Background
Antimicrobial peptides are produced by many living cells in animals and plants, and constitute integral components of innate host defense [
1‐
3]. Several neuropeptides also have antimicrobial activity such as neuropeptide Y (NPY) and Substance P (SP) [
4‐
7], and are known to be implicated in the microbiota–gut–brain axis [
8]. SP is an undecapeptide of the tachykinin family which is abundant in the skin [
9] and the gut [
10]. In the skin, SP is considered as a major mediator of inflammation. It contributes to the pathogenesis of numerous skin diseases, like psoriasis, atopic dermatitis and acne, and can modulate the virulence of
Bacillus cereus,
Staphylococcus aureus and
Staphylococcus epidermidis [
9,
11]. In the bowel, SP is expressed in both myenteric and submucosal plexuses and is found within intrinsic and extrinsic sensory neurons. Immune effector cells, such as monocytes, macrophages, eosinophils, and lymphocytes, also express SP. A role of various levels of SP in the pathophysiology of inflammatory bowel disease has been suggested but disparate results were reported depending of the studies [
10].
Enterococcus faecalis is a common resident of the gastrointestinal tract of humans and animals, and a wide-spread Gram-positive lactic acid bacterium that can be isolated from a variety of habitats, including fermented foods, human milk and vaginal secretions.
E. faecalis Symbioflor 1 is also used as probiotic for more than 20 years and has been reported to reduce the number of relapses in patients with chronic recurrent hypertrophic sinusitis, as well as the number and severity of relapses in patients with chronic recurrent bronchitis [
12]. Nevertheless, the use of enterococci as a probiotic or cheese starter remains controversial, due to biogenic amines production (tyramine and putrescine) that are toxic for human health [
13,
14] and the risks of transfer of antimicrobial resistance and virulence genes to human strains [
15,
16]. In fact, some strains, such as
E. faecalis V583, are known to be opportunistic pathogen. This bacterium originates from a patient suffering from a persistent bloodstream infection, and it can cause diseases like urinary tract infections, bacteremia, and infective endocarditis in immunocompromised patients [
17,
18]. These infections may be problematic because some
E. faecalis strains are resistant to many antibiotics including vancomycin, and Enterococci are now considered among the most prevalent nosocomial pathogens [
19,
20]. These multidrug-resistant (MDR) enterococci may colonize the patient after perturbation of the native flora by antibiotic treatment. Indeed, a recent study showed that commensal strains of
E. faecalis generally protect the gut by producing pheromone peptides that can kill MDR
E. faecalis V583 [
21].
The opportunistic pathogen
E. faecalis has extraordinary capacities to grow under adverse conditions and to colonize and survive in a large range of ecological niches including in macrophages [
22‐
24]. The mechanism by which this bacterium is able to cross the barrier from a commensal to become a pathobiont may be a thin line [
25] and is not well understood. In the host or after infection,
E. faecalis may be in contact with various eukaryotic molecules that have antimicrobial properties and help fighting against the pathogen or in the contrary can contribute to promote the bacterial colonization. Therefore, the aim of this study was to determine the effect of SP on
E. faecalis V583.
Discussion
Neuropeptides such as SP are important transmitters in the bidirectional gut–brain communication network that may influence the activity of the gastrointestinal microbiota and its interaction with the gut-brain axis [
8]. As SP is known to display antimicrobial activity against oral, respiratory, gut and skin bacteria [
5,
26] but also to conversely enhance virulence of
Bacillus cereus,
Staphylococcus aureus [
9] and
Pseudomonas fluorescens [
27], the aim of this study was to determine its effect on
E. faecalis V583.
El Karim et al. found that
E. faecalis NCTC 12697 is highly resistant to SP with a MIC >500 µg/ml [
26]. Similarly, our MIC analysis showed no antibacterial activity of SP against
E. faecalis V583 even for a high concentration of 1000 µg/ml (7 × 10
−4 M). However, confocal microscopy using LIVE/DEAD BacLight Bacterial Viability Assay revealed some dead cells for SP >10
−4 M and the bacteria seem to slightly agglutinate which was then confirmed by SEM microscopy.
