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An in vitro assay of the effect of lysine oxidation end-product, α-aminoadipic acid, on the redox status and gene expression in probiotic Lactobacillus reuteri PL503

An in vitro assay of the effect of lysine oxidation end-product, α-aminoadipic acid, on the redox... This study was designed to gain information about the underlying mechanisms of the effects of a food-occurring free oxi- dized amino acid, α-aminoadipic acid (AAA), on the probiotic Lactobacillus reuteri PL503. This bacterium was incubated in colonic-simulated conditions (37 °C for 24 h in microaerophilic conditions) and exposed to three food-compatible AAA concentrations, namely, 1 mM, 5 mM, and 10 mM. A control group with no AAA exposure was also considered. Each of the four experimental conditions was replicated three times and samplings were collected at 12, 16, 20, and 24 h. The down- regulation of the uspA gene by AAA (0.5-fold decrease as compared to control) suggests that AAA is identified as a potential chemical threat. The dhaT gene, implicated in the antioxidant defense, was found to be upregulated in bacteria treated with 1 and 5 mM AAA (up to twofold increase, as compared to control), which suggest the ability of the oxidized amino acid to impair the redox status of the bacterium. In fact, AAA caused an increased production of reactive oxygen species (ROS) and the accretion of post-translational changes (protein carbonylation) in L. reuteri (up to 13 nmol allysine/mg protein vs 1.8 nmol allysine/mg protein in control). These results suggest that probiotic bacteria identify oxidized amino acids as harmful species and activate mechanisms that may protect themselves and the host against their noxious effects. Keywords Oxidized amino acids · Oxidative stress · Probiotic bacterium · Protein oxidation · Transcripts Abbreviations 1,3-PDO Propane-1,3-diol 3-HPA 3-Hydroxypropionaldehyde Handling editor: D. Tsikas. AAA α-Aminoadipic acid AAS α-Aminoadipic semialdehyde * Mario Estévez ANOVA Analyses of variance mariovet@unex.es DNA Deoxyribonucleic acid Patricia Padilla ABA Aminobenzoic acid patriciapt@unex.es DPS 4,4′-Dipyridyldisulphide María J. Andrade DTPA Diethylenetriaminepentaacetic acid mjandrad@unex.es FLD Fluorescence detector Fernando J. Peña FMO Fluorescence minus one fjuanpvega@unex.es HPLC High-performance liquid chromatography Alicia Rodríguez MDA Malondialdehyde aliciarj@unex.es MES 2-(N-morpholino)ethanesulfonic acid Food Technology, IPROCAR Research Institute, University MRS Man Rogosa and Sharpe of Extremadura, 10003 Cáceres, Spain NADH Nicotinamide adenine dinucleotide Faculty of Veterinary Science, IPROCAR Research Institute, PBS Phosphate buffered saline solution Food Hygiene and Safety, University of Extremadura, PCR Polymerase chain reaction 10003 Cáceres, Spain ROS Reactive oxygen species Laboratory of Equine Reproduction and Equine RNA Ribonucleic acid Spermatology, University of Extremadura, 10003 Cáceres, TBARS Thiobarbituric-reactive substances Spain Vol.:(0123456789) 1 3 664 P. Padilla et al. TCA Trichloroacetic acid molecular mechanisms implicated in the responses of this TEP Tetraethoxypropane probiotic bacterium under specific pro-oxidant conditions UspA U niversal stress protein A are not well understood. According to some previous reports, the expression of the uspA and dhaT genes in L. reuteri is affected by the Introduction oxidative threat caused by reactive oxygen species (ROS) (Arcanjo et al. 2019). Usp proteins seem to be implicated in Protein oxidation is a post-translational modification induced the defense against DNA-damaging agents while the dhaT by reactive oxygen species (ROS) and other pro-oxidative gene encodes for a propane-1,3-diol (1,3-PDO) oxidoreduc- compounds, and plays an essential role in the pathogen- tase, which is involved in the protection of L. reuteri against esis of relevant degenerative diseases (Davies 2005). The oxidative stress (Arcanjo et al. 2019). In a preceding study, oxidative damage to proteins leads to depletion of original we investigated the molecular responses of this bacterium to amino acids and the formation, in its place of specific oxida- a major lipid oxidation product, malondialdehyde (MDA), at tion products (Davies 2005). Chemical species such as the concentrations between 5 and 100 μM (Padilla et al. 2021). α-aminoadipic semialdehyde (AAS), also known as allysine, Yet, the underlying mechanisms of the potential impact of and its end-product, the α-aminoadipic acid (AAA), are gen- oxidized amino acids on probiotic bacteria are unknown. erated by the oxidation of lysine through metal-catalyzed The aim of this study was to understand the molecular reactions (Stadtman and Oliver 1991). While both species mechanisms activated in L. reuteri in response to the poten- occur as intermediates in lysine metabolism, the accretion of tial harmful effects of AAA. To fulfil this objective, the such species in biological samples, including food systems, redox status (ROS generation and lipid and protein oxidation respond to a radical-mediated oxidation mechanism (Davies markers), and the expression of the uspA and dhaT genes in 2005; Stadtman and Oliver 1991). The chemical struc- L. reuteri challenged by increasing concentrations of AAA, tures and formation mechanisms of these oxidized amino was investigated. acids can be found in detail elsewhere (Luna et al. 2021). AAA has been identified in meat products, such as raw and cooked patties, cooked sausages and fermented meats, at Materials and methods levels ranging 50–200 µM (Utrera and Estévez 2012; Utrera et al. 2012). Recently, Estévez and Xiong (2019) collected Chemicals and raw material information about the scientific evidences of the potential harmful effects of dietary oxidized proteins and amino Chemicals and reagents used in this study were of Ameri- acids. AAA, in particular, has been found to exert, at food- can Chemical Society analytical grade and purchased from compatible concentrations of AAA (200 µM), mitochon- Sigma Chemicals (Sigma–Aldrich, Germany), Scharlab drial disturbance, oxidative stress, apoptosis, and necrosis S.L. (Spain), Pronadisa (Conda Laboratory, Spain), Applied in human intestinal and mice pancreatic cells (Díaz-Velasco Biosystems (USA), Epicentre (USA), and Acros Organics et al. 2020; Estaras et al. 2020). (Spain). L. reuteri PL503 was isolated from pig faeces and Among the assorted pathophysiological conditions then identified using 16S rRNA gene sequencing by Ruiz- induced by the intake of oxidized proteins and amino acids, Moyano et al. (2008). the disturbance of the microbiota has been found in both in vitro (Arcanjo et al. 2019) and in vivo (Goethals et al. Experimental setting 2020) studies. The protective role of microbiota is gain- ing interest since luminal oxidative stress in humans can L. reuteri PL503 was stored at − 80 °C in Man Rogosa and be counteracted by microbiota (Spyropoulos et al. 2011). Sharpe (MRS) broth with 20% (v/v) glycerol. To prepare In this regard, Lactobacillus reuteri, a natural colonizer of the working cultures, L. reuteri PL503 was cultivated twice the gastrointestinal tract in humans and animals, has been at 37 °C for 24 h in MRS broth supplemented with 0.5% used as a dietary supplement to enhance human gut health acetic acid 10% (v/v). A volume of 100  µL of such cul- (Shornikova et al. 1997), and its oral administration reduces ture of L. reuteri PL503 was inoculated in tubes of 5 mL gastrointestinal disorders and infections and contributes to of MRS broth containing different concentrations of free a balanced colonic microbiota (Shornikova et  al. 1997). AAA. In particular, four groups were considered based on L. reuteri has been reported to protect against oxidative the added concentration of free AAA: Control (L. reuteri), stress and inhibits the