Glyco-phenotyping of mutants of Lacticaseibacillus paracasei by lectin microarray

We previously identified a gene cluster of Lacticaseibacillus paracasei strain Shirota (YIT 9029) for cell surface long-chain polysaccharides (LCPS-1) biosynthesis, which modulates YIT 9029 activity to induce cytokine production in immune cells, and showed that a lectin microarray can be useful for distinguishing the profile of bacterial cell-surface polysaccharide (PS) structures. Therefore, we isolated disruptive mutant strains of 51 genes predicted to be involved in cell wall PS biosynthesis in YIT 9029. Their binding profiles to lectins in conjunction with their binding abilities to YIT 9029-specific monoclonal antibody (MAb) were compared. The mutants defective in binding to the MAb all had defects within the cps1 gene cluster. Some mutants partially bound to MAb, indicating that these genes may influence the synthesis and maturation of LCPS-1. Advanced lectin microarray analyzed the cell surface glycosylation properties of YIT 9029 and its mutants. YIT 9029 bound to a rhamnose-specific lectin CSA, and three additional lectins including an O-glycan binder (rDiscoidin II) and two mannose binders (rOrysata and rBanana). Lectin binding specificity was confirmed by a gene complementation assay for the cps1C gene and a carbohydrate inhibition assay. When the binding profiles of individual cps1A through cps1J knockout mutants were compared, typical and specific binding profiles patterns were observed, in which some similarities in the functions of each gene could be predicted. In conclusion, the combined use of lectin microarray and a YIT 9029 mutant strain library is a powerful tool for identifying unknown bacterial gene functions related to cell surface glycome. IMPORTANCEPreviously, only a limited number of methods have been available for studying mutations in bacterial cell surface polysaccharide structures in relation to gene function. In this study, we focused on the lectin-binding properties of Lacticaseibacillus paracasei YIT 9029 (wild-type; WT) and investigated the lectin-binding capabilities of 51 cell wall biosynthesis gene disruption strains using lectin microarrays. The results indicated that lectin-binding properties in gene-disrupted strains varied significantly with the presence or absence of LCPS-1, ranging from similar to WT to distinctly different. The use of lectin microarrays in conjunction with the YIT 9029 mutant library has been shown to be a highly effective method for identifying the functions of unknown bacterial genes related to cell-surface glycomes. This innovative approach to glycophenotyping allows for the determination of cell wall glycomes associated with bacterial gene functions using lectin microarrays.