New Insights into Beta-Lactam Resistance of Streptococcus pneumoniae: Serine Protease HtrA Degrades Altered Penicillin-Binding Protein 2x.

Reduced amounts of the essential penicillin-binding protein 2x (PBP2x) were detected in two cefotaxime-resistant Streptococcus pneumoniae laboratory mutants C405 and C606. These mutants contain two or four mutations in the penicillin-binding domain of PBP2x, respectively. The transcription of the pbp2x gene was not affected in both mutants; thus, the reduced PBP2x amounts were likely due to post-transcriptional regulation. The mutants carry a mutation in the histidine protein kinase gene ciaH, resulting in enhanced gene expression mediated by the cognate response regulator CiaR. Deletion of htrA, encoding a serine protease regulated by CiaR, or inactivation of HtrA proteolytic activity showed that HtrA is indeed responsible for PBP2x degradation in both mutants, and that this affects β-lactam resistance. Depletion of the PBP2xC405 in different genetic backgrounds confirmed that HtrA degrades PBP2xC405. A GFP-PBP2xC405 fusion protein still localized at the septum in the absence of HtrA. The complementation studies in HtrA deletion strains showed that HtrA can be overexpressed in pneumococcal cells to specific levels, depending on the genetic background. Quantitative Western blotting revealed that the PBP2x amount in C405 strain was less than 20% compared to parental strain, suggesting that PBP2x is an abundant protein in S. pneumoniae R6 strain.