GNA33 from Neisseria meningitidis serogroup B encodes a membrane‐bound lytic transglycosylase (MltA)

Abstract

In a previous study, we used the genome of serogroup B Meningococcus to identify novel vaccine candidates. One of these molecules, GNA33, is well conserved among Meningococcus B strains, other Meningococcus serogroups and Gonococcus and induces bactericidal antibodies as a result of being a mimetic antigen of the PorA epitope P1.2. GNA33 encodes a 48‐kDa lipoprotein that is 34.5% identical with membrane‐bound lytic transglycosylase A (MltA) from Escherichia coli. In this study, we expressed GNA33, i.e. Meningococcus MltA, as a lipoprotein in E. coli. The lipoprotein nature of recombinant MltA was demonstrated by incorporation of [3H]palmitate. MltA lipoprotein was purified to homogeneity from E. coli membranes by cation‐exchange chromatography. Muramidase activity was confirmed when MltA was shown to degrade insoluble murein sacculi and unsubstituted glycan strands. HPLC analysis demonstrated the formation of 1,6‐anhydrodisaccharide tripeptide and tetrapeptide reaction products, confirming that the protein is a lytic transglycosylase. Optimal muramidase activity was observed at pH 5.5 and 37 °C and enhanced by Mg2+, Mn2+ and Ca2+. The addition of Ni2+ and EDTA had no significant effect on activity, whereas Zn2+ inhibited activity. Triton X‐100 stimulated activity 5.1‐fold. Affinity chromatography indicated that MltA interacts with penicillin‐binding protein 2 from Meningococcus B, and, like MltA from E. coli, may form part of a multienzyme complex.

The Genome Revolution in Vaccine Research

“…Biochemical characterization of the recombinant protein, expressed and purified from E. coli, showed that GNA33 is able to degrade both insoluble murein sacculi and unsubstituted glycan strands. The results confirmed that GNA33 is a lytic transglycosylase with muramidase activity (Jennings et al, 2002). The recombinant protein elicits bactericidal antibody by mimicking a surface-exposed epitope of porin A (PorA) and confers passive protection against bacteremia in an infant rat model.…”

The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression

“…In a different approach, also aimed at increasing expression of GNA33 in a secreted form, the sequence encoding the mature portion of GNA33 was fused in-frame with the signal peptide sequence from another N. meningitidis lipoprotein, NMB1946 (Jennings et al, 2002). Substitution of the GNA33 signal peptide sequence with that of NMB1946 was found to give a level of GNA33 comparable to that obtained for the 'native' clone expressed in the presence of pLysS.…”

“…GNA33 encodes a protein of 441 aa with an N-terminal 20 aa signal sequence and shares 34?5 % sequence identity with the E. coli membrane-bound lytic transglycosylase A (MltA) (Jennings et al, 2002). Recently, functional homology of GNA33 to E. coli MltA has been confirmed by in vitro characterization of its muramidase and lytic transglycosylase activity (Jennings et al, 2002).…”

Correlation between Alterations of the Penicillin-binding Protein 2 and Modifications of the Peptidoglycan Structure in Neisseria meningitidis with Reduced Susceptibility to Penicillin G

“…Meningococcal PBP2 has been shown to interact with the lytic transglycosylase MltA (55). Furthermore, the E. coli homolog of meningococcal PBP2, PBP3, can form a complex with the lytic transglycosylase Slt70 and the low molecular weight PBP7/8 (56).…”