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    • Despite all these observations linking bacteria

    2018-10-23

    Despite all these observations linking bacteria to intrinsic serotonin synthesis, very little is understood regarding why, how, and what are the consequences of the microbiome\'s influence on the host\'s neurotransmitter levels and its manipulation, and vice versa. Most studies focus on the influence of the microbiome on changes to the host\'s production of these molecules, while very few examine the effects these molecules have on bacteria. It was observed that serotonin was able to stimulate growth of specific bacteria in culture (Oleskin et al., 1998), and some virulent bacteria can use neurotransmitters such as melanin inhibitor and norepinephrine to activate virulence genes (Clarke et al., 2006). Both of these phenomena are linked to bacterial quorum sensing (QS), which is, in bacteria, a phenomenon when populations reach a specific threshold they communicate with organisms in their surroundings by releasing small diffusible quorum sensing molecules (QSMs). The QSMs then bind to regulatory proteins, causing a conformational change and allowing the protein to bind to DNA and initiate the transcription of virulence factors (Fig. 1). Herein, we hypothesized that bacteria are able to interact with host serotonin molecules and exploit them as a bacterial QSMs. Specifically, through experiments conducted in vitro and in vivo in animal studies, we demonstrate for the first time that serotonin acts as a signaling molecule for the las regulatory QS system of Pseudomonas aeruginosa inducing, among other effects, serious pathogenicity in the host. P. aeruginosa has a well-studied QS network that relies on multiple QS pathways critical in activating Pseudomonas virulence including the las and rhl systems. This work helps explains how high levels of serotonin found in the gut, produced endogenously or by bacteria, can be linked to the host\'s health.
    Materials and Methods
    Results
    Discussion This study establishes serotonin\'s role as a bacterial quorum sensing molecule. Our cellular system data show that serotonin can activate the LasR QS pathway at μM concentrations (Fig. 3b). This finding is of great interest as physiological levels of serotonin in the digestive tract of healthy and diseased individuals is 10μM (Erspamer, 1966) and ~100μM (Miwa et al., 2001) respectively. The raised levels of serotonin in patients with Inflammatory Bowel Disease (IBD), combined with the insight that serotonin can activate QS pathways, may allow for better patient care and therapeutics. Additionally, the additive effects of serotonin combined with QSM (Fig. 3c) are relevant as bacteria exist naturally in the gut, thus it is highly likely that serotonin will co-exist with bacterial QSMs. Previous reports have shown that virulence factor production, such as biofilm formation and protease production, are quorum-dependent (Waters et al., 2008, Valiente et al., 2007), which supports the increase in these factors seen when serotonin was administered in both our in vitro (Fig. 4) and in vivo (Figs. 2, 5) experiments as serotonin activating the LasR QS system. In vitro, our results show that serotonin mimics the effects of exogenous QSM addition in JP2 cells\' biofilm formation (Fig. 4C–D), indicating that serotonin acts as a bacterial signaling molecule that is capable of activating QS-regulated phenotypes. Our developed Pseudomonas infection model demonstrated that serotonin was able to activate Pseudomonas virulence in vivo, within the intestines of the mice (Figs. 2, 5). Within these experiments, there are two points of particular interest: in the absence of serotonin the JP2 mutant that cannot generate its own QSMs was not able to establish an infection, and the effects of PAO1 infection alone were similar to the effects of JP2 with serotonin. When administered the same CFU of JP2 cells compared to PAO1 cells, the JP2 mutant Pseudomonas did not establish any detectable colonies (Fig. S3B). This lack of infection is supported by the SEM images (Fig. 5A) and histology data (Fig. 5B–C). As the JP2 mutant is only lacking in QSM synthesis, this inability to establish an infection both supports the current literature that Pseudomonas virulence is QS dependent and allows for the assessment of restoration of function, specifically the ability to establish an infection. Thus when we restore the infectivity of JP2 by administering serotonin (Fig. S3B), it indicates that serotonin is fulfilling the role of a QSM. The infection with PAO1 alone and JP2 with serotonin resulted in similar levels of CFUs harvested from the intestines (Fig. S1D, S3B), which further supports serotonin\'s role as a QSM. This similarity between PAO1 alone and JP2 with serotonin is also noted in the histology data (Figs. 2B–C, 5B–C), with both exhibiting intestinal epithelial damage and villi destruction. These data further demonstrate serotonin\'s ability to restore function to the JP2 mutant lacking QSMs, providing greater evidence for its role as a QSM.