacterial cell membrane. We investigated the efficacy of recombinant PiuA and PiaA proteins at eliciting protective immunity in mice against systemic infection with S. pneumoniae. Both recombinant PiuA and PiaA generated antibody responses that cross-reacted with each other but not with pneumolysin and reacted with identical proteins from nine different S. pneumoniae serotypes. Mice immunized with recombinant PiuA and PiaA were protected against systemic challenge to a degree similar to those immunized with an existing protein vaccine candidate, PdB (a genetically modified pneumolysin toxoid). Immunization with a combination of both PiuA and PiaA resulted in additive protection and was highly protective against systemic infection with S. pneumoniae. PiuA and PiaA are therefore promising additional candidates for a novel S. pneumoniae vaccine using protein antigens.

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; ATP-Binding Cassette Transporters; Bacterial Proteins; Electrophoresis, Polyacrylamide Gel; Histidine; Immunization, Passive; Iron; Lipoproteins; Mice; Mice, Inbred BALB C; Pneumococcal Infections; Pneumococcal Vaccines; Recombinant Fusion Proteins; Sodium Dodecyl Sulfate; Streptococcus pneumoniae; Vaccination; Vaccines, Synthetic