accretion of oxidation products in the 1 mM (L. reuteri + 1 mM AAA), 5 mM (L. reuteri + 5 mM lumen, according to the mechanisms related to its probiotic AAA), and 10 mM (L. reuteri + 10 mM AAA). They were effects (Amaretti et al. 2013). While the benefits of L. reuteri incubated at 37 °C for up to 24 h in microaerophilic condi- against oxidative stress are documented (Petrella 2016), the tions to simulate physiological conditions in the colon. The 1 3 An in vitro assay of the effect of lysine oxidation end‑product, α‑aminoadipic acid, on the redox… 665 concentrations of free AAA are those expected to be found SYBR Premix Ex Taq™ (Takara Bio Inc.), 0.625 µL of in the colon after gastrointestinal digestion of a severely ROX™ Reference Dye (Takara Bio Inc.), and 300 nM of processed muscle food (Utrera and Estévez 2012; Utrera each primer pair (Table 1). The qPCR program consisted of et al. 2012; Goethals et al. 2020). For each treatment, three an initial denaturation step at 95 °C for 10 min; 40 cycles at replicates were performed. During the incubation period, a denaturation temperature of 95 °C for 15 s and annealing/ samples were taken at 12, 16, 20, and 24 h. For counting extension temperatures of 55 °C and 60 °C for the 16S and viable cells, 100 µL of L. reuteri PL503 of each treatment target genes, respectively, during 30 s. After the final qPCR and sampling time were inoculated on MRS agar at the same cycle, a melting curve was included by heating the product sampling time and conditions as the experimental tubes. For from 60 to 99 °C and continuous measurement of the fluo- protein analyses, to avoid possible contamination from the rescence was performed to verify the qPCR products. All culture medium, two washes with phosphate buffered saline samples were analyzed in triplicate, including control sam- solution (PBS, pH 7.4) were made. ples consisting of adding sterile ultrapure water instead of −ΔΔC cDNA. The expression ratio was calculated using the 2 Gene expression studies method reported by Livak and Schmittgen (2001). The cali- brator sample corresponded to the value of the expression RNA extraction of the experimental group Control at each sampling time. The RNA extraction of each experimental group and incuba- Analytical procedures tion time was performed using the MasterPure™ RNA puri- fication kit (Epicentre), which includes DNase treatment. Analysis of ROS by flow‑cytometry The obtained RNA was eluted in 35 μL TE buffer and kept at − 80 °C until further use. RNA quantity (ng/µL) and qual- Flow cytometry detection of ROS (e.g., hydroxyl and ity (A /A ratio) were spectrophotometrically determined superoxide radicals) in L. reuteri PL503 was performed as 260 280 using the Nanodrop 2000 (Thermo Scientific, USA). determined using protocols described by Díaz-Velasco et al. (2020) with some minor modifications. In brief, samples Reverse transcription reaction of L. reuteri PL503 (1 × 10  ufc/mL) of each experimen- tal group and incubation time, were extended in 1 mL of The cDNA was synthesized using about 500  ng of total PBS, and stained with CellRox Deep Red (5 μM; Ther- RNA, according to the PrimeScript™ RT Reagent kit moFisher, USA) (excitation and emission wavelengths, 644 (Takara Bio Inc., Japan). The cDNA was stored at − 20 °C and 645 nm, respectively) for detecting the bacterium pro- until being used for the PCR reactions. ducing ROS, and Hoechst 33,342 (0.5 μM; Sigma–Aldrich) (excitation and emission wavelengths, 345 nm and 488 nm, Real‑time PCR analysis of gene expression respectively) to identify the bacterium and remove debris from the analysis. After thorough mixing, the cell sus- The uspA and dhaT genes were selected for relative expres- pension was incubated at room temperature for 25 min in sion studies using real-time PCR (qPCR), being used the 16S the dark, washed in PBS and immediately run on the flow gene as reference gene. The amplification was performed