Deciphering the substrate specificity of housekeeping and pilus-specific sortases from probiotic Lactococcus lactis

Sortases are cysteine transpeptidase enzymes that decorate the Gram-positive bacterial cell wall with various surface proteins and pili to facilitate the interactions with the host and environment for the colonization. While the sortases and sortase anchored proteins from Gram-positive pathogens have been the prime focus of the research in the past, sortases from many non-pathogenic bacteria, including Lactic Acid Bacteria (LAB) strains, have recently attracted attention for their potential applications in vaccine delivery and other clinical interventions. The LAB bacteria have beneficial effects on the host as they transiently co-exist with the gut microbiome and consequently affect the composition and metabolism of the gut microbiome and possibly human health. Two different types of sortases (A-type or housekeeping and C-type or pilus-specific) involve in the pilus biogenesis. The sortase assembled pili in these LAB bacteria play an essential role in mediating attachment to host gut mucosa. This initial adherence is often a prerequisite for colonization and beneficial effects. The C-type sortase catalyzes pilus polymerization by linking pilin subunits (i.e., building blocks of pili) in a head-to-tail fashion. In contrast, the A-type sortase anchors the covalently cross-linked pilus polymers on the cell wall. This study comprehensively characterizes A- and C-type sortases from probiotic Lactococcus lactis using biochemical, biophysical, and computational analyses. The purified recombinant sortases were active against their respective substrates containing sorting (LPXTG) motifs with variations in the affinity and specificity. Overall, this study provides insights into substrate recognition by A- and C-type sortases and highlights the critical residues that might play a significant role in the binding and recognition. This study represents the first step toward understanding sortases from probiotics involved in pili assembly. The findings that the A-type sortase of L. lactis recognizes an LLXTG sorting motif is unprecedented for a housekeeping sortase and has the potential to expand the repertoire of sortase application in peptide ligation and protein bioconjugations.

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Deciphering the substrate specificity of housekeeping sortase A and pilus-specific sortase C of probiotic bacterium Lactococcus lactis


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