SP is a cationic antimicrobial peptide that may kill bacteria by pore formation and/or probably also act as a metabolic inhibitor [
4]. To a certain extent, in our study, aggregation of
E. faecalis V583, may protect the bacteria from SP, but conversely in some cases aggregation/agglutination of bacteria is also known to be inherent of the mechanism of action of some antimicrobial peptides [
28,
29]. In addition, aggregation of bacteria is often associated with hydrophobicity, and these two parameters can help to adhere to different surfaces including intestinal epithelial cells [
30]. Therefore,
E. faecalis V583 was examined for its hydrophobicity according to exposure to SP. The results showed that a treatment of the bacteria with SP increased the hydrophobicity value. This allows us to hypothesize that SP in the gastrointestinal tract may promote adhesion of
E. faecalis and/or other gut microbes to the mucosa.
As
E. faecalis V583 is known to be an opportunistic pathogen, we examined some virulence properties of the bacteria following SP exposure. We found that lactic acid production was enhanced when
E. faecalis V583 is treated with SP. Lactic acid produced by the lactate dehydrogenase (LDH) enzyme, is implicated in multiple stress resistance and virulence in
E. faecalis [
23] and was also demonstrated to be a potential virulence factor in
Streptococcus [
31]. In our study, the increase of lactic acid production during exposure of the bacteria to SP didn’t result to a fall in pH. In fact, to protect the bacteria from its own acid production,
E. faecalis is able to decarboxylate amino acid, especially tyrosine in tyramine [
32‐
34]. Tyramine biosynthesis is transcriptionally induced at low pH [
35,
36] and this improves the fitness of
E. faecalis and
Enterococcus durans in acidic environments such as in the gastrointestinal tract [
37]. We showed that exposure to SP enhanced the production of tyramine by
E. faecalis V583 concomitantly with lactic acid, this may explain why no modification in pH was observed in our experiment. The biosynthesis of tyramine is a general species trait of
E. faecalis [
13]. Large amounts of this biogenic amine can cause toxicological effects [
14,
38] including migraines and hypertension and sometimes problems as serious as cerebral haemorrhages. Thus, production of tyramine by gut commensal
E. faecalis exposed to SP deserves more attention.
Subsequently, the pathogenicity of
E. faecalis V583 treated with SP was evaluated using Caco-2/TC7 intestinal epithelial cells. The results showed that the cytotoxicity of
E. faecalis V583 was enhanced with SP exposure and the permeability of differentiated Caco-2/TC7 cells increased rapidly, allowing the bacteria to translocate faster to the basolateral compartment. Linares et al. recently found that tyramine is toxic for HT29 intestinal cell cultures and induces necrosis [
14]. Thus, the increase of the cytotoxic effect of
E. faecalis V583, pretreated with SP, on Caco-2/TC7 cells, could be partially attributed to the overproduction of tyramine we noticed before.
In previous works, we showed that SP can modulate the swarm and proinflammatory potential of
P. fluorescens [
27] and strongly stimulated the cytotoxicity of
B. cereus [
9], so this neuropeptide, abundant in the skin and the gut, may promote pathogenicity in various bacteria. In fact, microbial endocrinology shows that, through their long coexistence with animals and plants, microorganisms have evolved sensors for detecting eukaryotic molecules [
39] and a bidirectional communication exists between the gut and its microflora [
40,
41].
Ef-Tu is a moonlighting protein [
42,
43] that has been found as the SP sensor in
Bacillus cereus [
9], and recently identified also as the SP-interacting protein in
S. aureus and
S. epidermidis [
11]. The putative binding site of SP in
E. faecalis is now under investigation in our laboratory and the preliminary results showed that Ef-Tu would indeed be the sensor. It remains also to determine which other bacteria within the gut microbiota expresses neuropeptide receptors or releases metabolites that are ligands for eukaryotic neuropeptide receptors. This may probably help to better understand the complex relationship between brain, gut and microbiota, and to found adequate therapy in intestinal disorders.