in cytometer. The analyses were conducted using a Cytoflex MicroAmp optical 96-well plates sealed with optical adhe- flow cytometer (Beckman Coulter, USA) equipped with vio- sive covers (Applied Biosystems) on a ViiA™ 7 Real-Time let, blue, and red lasers. The instrument was daily calibrated System (Applied Biosystems) using the SYBR Green tech- using specific calibration beads provided by the manufac- nology. Each well contained 2.5 µL of cDNA, 6.25 µL of turer. A compensation overlap was performed before each Table 1 Sequences of primers used for reverse transcription real-time PCR assays to conduct gene expression analyses Primers Gene Nucleotide sequence (5′-3′) Annealing tem- References perature uspALr-F1 uspA CTT GGG TAG CGT TCA CCA TT 60 °C Arcanjo et al. (2019) uspALr-R1 TGA AAA AGC GGT TGA CAC TG 60 °C Arcanjo et al. (2019) LS67 dhaT TGA CTG GAT CCT AAT TTG GTC CTG GTG TTA TTGC 60 °C Schaefer et al. (2010) LS68 TGA CTG AAT TCT TCC GGA TCT TAG GGT TAG G 60 °C Schaefer et al. (2010) Lr16S_F 16S rRNA CCG CTT AAA CTC TGT TGT TG 55 °C Arcanjo et al. (2019) Lr16S_R CGT GAC TTT CTG GTT GGA TA 55 °C Arcanjo et al. (2019) 1 3 666 P. Padilla et al. experiment; however, due to emission and excitation char- the pellet was treated with 6 M HCl and kept in an oven at acteristics of the combination of the used probes, spectral 110 °C for 18 h until completion of hydrolysis. The hydro- overlap was negligible. Files were exported as FCS files and lysates were dried in vacuo in a centrifugal evaporator. The analyzed using FlowJoV 10.5.3 Software for Mac OS (Ash- generated residue was reconstituted with 200 µL of milliQ land, USA). Unstained, single-stained, and Fluorescence water and then filtered through hydrophilic polypropylene Minus One (FMO) controls were used to determine com- GH Polypro (GHP) syringe filters (0.45  μm pore size, Pall pensations and positive and negative events, as well as to Corporation, USA) for HPLC analysis. set regions of interest. A Shimadzu ‘Prominence’ HPLC apparatus (Shimadzu Corporation, Japan), equipped with a quaternary solvent Synthesis of allysine standard compound delivery system (LC-20AD), a DGU-20AS on-line degasser, a SIL-20A auto-sampler, a RF-10A XL fluorescence detec- N-Acetyl-L-AAS (allysine) was synthesized from tor (FLD), and a CBM-20A system controller, was used. An Nα-acetyl-L-lysine using lysyl oxidase activity from egg aliquot (1 μL) from the reconstituted protein hydrolysates shell membrane following the procedure described by Aka- was injected and analyzed in the HPLC-FLD equipment. gawa et al. (2002). Briefly, 10 mM Nα -acetyl-L-lysine was Allysine–ABA was eluted in a Cosmosil 5C -AR-II RP- incubated at constant stirring with 5 g of egg shell mem- HPLC column (5 µm, 150 × 4.6 mm) equipped with a guard brane in 50 mL of 20 mM sodium phosphate buffer, pH 9.0 column (10 × 4.6 mm) packed with the same material (Phe- at 37 °C for 24 h. The egg shell membrane was then removed nomenex, PA, USA). The flow rate was kept at 1 mL/min by centrifugation and the pH of the solution adjusted to 6.0 and the temperature of the column was maintained constant using 1 M HCl. The resulting aldehydes were reductively at 30  °C. The eluate was monitored with excitation and aminated with 3 mM 4-aminobenzoic acid (ABA) in the emission wavelengths set at 283 and 350 nm, respectively. presence of 4.5 mM sodium cyanoborohydride (NaBH CN) Standards (0.1 μL) were run and analyzed under the same at 37 °C for 2 h with stirring. ABA derivatives were then conditions. Identification of both derivatized semialdehydes hydrolyzed by 50 mL of 12 M HCl at 110 °C for 10 h. The in the chromatograms was carried out by comparing their hydrolysates were evaporated at 40 °C in vacuo to dryness. retention times with those from the standard compounds. The resulting allysine–ABA was purified using silica gel The peak corresponding to allysine–ABA was manually column chromatography