The molecular characterization of the first autolytic lysozyme of Streptococcus pneumoniae reveals evolutionary mobile domains

Abstract

SummaryA biochemical approach to identify proteins with high af®nity for choline-containing pneumococcal cell walls has allowed the localization, cloning and sequencing of a gene (lytC ) coding for a protein that degrades the cell walls of Streptococcus pneumoniae. The lytC gene is 1506 bp long and encodes a protein (LytC) of 501 amino acid residues with a predicted M r of 58 682. LytC has a cleavable signal peptide, as demonstrated when the mature protein (about 55 kDa) was puri®ed from S. pneumoniae. Biochemical analyses of the pure, mature protein proved that LytC is a lysozyme. Combined cell fractionation and Western blot analysis showed that the unprocessed, primary product of the lytC gene is located in the pneumococcal cytoplasm whereas the processed, active form of LytC is tightly bound to the cell envelope. In vivo experiments demonstrated that this lysozyme behaves as a pneumococcal autolytic enzyme at 308C. The DNA region encoding the 253 C-terminal amino acid residues of LytC has been cloned and expressed in Escherichia coli. The truncated protein exhibits a low, but signi®cant, choline-independent lysozyme activity, which suggests that this polypeptide adopts an active conformation. Self-alignment of the N-terminal part of the deduced amino acid sequence of LytC revealed the presence of 11 repeated motifs. These results strongly suggest that the lysozyme reported here has changed the general building plan characteristic of the choline-binding proteins of S. pneumoniae and its bacteriophages, i.e. the choline-binding domain and the catalytic domain are located, respectively, at the Nterminal and the C-terminal moieties of LytC. This work illustrates the natural versatility exhibited by the pneumococcal genes coding for choline-binding proteins to fuse separated catalytic and substrate-binding domains and create new and functional mature proteins.

Cpl-7, a Lysozyme Encoded by a Pneumococcal Bacteriophage with a Novel Cell Wall-binding Motif*

“…N-terminal sequence determination showed that Met 1 was removed under the overexpression and purification conditions used. The specific activity of the purified protein on [ 3 H]cholinelabeled pneumococcal cell walls was 1.1 ϫ 10 5 units mg Ϫ1 under optimal conditions (25 mM sodium acetate, pH 5.5, 37°C), which was slightly lower than the value reported for the Cpl-1 lysozyme at equivalent conditions (6).…”

“…However, the cell wall binding region (CWBR) of Cpl-1, made of six tandem copies of ϳ20 amino acid residues plus a terminal tail, belongs to the family of pneumococcal choline-binding modules (5). Its lytic activity is thus conditioned by the presence of choline moieties in pneumococcal teichoic and lipoteichoic acids and becomes inhibited at high choline concentrations (IC 50 ϭ 2 mM) (1,6). In contrast, Cpl-7 degrades pneumococcal cell walls containing either choline or ethanolamine and retains full activity even in the presence of high concentrations of these amino alcohols (7).…”

The pgdA Gene Encodes for a PeptidoglycanN-Acetylglucosamine Deacetylase in Streptococcus pneumoniae

“…The LytB endo-␤-Nacetylglucosaminidase is involved in daughter cell separation (37), whereas the LytC 1,4-␤-N-acetylmuramidase increases the rate of autolysis at 30°C (38). The deacetylation of the glycan strand GlcNAc residues could be a way of controlling the activities of these potentially autolytic enzymes.…”

Ofloxacin-like Antibiotics Inhibit Pneumococcal Cell Wall-degrading Virulence Factors

“…9). Of these, the major autolysin (LytA) is an amidase (9), whereas the two minor peptidoglycan hydrolases (LytB and LytC) function as a glucosaminidase (10) and a lysozyme (11), respectively. LytA and LytC are responsible of the autolytic release of toxins like pneumolysin, which by irritating the host tissues allows pneumococcus to gain access to the bloodstream.…”