Methods
Bacterial strain, culture conditions and reagents
The human clinical isolate
E. faecalis V583 was used in our study [
18]. This strain was cultivated at 37 °C in Brain Heart Infusion (BHI) medium and stored at −80 °C in 50% (v/v) glycerol. SP was synthesized by PolyPeptide group. For each experiment, a stock solution of the peptide was freshly prepared in Milli-Q water and filter-sterilized.
Minimal inhibitory concentration (MIC) analysis
MIC was studied by microdilution in Mueller–Hinton (MH) broth using an inoculum of 105 colony forming units per ml. Microtiter plate containing triplicate twofold dilution series of 1000 µg/ml SP was incubated overnight at 37 °C. Turbidity was observed at the end of incubation.
LIVE/DEAD analysis by confocal microscopy
Viability of E. faecalis V583 treated or not with SP was analyzed using the LIVE/DEAD BacLight Bacterial Viability Kit for microscopy (L7007, Invitrogen). In this system, live bacteria stain with SYTO 9 to produce a green fluorescence whereas dead bacteria stain with propidium iodide (PI) to produce a red fluorescence. Briefly, 3 µl of the SYTO 9/PI mixture, prepared according to the manufacturer’s instruction, was added to the bacterial cells previously treated with 10−2, 10−4 or 10−6 M of SP for 2 h, and untreated cells were kept as control. The samples were incubated for 15 min in dark at room temperature and 5 µl of this sample was trapped in between coverslip and glass slide. The slide was viewed under a confocal laser-scanning microscope (LSM 710 CLSM, Zeiss), using 60× objective sequentially using fluorescence setting for FITC (green/SYTO 9, viable cells) and PI (red/PI, dead cells) filters, respectively, followed by phase contrast and bright field settings. SYTO 9 and PI images were merged and acquired using ZEN® software (Zeiss).
Scanning electron microscopy (SEM)
108 CFU/ml bacterial suspension was incubated with 10−4 M of SP in phosphate buffered saline (PBS) for 2 h. At the end of incubation, the bacteria were washed with PBS and fixed on coverslips with 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 2 h. After washing with Milli-Q water, bacteria were dehydrated with increasing ethanol concentration (50, 70, 95 and 100%). Dry coverslips were mounted on stubs and coated with 5 nm platinum (Quorum Technologies Q150T, Elexience, France). Bacteria were observed with a secondary detector in a Zeiss SEM Merlin Compact VP (Zeiss, France) operating at 5 kV.
Hydrophobicity
The hydrophobicity of
E. faecalis was evaluated by the microbial adhesion to solvent (MATS) test, according to Al Atya et al. [
44] and Kos et al. [
45] with a slight modification. Bacteria grown in BHI with or without SP (10
−4 or 10
−6 M) for 18 h at 37 °C, were harvested by centrifugation at 5000
g for 15 min, washed twice, and resuspended in PBS to approximately 10
8 CFU/ml. The absorbance of the cell suspension was measured at 600 nm (
A
0). One ml of solvent was added to 3 ml of cell suspension. After a 10 min preincubation at room temperature, the two-phase system was mixed by vortexing for 2 min. The aqueous phase was removed after 20 min of incubation at room temperature, and the absorbance at 600 nm (
A
1) was measured. Two apolar solvents were tested: xylene and toluene. Bacterial adhesion to these solvents reflects cell surface hydrophobicity. The percentage of bacterial adhesion to the solvents was calculated as 1 − (
A
1/
A
0) × 100.
Lactic acid and tyramine quantification
Lactic acid was quantified as recently described [
44]. Briefly,
E. faecalis were grown in BHI with or without 10
−6 M SP at 37 °C, and samples were withdrawn after 14 h of incubation, centrifuged (10,000
g, 4 °C, 10 min), and sterilized by filtration using Millipore filter (0.2 µm). The concentration of lactic acid in the samples was determined by HPLC spectra system P1000XR (Thermo, USA).
