Anchoring of heterologous proteins in multiple Lactobacillus species using anchors derived from Lactobacillus plantarum

<jats:title>Abstract</jats:title><jats:p>Members of the genus <jats:italic>Lactobacillus</jats:italic> have a long history in food applications and are considered as promising and safe hosts for delivery of medically interesting proteins. We have assessed multiple surface anchors derived from <jats:italic>Lactobacillus plantarum</jats:italic> for protein surface display in multiple <jats:italic>Lactobacillus</jats:italic> species, using a <jats:italic>Mycobacterium tuberculosis</jats:italic> hybrid antigen as test protein. The anchors tested were a lipoprotein anchor and two cell wall anchors, one non-covalent (LysM domain) and one covalent (sortase-based anchoring using the LPXTG motif). Thus, three different expression vectors for surface-anchoring were tested in eight <jats:italic>Lactobacillus</jats:italic> species. When using the LPXTG and LysM cell wall anchors, surface display, as assessed by flow cytometry and fluorescence microscopy, was observed in all species except <jats:italic>Lactobacillus acidophilus</jats:italic>. Use of the cell membrane anchor revealed more variation in the apparent degree of surface-exposure among the various lactobacilli. Overproduction of the secreted and anchored antigen impaired bacterial growth rate to extents that varied among the lactobacilli and were dependent on the type of anchor. Overall, these results show that surface anchors derived from <jats:italic>L</jats:italic>. <jats:italic>plantarum</jats:italic> are promising candidates for efficient anchoring of medically interesting proteins in other food grade <jats:italic>Lactobacillus</jats:italic> species.</jats:p>