Evidence for PMAT- and OCT-like biogenic amine transporters in a probiotic strain of Lactobacillus: Implications for interkingdom communication within the microbiota-gut-brain axis

Abstract

The ability of prokaryotic microbes to produce and respond to neurochemicals that are more often associated with eukaryotic systems is increasingly recognized through the concept of microbial endocrinology. Most studies have described the phenomena of neurochemical production by bacteria, but there remains an incomplete understanding of the mechanisms by which microbe- or host-derived neuroactive substances can be recognized by bacteria. Based on the evolutionary origins of eukaryotic solute carrier transporters, we hypothesized that bacteria may possess an analogous uptake function for neuroactive biogenic amines. Using specific fluorescence-based assays, Lactobacillus salivarius biofilms appear to express both plasma membrane monoamine transporter (PMAT)- and organic cation transporter (OCT)-like uptake of transporter-specific fluorophores. This phenomenon is not distributed throughout the genus Lactobacillus as L. rhamnosus biofilms did not take up these fluorophores. PMAT probe uptake into L. salivarius biofilms was attenuated by the protonophore CCCP, the cation transport inhibitor decynium-22, and the natural substrates norepinephrine, serotonin and fluoxetine. These results provide the first evidence, to our knowledge, for the existence of PMAT- and OCT-like uptake systems in a bacterium. They also suggest the existence of a hitherto unrecognized mechanism by which a probiotic bacterium may interact with host signals and may provide a means to examine microbial endocrinology-based interactions in health and disease that are part of the larger microbiota-gut-brain axis.

Fig 1. Uptake of transporter fluorophore reporters into Lactobacillus biofilms depends upon bacterial species and ambient temperature.

Bacterial biofilms in flat clear-bottomed 96 well microplates were prepared as described in Materials and Methods. Following a 6 or 24 hour incubation of L. salivarius or L. rhamnosus to promote early or established biofilm formation, the indicated fluorophore, either IDT307 (probe for PMAT) or ASP⁺ (probe for OCTs), was added as per kit directions (IDT307) and a final concentration of 4 μM (ASP⁺), respectively, to appropriate wells and then immediately measured for fluorescence as described. For plates measured at 37°C (panels A and B), fluorophore solutions were pre-warmed and changes in relative fluorescence units (RFU) in each well of the plate were measured at 37°C over a 6 hour time interval. For plates measured at 25°C (panels C and D), the plate was removed from the incubator and placed on a bench-top surface for 1 hour to allow it to reach room temperature prior to fluorophore addition of the at room temperature. Following fluorophore addition, RFUs were measured in the plate at 25°C over a six hour time interval. Results represent mean ± S.E.M of quadruplicate wells and are representative of a minimum of 3 separate experiments. For some points, the S.E.M. error bars are obscured by the symbol.

Fig 2. Effects of decynium-22, a cation transport inhibitor, on fluorophore uptake into L. salivarius biofilms.

L. salivarius biofilms in flat clear-bottomed 96 well microplates were prepared as described in Materials and Methods. Following a 6 hour incubation of L. salivarius culture to form an early biofilm, the medium in each well was gently removed and 100 μl of a pre-warmed solution containing decynium-22 (D-22) was added to achieve a final media concentration of 20 μM (control wells contained the same solvent percentage as did the D-22 wells, see Materials and methods). The plate was then immediately placed back into a 37°C incubator for 30 minutes after which time it was removed and 100 μl of pre-warmed fluorophore was added to appropriate wells. The plate was then immediately placed into the fluorescence reader and measured at 37°C. Results represent mean ± S.E.M of triplicate wells and are representative of at least 2 separate experiments. For some points, the S.E.M. error bars are obscured by the symbol. AUC, area under the curve.

Fig 3. Ability of biogenic amine neurotransmitters and the metabolic inhibitor nitrate to inhibit IDT307 uptake into L. salivarius biofilms.

L. salivarius biofilms in flat clear-bottomed 96 well microplates were prepared as described in Materials and Methods. Following 6 hours of incubation, the medium in each well was gently removed and 100 μl of a pre-warmed solution of the natural transporter substrates norepinephrine (NE) and serotonin (SER), alone or in equimolar combination, was added to appropriate wells to achieve a final biogenic amine concentration of 100 microM. The metabolic inhibitor sodium nitrite (NaNO₂) was also added to appropriate wells to achieve a final concentration of 100 microM. The plate was then immediately placed back into a 37°C incubator for 30 minutes after which time it was removed and 100 μl of pre-warmed fluorophore as added to appropriate wells. The plate was then immediately placed into the fluorescence reader and measured at 37°C. Results represent mean ± S.E.M of triplicate wells and are representative of at least 2 separate experiments. The results for the NE only condition overlap those of the NE/SER condition. For some points, the S.E.M. error bars are obscured by the symbol.

Fig 4. Ability of the protonophore CCCP to inhibit IDT307 uptake into L. salivarius biofilms.

L. salivarius biofilms in flat clear-bottomed 96 well microplates were prepared as described in Materials and Methods. Following 24 hours of incubation, the medium in each well was gently removed and 100 μl of a pre-warmed solution of the proton ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) dissolved in IMDM (prepared as described in Materials and methods) or control IMDM with or without ethanol at an identical concentration present in CCCP-containing medium was added to appropriate wells. The plate was then immediately placed back into a 37°C incubator for 20 minutes after which time it was removed and 100 μl of pre-warmed IDT307 fluorophore was added to wells. The final concentration of CCCP was 10 μM following addition of IDT307 and the concentration of ethanol was 0.5%. The plate was then immediately placed into the fluorescence reader and measured at 37°C. Results represent mean ± S.E.M of triplicate wells. For some points, the S.E.M. error bars are obscured by the symbol.

Fig 5. Concentration-dependent inhibition of IDT307 and ASP⁺ uptake by fluoxetine into early L. salivarius biofilms.

L. salivarius biofilms in flat clear-bottomed 96 well microplates were prepared as described in Materials and Methods. Following 6 hours of incubation, the medium in each well was gently removed and 100 μl of a pre-warmed solution of fluoxetine dissolved in IMDM in the increasing concentrations shown in Fig 5 or IMDM alone was added to appropriate wells. The plate was then immediately placed back into a 37°C incubator for 30 minutes after which time it was removed and 100 μl of pre-warmed fluorophore was added to appropriate wells. The plate was then immediately placed into the fluorescence reader and measured at 37°C. Results represent mean ± S.E.M of triplicate wells and are representative of at least 2 separate experiments. For some points, the S.E.M. error bars are obscured by the symbol.