Elucidating the Mechanisms of Cell-to-Cell Crosstalk in Probiotics Co-culture: A Proteomics Study of Limosilactobacillus reuteri ZJ625 and Ligilactobacillus salivarius ZJ614.

Limosilactobacillus reuteri ZJ625 and Ligilactobacillus salivarius ZJ614 are potential probiotic bacteria with improved benefits when administered to the host as a multi-strain preparation. To elucidate the mechanisms of cell-to-cell crosstalk between these two strains, we studied their intracellular and extracellular proteomes in co-culture by liquid-chromatography mass-spectrometry (LC-MS) using Dionex Nano-RSLC and fusion mass spectrometer. The experiment consisted of five biological replicates, and samples were collected during the mid-exponential growth phase. The quantitative proteomic profiles revealed several differentially expressed proteins (DEPs), which are down- or up-regulated between and within groups for both the intracellular and extracellular proteomes. These DEPs include proteins synthesising autoinducer-2, a sensor compound for cell-to-cell bacterial crosstalk during quorum sensing in mixed culture. Other important DEPs identified include enolase, phosphoglycerate kinase, and l-lactate dehydrogenase, which play roles in carbohydrate metabolism. Proteins associated with transcription, ATP production and transport across the membrane, DNA repair, and those with the potential to bind to the host epithelium were also identified. The post-translational modifications associated with the proteins include oxidation, deamidation, and ammonia loss. Importantly, this study revealed a significant expression of S-ribosylhomocysteine lyase (luxS) involved in synthesising autoinducer-2 that plays important roles in quorum sensing, aiding bacterial cell-to-cell crosstalk in co-cultures. The proteome of L. salivarius ZJ614 was most affected when co-cultured with L. reuteri ZJ625. In contrast, omitting some medium components from the defined medium exerted more effects on L. reuteri ZJ625 than L. salivarius ZJ614.