and ethyl acetate/acetic acid/water integrated from the FLD chromatograms and the resulting (20:2:1, v/v/v) as elution solvent. The purity of the resulting areas plotted against an ABA standard curve with known solution (70%) and authenticity of the standard compounds concentrations that ranged from 0.1 to 0.5 mM (Utrera et al. obtained following the aforementioned procedures were 2011). Results were expressed as nmol of allysine per mg checked using MS and H NMR (Estévez et al. 2009). of protein. Quantification of allysine Analysis of Schiff bases Allysine was quantified in bacterial protein as a marker of The formation of Schiff bases in bacterial protein was oxidation-induced post-translational modification, according assessed in each experimental group and incubation time to the method described by Utrera et al. (2011). Five hun- by fluorescence spectroscopy. Prior to the analysis, reac - dred μL of each experimental group and incubation time of tion mixtures were diluted (1:20) with 8 M urea in 100 mM culture were dispensed in 2 mL microtubes and treated with sodium phosphate buffer, pH 7. Diluted samples were dis- cold 10% (v/v) trichloroacetic acid (TCA) solution. Each pensed in spectrofluorometric cuvettes and excited at 350 nm microtube was vortexed and then subjected to centrifugation using a LS-55 Perkin–Elmer fluorescence spectrometer at 600 × g for 5 min at 4 °C. The supernatants were removed, (PerkinElmer, UK). The fluorescence emitted by Schiff and the pellets were incubated with the following freshly bases was recorded at 450 nm. The excitation and emission prepared solutions: 0.5  mL 250  mM 2-(N-morpholino) slit widths were set at 10 nm and the speed of data collection ethanesulfonic acid (MES) buffer pH 6.0 containing 1 mM while scanning was of 500 nm per min. The height of the diethylenetriaminepentaacetic acid (DTPA), 0.5 mL 50 mM peaks corresponding to Schiff bases spectra was recorded. ABA in 250 mM MES buffer pH 6.0 and 0.25 mL 100 mM After taking into consideration the applied dilutions, the NaBH CN in 250 mM MES buffer pH 6.0. After vortexing, results were expressed as fluorescence units. the tubes were incubated in a water bath at 37 °C for 90 min. The samples were stirred every 15 min. The samples were Analysis of protein thiols then treated with a cold 50% TCA (v/v) solution and cen- trifuged at 1200 × g for 10 min. The pellets were washed Thiols from sulfur-containing amino acids in bacterial pro- twice with 10% TCA and diethyl ether-ethanol (1:1). Finally, teins were quantified in accordance to the method reported 1 3 An in vitro assay of the effect of lysine oxidation end‑product, α‑aminoadipic acid, on the redox… 667 by Rysman et al. (2014). A volume of 250 µL of each L. a standard curve with tetraethoxypropane (TEP). The results reuteri PL503 experimental group and incubation time, was are expressed as mg TBARS per L of sample. washed twice with PBS and ethanol:ethyl acetate (1:1) to avoid possible contamination with thiols from the medium. Statistical analysis Upon centrifugation (600 × g/5 min), the pellet was resus- pended in 250 µL of guanidine hydrochloride, treated with True replicates (n = 3) were subjected to duplicate analyses 250 µL of 4,4′-dipyridyldisulphide (DPS) in 12 mM HCl and and data were collected and subjected to statistical analysis. dispensed in a spectrophotometric cuvette. Absorbance was Earlier, the data were analyzed for normality (Shapiro–Wilk measured at 324 nm against a blank sample in which DPS test) and homoscedasticity (Bartlett test). The effects of was replaced with an equivalent volume of guanidine hydro- AAA concentration and incubation times were studied using chloride. Quantification was made by preparing a standard analyses of variance (ANOVA) (SPSS v. 15.5). The effect of curve with cysteine. The results were expressed as µmol of AAA on the gene expression (ΔΔC values) was analyzed free thiol groups per mg of