Final Report Summary - NMEN-PBP (Functional organisation of penicillin binding proteins of Neisseria meningitidis)
Neisseria meningitides (Nm) is a leading cause of bacterial meningitis and septicaemia worldwide with a high case-fatality rate. Although penicillin remains the drug of choice in the treatment of invasive meningococcal disease (IMD), an increasing number of meningococcal with reduced susceptibility to penicillin have been emerging worldwide. Reduced susceptibility or resistance to penicillin in meningococcal is connected to alterations in penicillin binding protein 2, PBP2. PBPs are involved in the late stages of peptidoglycan (PG) biosynthesis and therefore PBPs alterations might result in PG modifications and hence affect PG-dependent signalling. Until recently it was thought that N. meningitides strains own only three PBPs, PBP1, PBP2 and PBP3. However, genomic analysis of the complete sequence of meningococcal strains revealed the presence of two other putative genes encoding PBP4 and PBP5 which can possess D, D-carboxypeptidase and endopeptidase activity. And indeed the results obtained during the previous Marie Curie IEF from the PG structure study as well as from animal model provide the first indication that putative PBP4 and PBP5 are functional in N. meningitides, play a role in the PG synthesis and influence the virulence of meningococcal isolates. It has been proposed for some bacteria that different enzymes involved in PG biosynthesis might act in concert in vivo.
The aim of the project was to analyze the interactions between penicillin binding proteins and to get deeper knowledge concerning functional organisation of PBPs of N. meningitides. We wanted to know if some PBPs may act in PG recycling as a complex. Despite the fact that PBP2 is a class B high molecular weight PBP, our results suggested that the meningococcal PBP2 functioned in vivo as a D, D-carboxypeptidase. However, several attempts to detect in vitro a D, D-carboxypeptidase activity using recombinant PBP2 failed so far. Alternatively, the decreased D, Dcarboxypeptidase activity associated to altered PBP2 function could be an indirect effect. In Escherichia coli, the meningococcal PBP2 homologue, PBP3, was shown to interact with the low molecular weight PBP7/8 which are true D, D-carboxypeptidases. Meningococcal have three low molecular weight PBPs, PBP3 identifiable in fluorographic PBP assays, and PBP4 and 5 revealed by sequence homology analysis. Hence, we tested whether the differences in PG composition between penicillin susceptible wild type strain and its isogenic mutant with decreased susceptibility to penicillin were indirectly related to PBP3, 4, and 5. We constructed derivatives of wild type strain and mutant with decreased susceptibility to penicillin, in which the three genes pbp3, pbp4, and pbp5 were inactivated and we analyzed the PG composition. Both derivatives produced identical PG composition characterised by a very high percentage of pentapeptide-carrying muropeptides. These results suggested that the PBP2 related effects observed on the PG metabolism were probably indirect and mediated by the low molecular weight PBPs. Thus, we tested whether recombinant PBP2 and the major D, D-carboxypeptidase, PBP3, formed a stable protein complex. Indeed, PBP2 and PBP3 associated strongly.
We also constructed pbp3, 4 and 5 deletion mutants of N. meningitides lacking amino acids 266-396 or 402-466, 219-274 and 265-381 or 143-263, respectively. The deletions impaired D, D-carboxypeptidase domains of PBPs. These mutants will be used in further studies to resolve functional organisation of PBPs of N. meningitides.
Electron microscopy studies were performed to reveal if changes in PG structure influence meningococcal cell morphology. Such experiments performed with wild type strain and triple knock-out mutant of pbp 3, pbp 4 and pbp 5 did not show any differences in cell morphology and therefore, consequently, we decided not to continue such studies for isogenic meningococcal mutants with different in frame-deleted alleles of pbps.
The project used a multidisciplinary approach applying bacteriological, molecular and biochemical methods as well as electron microscopy and gave a possibility to determine interactions between different PBPs of meningococci. It allowed to construct mutants necessary for further testing of a role of different parts/domains of PBPs through the assessment of their influence on structural modification of N. meningitidis PG. Thank to that project the host institution had an opportunity to strengthen cooperation with Neisseria Unit at Institute Pasteur and also to establish cooperation with the Nicolaus Copernicus University in Torun. We plan to continue research with both Partners. During the term, researcher of the Project, Anna Skoczynska did her habilitation and started to be head of the department of Epidemiology and Clinical Microbiology.
The aim of the project was to analyze the interactions between penicillin binding proteins and to get deeper knowledge concerning functional organisation of PBPs of N. meningitides. We wanted to know if some PBPs may act in PG recycling as a complex. Despite the fact that PBP2 is a class B high molecular weight PBP, our results suggested that the meningococcal PBP2 functioned in vivo as a D, D-carboxypeptidase. However, several attempts to detect in vitro a D, D-carboxypeptidase activity using recombinant PBP2 failed so far. Alternatively, the decreased D, Dcarboxypeptidase activity associated to altered PBP2 function could be an indirect effect. In Escherichia coli, the meningococcal PBP2 homologue, PBP3, was shown to interact with the low molecular weight PBP7/8 which are true D, D-carboxypeptidases. Meningococcal have three low molecular weight PBPs, PBP3 identifiable in fluorographic PBP assays, and PBP4 and 5 revealed by sequence homology analysis. Hence, we tested whether the differences in PG composition between penicillin susceptible wild type strain and its isogenic mutant with decreased susceptibility to penicillin were indirectly related to PBP3, 4, and 5. We constructed derivatives of wild type strain and mutant with decreased susceptibility to penicillin, in which the three genes pbp3, pbp4, and pbp5 were inactivated and we analyzed the PG composition. Both derivatives produced identical PG composition characterised by a very high percentage of pentapeptide-carrying muropeptides. These results suggested that the PBP2 related effects observed on the PG metabolism were probably indirect and mediated by the low molecular weight PBPs. Thus, we tested whether recombinant PBP2 and the major D, D-carboxypeptidase, PBP3, formed a stable protein complex. Indeed, PBP2 and PBP3 associated strongly.
We also constructed pbp3, 4 and 5 deletion mutants of N. meningitides lacking amino acids 266-396 or 402-466, 219-274 and 265-381 or 143-263, respectively. The deletions impaired D, D-carboxypeptidase domains of PBPs. These mutants will be used in further studies to resolve functional organisation of PBPs of N. meningitides.
Electron microscopy studies were performed to reveal if changes in PG structure influence meningococcal cell morphology. Such experiments performed with wild type strain and triple knock-out mutant of pbp 3, pbp 4 and pbp 5 did not show any differences in cell morphology and therefore, consequently, we decided not to continue such studies for isogenic meningococcal mutants with different in frame-deleted alleles of pbps.
The project used a multidisciplinary approach applying bacteriological, molecular and biochemical methods as well as electron microscopy and gave a possibility to determine interactions between different PBPs of meningococci. It allowed to construct mutants necessary for further testing of a role of different parts/domains of PBPs through the assessment of their influence on structural modification of N. meningitidis PG. Thank to that project the host institution had an opportunity to strengthen cooperation with Neisseria Unit at Institute Pasteur and also to establish cooperation with the Nicolaus Copernicus University in Torun. We plan to continue research with both Partners. During the term, researcher of the Project, Anna Skoczynska did her habilitation and started to be head of the department of Epidemiology and Clinical Microbiology.