Abstract
In the complex interplay of beneficial bacteria with the host, there are few examples of bacterial metabolites and effector molecules that have been consistently identified. Protective effects on the intestinal epithelium have been ascribed to P40 and P75, two well characterized cell wall muramidases, present in the culture supernatant of strains belonging to the taxon Lactobacillus casei/paracasei/rhamnosus. This work reports that Lactobacillus casei BL23 extracellular vesicles (BL23 EVs) have a small size (17–20 nm or 24–32 nm, depending on the method used) and contain lipoteichoic acid (LTA). Interestingly, all detected P40 and most of P75 were associated to EVs and possibly located at their external surface, as shown by proteinase K digestion. Biosensor assays showed that both proteins bind LTA and vesicles, suggesting that they could bind to ligands like LTA present on BL23 EVs. Native BL23 EVs have a moderate proinflammatory effect and they were able to induce phosphorylation of the epidermal growth factor receptor (EGFR), showing an effect similar to purified P40 and P75 and leading to the conclusion that the activity described in the supernatant (postbiotic) of these bacteria would be mainly due to P40 and P75 bound to EVs.
Highlights
In the complex interplay of beneficial bacteria with the host, there are few examples of bacterial metabolites and effector molecules that have been consistently identified
BL23 EVs were isolated by ultracentrifugation from culture supernatant, filtered through 0.45 μm microbiological filter, and they were observed by transmission electron microscopy (TEM) negative staining (Fig. 1A,B)
We could detect in recombinant BL23 EVs both green and red fluorescence (Fig. 1D,E), indicating the presence of these fluorescent proteins in EVs which in the parental cell are localized in the cytosolic compartment
Summary
In the complex interplay of beneficial bacteria with the host, there are few examples of bacterial metabolites and effector molecules that have been consistently identified. Two secreted cell wall muramidases that contain a CHAP (NlpC/P60) domain have been characterized in BL23, P40 and P75, and homologs to these proteins are exclusively present in the L. casei taxon, that comprises mainly the species L. casei, L. paracasei and L. rhamnosus[26]. Their ability to stimulate growth and survival of epithelial cells was first described in L. rhamnosus GG27. These proteins proved to have anti-apoptotic and anti-inflammatory effects mediated by the activation of the EGFR/ Akt pathway. We have managed to explain, first how P40 and P75 can be secreted and associated to EVs, and that L. casei BL23 derived EVs have an equivalent activity to P40 and P75, on the EGFR/Akt pathway in IEC
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