Evidence for the N-Acetylglucosaminidase Activity of a Cell Wall-associated Autolysin ISPC and its Suitability as a Diagnostic Marker for 'Listeria Monocytogenes' Serotype 4B

• Main Author: Ronholm, Jennifer
• Other Authors: Lin, Min
• Language: en
• Published: Université d'Ottawa / University of Ottawa 2013
• Subjects: listeria; peptidoglycan hydrolase; food safety; antibody
• Online Access: http://hdl.handle.net/10393/23629; http://dx.doi.org/10.20381/ruor-6297

Listeria monocytogenes is the etiological agent of a life-threatening, opportunistic infection caused by the ingestion of contaminated foods. Although L. monocytogenes is divided into 13 serotypes, 98% of human illness is caused by serotype 1/2a, 1/2b and 4b strains, with serotype 4b accounting for almost all the major outbreaks of human listeriosis. The principle objective of this work was to develop surface-binding monoclonal antibodies (MAbs) highly specific for serotype 4b, as well as characterize their antigen targets to aid in the detection and isolation of serotype 4b strains using an antibody based procedure. To create such antibodies, mice were immunized with formalin killed whole cells of L. monocytogenes serotype 4b strain LI0521. A total of 15 MAbs reactive to serotype 4b isolates were shown to recognize a ~77 kDa surface antigen subsequently identified by mass spectrometry as surface associated autolysin, IspC. Epitope mapping experiments further revealed that each of the 15 MAbs bound to the cell wall binding GW domain of IspC and can be essentially divided into 4 major groups based on epitope localization. ELISA analysis of the reactivity of each of the MAbs with various L. monocytogenes serotypes indicated that several MAbs were 100% specific for serotype 4b isolates. Surface plasmon resonance experiments showed that the affinity constants for each of these MAbs fell within the range of 1.0 x 10-7 to 6.4 x 10-9 M. To determine whether IspC, shown to be well conserved among various serotype 4b strains, is a useful diagnostic marker with antibody-based methods, the expression of IspC was assessed in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of ispC gene was identified immediately upstream of the ispC open reading frame by constructing the promoterless lacZ gene fusion with the putative ispC promoter region and by 5'RACE analysis. Data obtained with the lacZ reporter gene system and immunofluorescent microscopy revealed that IspC is expressed on the cell surface under all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source and enrichment media) that allow for cellular division, although the level of ispC gene expression varies. In addition, a significant effort were put into elucidating the hydrolytic bond specificity of IspC by HPLC and mass spectrometry analysis of muropeptides released from IspC-mediated hydrolysis of L. monocytogenes peptidoglycan (PG). The results demonstrated that IspC functions as an N-acetylglucosaminidase capable of cleaving the β-1,4-glycosidic bond of the PG glycan strand. Furthermore, IspC was more efficient at hydrolysing fully Nacetylated PG from a PG deacetylase gene (pgdA) deletion mutant of L. monocytogenes than partially de-N-acetylated wild-type PG, indicating that modification of PG by de-Nacetylation of GlcNAc residues renders PG resistant to IspC hydrolysis. In conclusion, the surface autolysin IspC with the N-acetylglucosaminidase activity is a novel diagnostic marker for the 4b serotype strains, which can be explored , in conjunction with specific MAbs developed here, for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental and clinical samples with the need for minimal or no culture enrichment.