Contribution of sortase SrtA2 to Lactobacillus casei BL23 inhibition of Staphylococcus aureus internalization into bovine mammary epithelial cells

Abstract

Probiotics have been considered as a promising strategy to prevent various diseases in both humans and animals. This approach has gained interest in recent years as a potential means to control bovine mastitis. In a previous study, we found that several L. casei strains, including BL23, were able to inhibit the internalization of S. aureus, a major etiologic agent of mastitis, into bovine mammary epithelial cells (bMEC). This antagonism required a direct contact between L. casei and bMEC or S. aureus, suggesting the inhibition relied on interactions between L. casei cell surface components and bMEC. In this study, we have investigated the impact of some candidates which likely influence bacteria host cell interactions. We have shown that L. casei BL23 fbpA retained its inhibitory potential, indicating that L. casei BL23 antagonism did not rely (solely) on competition between S. aureus and L. casei fibronectin-binding proteins for adhesion to bMEC. We have then investigated the impact of four sortase mutants, srtA1, srtA2, srtC1 and srtC2, and a double mutant (srtA1-srtA2) on L. casei BL23 inhibitory potential. Sortases are responsible for the anchoring on the bacterial cell wall of LPXTG-proteins, which reportedly play an important role in bacteria-host cell interaction. All the srt mutants tested presented a reduced inhibition capacity, the most pronounced effect being observed with the srtA2 mutant. A lower internalization capacity of L. casei srtA2 into bMEC was also observed. This was associated with several changes at the surface of L. casei BL23 srtA2 compared to the wild type (wt) strain, including altered abundance of some LPXTG- and moonlighting proteins, and modifications of cell wall structure. These results strongly support the role of sortase A2 in L. casei BL23 inhibition against S. aureus internalization. Deciphering the contribution of the cell surface components altered in srtA2 strain in the inhibition will require further investigation.

Internalization, distribution, and activity of peptide H2 against the intracellular multidrug-resistant bovine mastitis-causing bacterium Staphylococcus aureus

“…The proportion of cells with fluorescence labeling of H2 and NZ2114 were reduced by 13.9% and 3.4%, respectively, and the fluorescence intensity of the cells decreased sharply (Fig. 4C,D), indicating that low temperature may have an inhibitory effect on peptide uptake 28 . In the inhibitor-treated groups, the fluorescence intensity of the cells treated with chlorpromazine (an inhibitor of clathrin-mediated endocytosis) decreased sharply; however, the fluorescence intensity did not change following treatment with amiloride (an inhibitor of macropinocytosis), methyl-β-cyclodextrin (MβCD) (a disruptor of lipid rafts), and nocodazole (an inhibitor of microtubule polymerization) (Fig.…”

Lactobacillus casei extracellular vesicles stimulate EGFR pathway likely due to the presence of proteins P40 and P75 bound to their surface

“…Also, EVs from probiotic bacteria have comparable effects to whole bacteria, increasing the number of T reg cells, in case of L. rhamnosus JB-1 9 , and promoting the survival of Caenorhabditis elegans exposed to vancomycin-resistant E. faecium by L. plantarum WCFS1 10 . Analysis of EVs from probiotic lactobacilli, like Lactobacillus rhamnosus , L. plantarum , L. reuteri and L. casei , showed that they contained glycolytic enzymes, chaperonins 9 , 11 – 14 and ligase functions 15 . In addition to proteins and membrane phospholipids, EVs from Gram-positive bacteria also contain lipoteichoic acid (LTA).…”

Heat inactivation partially preserved barrier and immunomodulatory effects of Lactobacillus gasseri LA806 in an in vitro model of bovine mastitis

“…Various cell surface components, such as proteins, peptidoglycans, teichoic acids and polysaccharides contribute to the interaction of LAB with epithelial cells (Piqué et al, 2019;Taverniti and Guglielmetti, 2011). A L. casei BL23 srtA2 mutant, srtA2 encoding a sortase, was less effective in inhibiting S. aureus adhesion to and internalisation into bMECs (Souza et al, 2017). This characteristic was associated with an altered abundance of LPXTG and moonlighting proteins at the surface of the srtA2 mutant strain as well as with modifications of the cell wall structure.…”