Serotonin Exposure Improves Stress Resistance, Aggregation, and Biofilm Formation in the Probiotic Enterococcus faecium NCIMB10415

Abstract

The role of the microbiota–gut–brain axis in maintaining a healthy status is well recognized. In this bidirectional flux, the influence of host hormones on gut bacteria is crucial. However, data on commensal/probiotics are scarce since most reports analyzed the effects of human bioactive compounds on opportunistic strains, highlighting the risk of increased pathogenicity under stimulation. The present investigation examined the modifications induced by 5HT, a tryptophan-derived molecule abundant in the intestine, on the probiotic Enterococcus faecium NCIMB10415. Specific phenotypic modifications concerning the probiotic potential and possible effects of treated bacteria on dendritic cells were explored together with the comparative soluble proteome evaluation. Increased resistance to bile salts and ampicillin in 5HT-stimulated conditions relate with overexpression of specific proteins (among which Zn-beta-lactamases, a Zn-transport protein and a protein involved in fatty acid incorporation into the membrane). Better auto-aggregating properties and biofilm-forming aptitude are consistent with enhanced QS peptide transport. Concerning interaction with the host, E. faecium NCIMB10415 enhanced dendritic cell maturation, but no significant differences were observed between 5HT-treated and untreated bacteria; meanwhile, after 5HT exposure, some moonlight proteins possibly involved in tissue adhesion were found in higher abundance. Finally, the finding in stimulated conditions of a higher abundance of VicR, a protein involved in two-component signal transduction system (VicK/R), suggests the existence of a possible surface receptor (VicK) for 5HT sensing in the strain studied. These overall data indicate that E. faecium NCIMB10415 modifies its physiology in response to 5HT by improving bacterial interactions and resistance to stressors.

Norepinephrine and Serotonin Can Modulate the Behavior of the Probiotic Enterococcus faecium NCIMB10415 towards the Host: Is a Putative Surface Sensor Involved?

“…The hormones have augmented all the three parameters; in detail, the treatment with 100 µM NE enhances the three of them by about 50%, while 50 µM 5HT increases twofold the biovolume and the maximum thickness with respect to the control, while the average thickness is augmented by about 40%. These results strongly confirm the CV assay outcomes concerning the boosting effect of the molecules and its measurement; indeed, 5HT shows a more intense effect compared to NE, precisely as demonstrated with the CV test [ 22 , 23 ].…”

“…Our group already assessed the biofilm formation ability of E. faecium NCIMB10415 with the crystal violet method in control and stimulated conditions, and both molecules caused a significant enhancement of biomasses after the treatment [ 22 , 23 ]. The CLSM technique was chosen to validate the results obtained with the CV staining since this method is not considered precise.…”

“…Adhesion on intestinal epithelial cells is considered an essential trait of probiotics, as adherent bacteria may overcome peristaltic movements and permanently colonize the gut epithelium by forming a biofilm structure where they can persist and exert their positive effects on human health [ 50 ]. An adhesion-independent biofilm enhancement was previously assessed by crystal violet staining in this strain after treatment with both NE [ 22 ] and 5HT [ 23 ]. The confocal microscope analysis of the biomasses validated these findings since the biovolumes, and the maximum and average thickness were higher than those under control conditions.…”

“…E. faecium NCIMB10415 ( E. faecium SF68) was grown at 37 °C in static conditions in a rich Chemically Defined Medium (CDM) to ensure highly reproducible results, as reported elsewhere [ 22 ]. 100× solutions of NE (L-Norepinephrine (+)-bitartrate salt monohydrate—Sigma Aldrich, St. Louis, MO, USA) and 5HT (serotonin hydrochloride—Sigma Aldrich) were freshly prepared in CDM, filter sterilized and diluted to obtain a final concentration that would reflect over secretion in the gut, 100 µM and 50 µM, respectively (for further details see [ 22 , 23 ]). For each experiment, except for biofilm formation, treatments, and control conditions, bacteria were harvested at late exponential phase (~4 h, see [ 22 , 23 ]).…”

“…For example, host-derived signals in the gut, such as norepinephrine (NE) and serotonin (5HT), have been reported to trigger the virulence of some intestinal pathogens including E. faecalis [ 19 , 20 , 21 ], but those conditions can also modify the probiotic properties of exogenously administered enterococci [ 21 ]. Previous research tested how NE and 5HT can influence the basal physiological features (growth and protein profiles, resistance to bile salts, and antibiotics) of the probiotic E. faecium NCIMB10415 ( E. faecium SF68) and its capability to auto-aggregate and form biofilm [ 22 , 23 ]. Curiously, the proteomic results of 5HT treatment suggested that the effects triggered by this molecule could be mediated by a sensor, primarily described as human signal receptors in prokaryotes [ 24 ].…”