We identified and biochemically characterized a novel surface-localized
autolysin from Listeria monocytogenes serotype 4b, an 86-kDa
protein consisting of 774
amino acids and known from our previous studies as the target (designated IspC) of the humoral immune response to listerial
infection. Recombinant IspC, expressed in Escherichia coli, was purified and used to raise specific rabbit polyclonal
antibodies for
protein characterization. The native IspC was detected in all growth phases at a relatively stable low level during a 22-h in vitro culture, although its gene was transiently transcribed only in the early exponential growth phase. This and our previous findings suggest that IspC is upregulated in vivo during
infection. The
protein was unevenly distributed in clusters on the cell surface, as shown by immunofluorescence and immunogold electron microscopy. The recombinant IspC was capable of hydrolyzing not only the cell walls of the gram-positive bacterium Micrococcus lysodeikticus and the gram-negative bacterium E. coli but also that of the IspC-producing strain of L. monocytogenes serotype 4b, indicating that it was an
autolysin. The IspC
autolysin exhibited
peptidoglycan hydrolase activity over a broad pH range of between 3 and 9, with a pH optimum of 7.5 to 9. Analysis of various truncated forms of IspC for cell wall-hydrolyzing or -binding activity has defined two separate functional domains: the N-terminal catalytic domain (
amino acids [aa] 1 to 197) responsible for the hydrolytic activity and the C-terminal domain (aa 198 to 774) made up of seven GW modules responsible for anchoring the
protein to the cell wall. In contrast to the full-length IspC, the N-terminal catalytic domain showed hydrolytic activity at acidic pHs, with a pH optimum of between 4 and 6 and negligible activity at alkaline pHs. This suggests that the cell wall binding domain may be of importance in modulating the activity of the N-terminal
hydrolase domain. Elucidation of the biochemical properties of IspC may have provided new insights into its
biological function(s) and its role in pathogenesis.