Domain Function Dissection And Substrate Binding Properties Of Listeria Monocytogenes P60 Protein

Listeria monocytogenes (Lm) is a typically zoonotic foodborne pathogen and widely distributed in the environments. It has become a major concern for the public due to its strong pathogenicity. p60 protein is an extracellular protein produced by Lm and has been shown to be required for Lm virulence. Contribution of p60 protein in L. monocytogenes pathogenesis is tentatively linked to cell division, host cell invasion, maintenance of bacterial cell surface architecture and release of immunologically active cell wall components. Therefore, p60 protein has also been named invasion-associated protein (lap).Based on the prediction of p60 protein conformation, p60 protein is predicted to contain two independent structural domains, N-terminal domain and C-terminal domain, which can be split in the amino acid residue 270. Amino acid sequence analysis demonstrated that there are two LysM motifs separated by an SH3 motif in the N-terminal portion and an NlpC/P60 motif in the C-terminal portion of the protein. However, the active functions of these domains in p60 protein still remain obscure. In this study, we expressed and purified seven recombinant proteins, including full-length p60 protein, LysMl domain, LysM2 domain, SH3 domain, C-terminal domain, LysMl and SH3 domain containing region of p60 protein and the LysMl-truncated version of p60 protein. The binding activities of these recombinant proteins were detected directly by Insoluble Pulldown approach. Our study showed, unequivocally, only N-terminal domain was involved in substrate recognition and binding. In addition, all the domains present in the N-terminal, possessed the binding activity; whereas only weak binding activity of LysMl was detected. Subsequently, the C-terminal domain was identified as catalytic element by the approach of renaturing gel electrophoresis. We also found that the binding activity of the LysM1 and SH3-domain containing region of p60 protein is not the simple sum of activities of LysM1 and SH3, indicating a potential synergistic effect. Moreover, the binding and bacteriolytic activity of the LysMl-truncated version of p60 protein was examined by the comprehensive application of Insoluble Pulldown approach and turbidimetric method. The results suggested that the deletion of LysMl domain can decrease the binding and bacteriolytic activity of p60 protein for about 10% and 70%, respectively.Previous studies showed that p60 protein is a peptidoglycan hydrolase. It can help Lm to escape the innate immunity of host by hydrolyzing the cell wall, to make sure that Lm can survive and propagate in the host and eventually leading to severe listeriosis. As substrate recognition and binding is the primary prerequisite for the bacteriolytic activity of p60 protein, comprehensive study on substrate binding properties of p60 protein become another crucial aspect of our work. Here, we proved that p60 protein is not a metalloprotein and there is no disulfide bond in this protein. The maximal binding activity of p60 protein was at pH 7.5 and 36℃, and the binding activity was enhanced by11%,15% and 21% at the presence of 10 mM Na+, K+, and Ca2+, respectively. Additionally, we found that as a bacterial LysM protein, p60 protein does not distinguish between MurNAc-GlcNAc polymers and GlcNAc polymers. Furthermore, we concluded that the amide group at the C2’position of the GlcNAc residues plays . a crucial role in the binding. Finally, the results from size-exclusion chromatography indicated that p60 protein is a monomer in buffer.