B. J. Pearce

Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae

Pneumococcus has been shown to bind to epithelial cells of the nasopharynx and lung, and to endothelial cells of the peripheral vasculature. To characterize bacterial elements required for attachment to these cell types, a library of genetically altered pneumococci with defects in exported proteins was screened for the loss of attachment to glycoconjugates representative of the nasopharyngeal cell receptor, type II lung cells (LC) and human endothelial cells (EC). A mutant was identified which showed a greater than 70% loss in the ability to attach to all cell types. This mutant also showed decreased adherence to the glycoconjugates containing the terminal sugar residues GalNAcβ1‐3Gal, GalNAcβ1‐4Gal and the carbohydrate GlcNAc, which are proposed components of the pneumococcal receptors specific to the surfaces of LC and EC. Analysis of the locus altered in this mutant revealed a gene, spxB, that encodes a member of the family of bacterial pyruvate oxidases which decarboxylates pyruvate to acetyl phosphate plus H2O2 and CO2. This mutant produced decreased concentrations of H2O2 and failed to grow aerobically in a chemically defined medium, unless supplemented with acetate which presumably restores acetyl phosphate levels by the action of acetate kinase, further suggesting that spxB encodes a pyruvate oxidase. The addition of acetate to the growth medium restored the adherence properties of the mutant indicating a link between the enzyme and the expression of bacterial adhesins. A defect in spxB corresponded to impaired virulence of the mutant in vivo. Compared to the parent strain, an spxB mutant showed reduced virulence in animal models for nasopharyngeal colonization, pneumonia, and sepsis. We propose that a mutation in spxB leads to down‐regulation of the multiple adhesive properties of pneumococcus which, in turn, may correlate to diminished virulence in vivo

Genetic identification of exported proteins in Streptococcus pneumoniae

A strategy was developed to mutate and genetically identify exported proteins in Streptococcus pneumoniae. Vectors were created and used to screen pneumococcal DNA in Escherichia coli and S. pneumoniae for translational gene fusions to alkaline phosphatase (PhoA), Twenty five PhoA+ pneumococcal mutants were isolated and the loci from eight of these mutants showed similarity to known exported or membrane‐associated proteins. Homologues were found to: (i) protein‐dependent peptide permeases, (ii) penicillin‐binding proteins, (iii) Cip proteases, (iv) two‐component sensor regulators, (v) the phospho‐enolpyruvate:carbohydrate phosphotransferase permeases, (vi) membrane‐associated dehydrogenases, (vii) P‐type (E1E2‐type) cation transport ATPases, (viii) ABC transporters responsible for the translocation of the RTX class of bacterial toxins. Unexpectedly one PhoA+ mutant contained a fusion to a member of the DEAD protein family of ATP‐dependent RNA helicases suggesting export of these proteins.

Peptide permeases modulate transformation in Streptococcus pneumoniae

To Identify elements participating In the process of transformation, a bank of genetically altered mutants of Streptococcus pneumoniae with defects in exported proteins was assessed for a decrease in transformation efficiency. One mutant consistentiy transformed 10‐foid less than the parent strain. Sequence analysis and reconstitution of the altered locus revealed a gene, plpA (permease‐like protein), which encodes a putative substrate‐binding protein belonging to the family of bacterial permeases responsible for peptide transport. The derived amino acid sequence for this gene was 80% similar to AmiA, a peptide‐binding protein homologue from pneumococcus, and 50% similar over 230 amino acids to SpooKA which is a regulatory element in the process of transformation and sporulation in Bacillus subtilis. PIpA fusions to alkaline phosphatase (PhoA) were shown to be membrane associated and labelled with [3H]‐palmitic acid, which probably serves as a membrane anchor. Experiments designed to define the roles of the pIpA and ami determinants in the process of transformation showed that: (i) mutants with defects in plpA were >90% transformation deficient while ami mutants exhibited up to a fourfold increase in transformation efficiency; (ii) compared to the parental strain, the onset of competence in an ami mutant occurred earlier in logarithmic growth, whereas the onset was delayed in a plpA mutant; and (ill) the plpA mutation decreases the expression of a competence‐regulated locus. Since the permease mutants would fail to bind specific ligands, it seems likely that the substrate‐permease interaction modulates the process of transformation.