For tyramine analysis, the bacterial samples were prepared similarly, except that tyrosine (800 mg/l) was added in the BHI medium [
34]. The quantification was performed by mixing 20 µl of bacterial samples with 80 µl of perchloric acid (0.2 M) and 5 µl of 1.7 diaminoheptane (6.4 mg/ml), shaking until complete homogenization and centrifuging at 8000
g, 4 °C, 5 min. The steps of derivatization using dansyl chloride and purification are described in Duflos et al. [
46]. Hitachi Elite LaChrom HPLC System was used to quantify tyramine with Kromasil column (C18, 5 µm 100 Å; 25 cm × 4.6 mm).
Caco-2/TC7 cells and culture
The eukaryotic cells used in our study were the intestinal Caco-2/TC7 cell line, a late passage of Caco-2 (P-198), which has been previously recommended for its performance and reproducibility [
47]. Cells were routinely grown at 37 °C in 5% CO
2–95% air atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 15% heat-inactivated foetal calf serum, and Penicillin/Streptomycin. For experimental assays, the cells were seeded in 24-well tissue culture plates (for cytotoxicity assay and interleukine-8 (IL-8) quantification) or on insert (3 µm pore size) for 21 days to ensure epithelial differentiation (for transepithelial electrical resistance (TER) measurement and translocation assay). At the end of differentiation, the mean TEER value measured for control cells was approximately 700 Ω/cm
2.
Bacterial treatment with SP and infection of Caco-2/TC7 cells
For all the following experiments conducted on Caco-2/TC7 cells, only a dose of 10−6 M SP was used, which is more relevant to the physiological concentration in the gut. SP was added to the cultures of E. faecalis V583 at the beginning of the exponential growth phase. The bacteria were exposed for 2 h with the peptide, then centrifuged for 5 min at 8000g, washed and resuspended in cell culture medium without antibiotics and serum. Caco-2/TC7 cells were infected at a MOI (multiplicity of infection) of 100:1 with the bacteria treated or not with SP, and incubated for 15 h at 37 °C, CO2 5%, unless otherwise stated.
Cytotoxicity and quantification of IL-8
The cytotoxicity of E. faecalis V583 treated or not with SP was estimated by enumeration of the remaining Caco-2/TC7 viable cells, 4 and 24 h after infection, using the neutral red (NR) uptake assay. Only viable cells will take up NR by active transport and incorporate the dye into lysosomes. Following infection, Caco-2/TC7 cells were washed once in PBS and incubated with NR (50 µg/ml) for 1 h at 37 °C, CO2 5%. After incubation, the cells were rinsed once in PBS, and NR incorporated in viable cells was extracted with a solution of 50% (v/v) ethanol and 1% (v/v) acetic acid for 10 min before measuring optical density at 550 nm.
The level of IL-8 was quantified in the supernatant of Caco-2/TC7 cells, 14 h after infection, using the CXCL8 ELISA Quantikine kits (R&D systems) according to the manufacturer’s protocol.
Transepithelial electrical resistance (TER)
The effect of E. faecalis V583, treated or not with SP, on the TER of Caco-2/TC7 monolayers was measured at 0, 4, 18 and 24 h, using the Millicell Electrical Resistance System. For each condition tested, the TER values were expressed as percentages of the initial level measured in the insert.
Bacterial translocation
After 4 and 24 h infection of the Caco-2/TC7 inserts, aliquots of 100 µl of the basolateral compartment were collected and the number of bacteria that crossed the epithelial monolayers was determined by serial dilution and plating onto BHI agar.
Statistical analysis
All the assays were performed from at least three independent replicates. GRAPHPAD PRISM software and Student’s t test were used to compare the data statistically.
Authors’ contributions
MF, DD, PD and NC designed the study. KB, VB, SS, RB, AN, AZ, MBJ and GD carried out the experiments. NC wrote the manuscript. All authors read and approved the final manuscript.