Identification of IspC, an 86-kDa protein target of humoral immune response to infection with Listeria monocytogenes serotype 4b, as a novel surface autolysin

Animal Diseases Research Institute, Nepean, Ontario K2H 8P9, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

^* To whom correspondence should be addressed. Email: linm{at}inspection.gc.ca.

	Abstract

We identify and biochemically characterize a novel surface localized autolysin from Listeria monocytogenes serotype 4b, an 86-kDa protein consisting of 774 amino acids known as the target (designated IspC) of humoral immune response to listerial infection from our previous studies. 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. 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 not only capable of hydrolyzing 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 it was an autolysin. The IspC autolysin exhibited peptidoglycan hydrolase activity over a broad pH range between 3 and 9 with a pH optimum of pH 7.5-9. 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 (aa 1-197) responsible for the hydrolytic activity and the C-terminal domain (aa 198-774) 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 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.