Functional analysis of the Lactobacillus casei BL23 sortases

Gram-positive bacteria use sortases to attach surface proteins to their cell walls. These enzymes are essential for adhesion and other surface-related functions. Lactobacillus casei BL23 contains four sortase genes, but their roles remain unclear. Prior research has shown that sortases are involved in adhesion and biofilm formation in other Lactobacillus species. However, no prior work had resolved how these enzymes function in L. casei BL23. This uncertainty drove the current investigation. The study aimed to clarify which sortases are functionally important in this strain. The absence of clear data on sortase activity in L. casei BL23 motivated the use of genetic disruption and phenotypic analysis. Understanding sortase roles could help in engineering probiotic bacteria for improved adhesion properties.

The study aimed to determine the functional roles of four sortase genes in Lactobacillus casei BL23. Researchers focused on whether these enzymes contribute to surface protein anchoring and bacterial adhesion. They hypothesized that mutations in these genes would reveal their importance. The specific problem addressed was the lack of clarity about which sortases are essential for L. casei BL23 physiology. The motivation was to identify which sortases are housekeeping and which have redundant or complementary roles. The researchers sought to test this by constructing gene disruptions and analyzing phenotypic changes. The goal was to determine how each sortase contributes to cell wall protein anchoring and adhesion. This approach could help in understanding how to manipulate L. casei BL23 for probiotic applications.

The researchers used genetic disruption to create strains lacking individual or multiple sortase genes. They analyzed the transcription of each sortase gene using RNA-based methods. Bacterial surface hydrophobicity was measured to assess cell surface properties. Whole-cell enzyme activities were tested, including β-N-acetyl-glucosaminidase and cell wall proteinase. Adhesion to Caco-2 and HT-29 intestinal cells was evaluated using in vitro assays. A reporter protein, staphylococcal NucA fused to a cell wall sorting sequence, was used to monitor anchoring efficiency. Strains with mutations in srtC1 and srtC2 were tested for phenotypic changes. The study combined genetic, biochemical, and functional assays to assess sortase roles.

Mutations in srtA1 reduced bacterial surface hydrophobicity and enzyme activities. The srtA1 srtA2 double mutant showed even greater decreases in β-N-acetyl-glucosaminidase and proteinase activities. Adhesion to intestinal cells was also reduced in the double mutant. Anchoring of the NucA reporter protein was affected in srtA mutants but not in srtC mutants. Only srtA1 mutations produced detectable phenotypes. The srtC1 and srtC2 mutations had no observable effects on cell function. The srtA1 mutant showed diminished cell wall proteinase activity compared to wild-type strains. The srtA1 srtA2 strain had a 60% reduction in adhesion to Caco-2 cells compared to wild-type.

The findings suggest that SrtA1 is the primary sortase in L. casei BL23. SrtA2 appears to have redundant or complementary functions that become evident when SrtA1 is absent. The srtC1 and srtC2 genes do not seem to play detectable roles in this strain. These conclusions are based on the observed phenotypic changes in srtA mutants. The researchers propose that SrtA1 is responsible for anchoring cell wall proteins. The redundancy between SrtA1 and SrtA2 may explain the partial effects seen in the double mutant. The lack of phenotypic change in srtC mutants suggests they are not essential for the tested functions. The study provides evidence for differential roles among sortase isotypes in L. casei BL23.