Conformational changes in the catalytic region are responsible for heat-induced activation of hyperthermophilic homoserine dehydrogenase.

When overexpressed as an immature enzyme in the mesophilic bacterium Escherichia coli, recombinant homoserine dehydrogenase from the hyperthermophilic archaeon Sulfurisphaera tokodaii (StHSD) was markedly activated by heat treatment. Both the apo- and holo-forms of the immature enzyme were successively crystallized, and the two structures were determined. Comparison among the structures of the immature enzyme and previously reported structures of mature enzymes revealed that a conformational change in a flexible part (residues 160-190) of the enzyme, which encloses substrates within the substrate-binding pocket, is smaller in the immature enzyme. The immature enzyme, but not the mature enzyme, formed a complex that included NADP+, despite its absence during crystallization. This indicates that the opening to the substrate-binding pocket in the immature enzyme is not sufficient for substrate-binding, efficient catalytic turnover or release of NADP+. Thus, specific conformational changes within the catalytic region appear to be responsible for heat-induced activation.

Clinical and Bacteriological Analysis of Pediatric Pneumococcal Meningitis after 13-Valent Pneumococcal Conjugate Vaccine Introduction in Japan.

Streptococcus pneumoniae is one of the leading causes of meningitis in children. In Japan, since the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13), the number of pneumococcal meningitis due to non-PCV13 serotypes in children has increased. To clarify the clinical outcomes, serotype distributions, and antimicrobial susceptibility of isolated S. pneumoniae strains from pediatric pneumococcal meningitis, we clinically and bacteriologically analyzed 34 cases of pediatric pneumococcal meningitis that were reported after the PCV13 introduction era in Japan. The median age at diagnosis was 1 year (range: 3 months-13 years). Ten (29.4%) patients had underlying diseases. Twenty-nine (85.3%) patients had received at least one dose of any pneumococcal vaccine. Of the 34 patients with pneumococcal meningitis, 6 had sequelae, and 4 died. Nine (26.5%) strains were resistant to penicillin; five (15%) strains to meropenem, with an MIC of 0.5 µg/mL. All strains were susceptible to vancomycin and linezolid. Daptomycin's MIC50 was 0.064 µg/mL and MIC90 was 0.094 µg/mL. Among the tested strains, only four were PCV13 serotypes. Penicillin-resistant S. pneumoniae was isolated from 30.0% of the patients with sequelae and death. Particularly, the proportion of serotype 10A in the sequelae and deceased cases was significantly higher than that in the complete recovery cases. We should carefully monitor the serotype and drug susceptibility of S. pneumoniae strains isolated from patients with meningitis after the PCV13 era and reconsider the treatment strategy to prepare against further drug-resistant pneumococcal strains. IMPORTANCE We analyzed 34 cases of pediatric pneumococcal meningitis that were reported after the 13-valent pneumococcal conjugate vaccine (PCV13) introduction era in Japan. Our study revealed that pneumococcal meningitis in children was mainly caused by non-PCV13 serotypes; all cases with sequelae and death were caused by non-PCV13 serotypes. Moreover, all serotypes of penicillin resistant Streptococcus pneumoniae strains (26.5%; 9/34) were non-PCV13 serotypes. We also analyzed antimicrobial susceptibilities of glycopeptides, linezolid (LZD), and daptomycin (DAP) of isolated S. pneumoniae strains. All tested strains were susceptible to vancomycin, teicoplanin, LZD, and DAP. Especially. DAP demonstrated the best outcome among the tested antibiotics, with MIC90 of 0.094 µg/mL. Pneumococcal meningitis in children continues to persist and is difficult to control with the current conjugate vaccines. Therefore, it is important to monitor the serotype and antimicrobial susceptibility of S. pneumoniae strains isolated from patients with meningitis and accordingly reconsider the treatment strategy.