Biochemical and genetic characterization of IspC, an immunogenic surface protein with peptidoglycan hydrolase activity essential for virulence of Listeria monocytogenes

IspC is a putative peptidoglycan hydrolase that is likely surface localized, as predicted from its deduced amino acid sequence. The biochemical properties and biological function (s) of IspC and its role in pathogenesis remain to be elucidated.;To determine if this enzyme has any biological functions and/or plays a role in virulence, an ispC in-frame deletion mutant was created from the wild type L. monocytogenes. This Delta ispC mutant exhibited complete abrogation of expression of IspC and had no defect in the in vitro growth, colony and microscopic morphologies, or biochemical characteristics. Comparison of bacteriolytic profiles of the surface protein extracts of the wild type and the mutant strains indicated that IspC was a minor autolysin in vitro. Lack of IspC led to attenuating the virulence of L. monocytogenes in mice, evidenced by a significant reduction of bacterial counts in liver and brain and no mortality compared with the wild type. Further, the data from the assays using various eukaryotic cells for adhesion, invasion, actin tail formation, plaque formation, and intracellular growth indicated that IspC expression was required for cell-type dependent adhesion and invasion, early-stage actin tail formation and late-stage intracellular survival and growth. The findings that (i) the mutant was impaired for adhesion to certain eukaryotic cells, (ii) both purified IspC and its C-terminal CWBD were capable of binding sheep choroid plexus (SCP) epithelial cells and Vero cells, and (iii) both purified IspC and its C-terminal CWBD were capable of binding to GAG heparin (extensively used as a representative glycosaminoglycan) in vitro supported the role of IspC as an adhesin in virulence. The Delta ispC mutant exhibited a marked defect in adhesion and invasion for SCP cells but not human brain microvascular endothelial cells (HBMEC), suggesting that IspC is necessary for crossing the blood-cerebrospinal fluid barrier. Proteomic and immunological analysis showed a reduced surface expression of some known or putative virulence factors (e.g., ActA, InIC2, and a flagellin homologue FlaA) due to the IspC deficiency. Together, these results demonstrate that IspC, expressed as a minor autolysin in vitro, is not important for cell division or separation but essential for full virulence of L. monocytogenes in vivo.;In conclusion, IspC plays a dual role in L. monocytogenes virulence, i.e., promoting the bacterium-host cell interaction through the adhesive properties of its C-terminal CWBD (direct role as an adhesin) and regulating the surface display of other virulence factors (indirect role) presumably by the autolytic activity of its N-terminal catalytic domain. The study has shed new light on our understanding of molecular mechanisms by which a minor surface peptidoglycan hydrolase contributes to bacterial pathogenesis. (Abstract shortened by UMI.);The IspC protein was expressed in Escherichia coli and purified to electrophoretic homogeneity by chromatographic methods. This resulted in sufficient amount of purified recombinant IspC (rIspC) required for production of rabbit polyclonal antibodies to IspC (RalphaIspC) and for biochemical and structural studies. N-terminal sequencing and mass spectrometry analysis showed that IspC contained a 23-residue N-terminal peptide being processed in E. coli. Circular dichroism and Raman spectroscopy analysis of purified rIspC revealed that the dominant secondary structure for the protein is beta-sheet, consistent with the prediction by the PSIPRED method. Renaturing SDS-PAGE analysis demonstrated that IspC was capable of hydrolyzing bacterial cell wall substrates from several bacterial sources including L. monocytogenes itself, indicating IspC is an autolysin. 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 the 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. Analysis of various truncated forms of IspC has defined two separate functional domains: the N-terminal catalytic domain (aa 24 to 197) responsible for the hydrolytic activity and the C-terminal domain (aa 198 to 774) made up of seven GW (glycine-tryptophan dipeptide) modules responsible for anchoring the protein to the cell wall. The IspC autolysin exhibited peptidoglycan hydrolase activity over a broad pH range between 3 and 9 with an alkaline pH optimum of 7.5 to 9. Interestingly, the separated 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 (CWBD) may be of importance in modulating the activity of the N-terminal enzyme domain.