Functional Analysis of the Lactobacillus casei BL23 Sortases

Abstract

bSortases are a class of enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria. Lactobacillus casei BL23 harbors four sortase genes, two belonging to class A (srtA1 and srtA2) and two belonging to class C (srtC1 and srtC2). Class C sortases were clustered with genes encoding their putative substrates that were homologous to the SpaEFG and SpaCBA proteins that encode mucus adhesive pili in Lactobacillus rhamnosus GG. Twenty-three genes encoding putative sortase substrates were identified in the L. casei BL23 genome with unknown (35%), enzymatic (30%), or adhesion-related (35%) functions. Strains disrupted in srtA1, srtA2, srtC1, and srtC2 and an srtA1 srtA2 double mutant were constructed. The transcription of all four sortase encoding genes was detected, but only the mutation of srtA1 resulted in a decrease in bacterial surface hydrophobicity. The ␤-Nacetyl-glucosaminidase and cell wall proteinase activities of whole cells diminished in the srtA1 mutant and, to a greater extent, in the srtA1 srtA2 double mutant. Cell wall anchoring of the staphylococcal NucA reporter protein fused to a cell wall sorting sequence was also affected in the srtA mutants, and the percentages of adhesion to Caco-2 and HT-29 intestinal epithelial cells were reduced for the srtA1 srtA2 strain. Mutations in srtC1 or srtC2 result in an undetectable phenotype. Together, these results suggest that SrtA1 is the housekeeping sortase in L. casei BL23 and SrtA2 would carry out redundant or complementary functions that become evident when SrtA1 activity is absent.

Noroviral P-Particles as an In Vitro Model to Assess the Interactions of Noroviruses with Probiotics

“…However, in the exclusion and displacement assays other interactions may play a role, such as the affinity of P-particles for bacteria attached to the cultured cells, which may led to higher P-particle retention on the HT-29 surface. We showed that L. casei BL23 and E. coli Nissle 1917 were indeed binding to HT-29 cells (Figure 8), although the binding was low, as has been previously reported [42]–[43]. Under our conditions a better adhesion was detected in L. casei BL23 compared to E. coli Nissle 1917.…”

The Role of Cell Surface Architecture of Lactobacilli in Host-Microbe Interactions in the Gastrointestinal Tract

“…LspA, of L. salivarius UCC118, is the fourth characterised sortase-dependent protein that also binds mucus and is known to mediate adhesion of this species to intestinal epithelial cells [109, 128]. Recent studies with L. casei BL23 sortases and SrtA mutants suggest that SrtA might be involved in adhesion of this strain to Caco-2 and HT29 cells [117]. Although most sortase-dependent proteins of lactobacilli seem to have mucus-binding capacity, not all of them have affinity to mucus components and the function of putative lactobacilli sortase-dependent proteins remains unclear [16].…”

Relevance and application of sortase and sortase-dependent proteins in lactic acid bacteria

“…Genes encoding housekeeping sortase enzymes ( srtA ) have been identified in the genomes of LAB including L. acidophilus (Buck et al, 2005), Lactobacillus salivarius UCC118 (van Pijkeren et al, 2006), Lactobacillus johnsonii NCC533 (Denou et al, 2008), L. rhamnosus GG (Kankainen et al, 2009), Lactococcus lactis IL1403 (Dieye et al, 2010), Lactobacillus casei BL23 (Munoz-Provencio et al, 2012), L. plantarum (Remus et al, 2013), and Lactobacillus crispatus ST1 (Edelman et al, 2012). Additionally, genes encoding class C sortase proteins ( srtC ) have been identified in both L. rhamnosus GG (Kankainen et al, 2009) and L. casei BL23 (Munoz-Provencio et al, 2012). The genomic context of the srtA locus varies widely among species ( Figure 2A ); however, srtC genes have been found to cluster with their targets ( spa genes) as was observed in both L. rhamnosus GG ( Figure 2B ) and L. casei BL23 (Munoz-Provencio et al, 2012).…”

“…Additionally, genes encoding class C sortase proteins ( srtC ) have been identified in both L. rhamnosus GG (Kankainen et al, 2009) and L. casei BL23 (Munoz-Provencio et al, 2012). The genomic context of the srtA locus varies widely among species ( Figure 2A ); however, srtC genes have been found to cluster with their targets ( spa genes) as was observed in both L. rhamnosus GG ( Figure 2B ) and L. casei BL23 (Munoz-Provencio et al, 2012). An unrooted phylogenetic tree shows the relationships between the sortase protein sequences identified in the aforementioned species of LAB as well as those present in other LAB species including L. lactis subsp.…”