S. pneumoniae is an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth, S. pneumoniae produces high levels of H2O2. Since S. pneumoniae lacks catalase, the question of how it controls H2O2 levels is of critical importance. The psa locus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase, tpxD. This study shows that tpxD encodes a functional thiol peroxidase, involved in the bacterial responses to oxidative stress.
Kinetic experiments showed that recombinant TpxD removed H2O2 efficiently. However, in vivo experiments revealed that TpxD detoxifies only a fraction of H2O2 generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD-cysteine58 undergoes selective oxidation in vivo, under conditions where H2O2 is formed, confirming its thiol peroxidase activity. TpxD expression and synthesis were significantly increased in cells grown under aerobic vs. anaerobic conditions. Challenging wild type (WT) strains with H2O2 resulted in tpxD up-regulation, while psaBCA expression was oppositely affected. However, challenging DtpxD-mutants with H2O2 did not affect psaBCA, implying that TpxD is involved in the regulation of the psa operon, in addition to its scavenging activity. Furthermore, microarray analysis revealed that challenging bacteria with H2O2 resulted in up-regulation of 43 genes and down-regulation of 105 genes in the WT strain, but had almost no effect on the DtpxD-mutant, indicating for the first time in prokaryotic cells, that TpxD serves as a sensor of H2O2, mediating bacterial responses at the transcriptional level.
The findings of this study suggest that TpxD constitutes a component of the microbe's fundamental strategy to fine-tune cellular processes in response to H2O2.