protein. using the paired Students’ t test (SPSS v. 15.5). The statisti- cal significance was set at p ≤ 0.05. Analysis of thiobarbituric‑reactive substances Results The quantification of MDA and other thiobarbituric-reactive substances (TBARS) in all experimental groups and incuba- Relative expression of the uspA gene tion times, was made in accordance to the method described by Ganhao et al. (2011). An aliquot of 200 µL of L. reu- The relative expression of the L. reuteri PL503 uspA gene teri PL503 experimental group was treated with 500 µL of during the incubation assay in the presence of different thiobarbituric acid (0.02 M) and 500 µL of TCA (10%) and concentrations of AAA is shown in Fig. 1a. A significant incubated during 20 min at 90 °C. After cooling, a 5 min downregulation of the uspA gene was particularly observed centrifugation at 600 × g was made and the supernatant was at 12 h of incubation in the presence of 5 and 10 mM of measured at 532 nm. Quantification was made by preparing AAA (0.22- and 0.40-fold decrease, respectively) as well Fig. 1 Relative expression (a) ΔΔC (2- ) of the uspA (a) and dhaT (b) genes in Lactobacil- lus reuteri PL503 grown in the presence of increasing concen- trations (0, 1, 5, and 10 mM) of α-aminoadipic acid (AAA) for up to 24 h. Black line at ΔΔC 2- = 1 denotes standardized expression rate for CONTROL group (0 mM) at each sampling ΔΔC time (calibrator). 2- < 1 denotes suppression of the expression of the target gene; ΔΔC 2- > 1 denotes activation of the expression of the target gene. Asterisks on top of bars (b) denote significant differences between such treatment and the control within a particu- lar sampling time (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) 1 3 668 P. Padilla et al. as at 16 h sampling with 10 mM (0.38-fold decrease). An TBARS/L vs. 1.10 mg TBARS/L). At 24 h, the concentra- upregulation of the gene (1.43-fold increase) was observed tion of TBARS in control samples (1.17 mg TBARS/L) was at 24 h when the bacterium was exposed to the highest AAA significantly higher than in the bacterium challenged with concentration. AAA (ranging from 1.05 to 1.10 mg TBARS/L). Relative expression of the dhaT gene Quantification of allysine The relative expression of the L. reuteri PL503 dhaT gene The changes of the concentration of allysine in L. reuteri during the incubation assay in the presence of different PL503 during the incubation period is shown in Fig.  3b. concentrations of AAA is shown in Fig. 1b. A significant Compared to control, the exposure to AAA caused a sig- downregulation of the gene was found at 12 h in the pres- nificant increase in the concentration of allysine in proteins ence of 5 mM (0.42-fold decrease) and at 16 h in the pres- from L. reuteri PL503 for 20 h. At that sampling point, the ence of both 1 mM and 10 mM of AAA (0.75- and 0.81- concentration of allysine in control samples (1.8 nmol/mg fold decreases, respectively). Nonetheless, in the two final protein) was significantly lower than in those treated with sampling times, an upregulation of the relative transcription 1, 5, and 10 mM AAA (11.7, 10.4, and 8.8 nmol/mg pro- of the dhaT gene was observed. In particular, significant tein, respectively). At 24 h sampling, the behavior varied changes were found in the presence of 5 mM of AAA at 20 h between groups. In L. reuteri challenged with 1 and 5 mM (1.98-fold increase) and 1 mM of AAA at 24 h (1.83-fold of AAA, the increase of allysine was constant during the increase). complete assay reaching the highest concentration at 24 h (12.0 and 13.5 nmol/mg protein, respectively). On the other ROS generation by flow‑cytometry analyses hand, when the bacterium was exposed to the highest AAA concentration (10 mM) allysine peaked at 20 h, after which The incubation of L. reuteri PL503 in the presence of AAA a decrease was observed at the end of the incubation period led to an increased production of ROS as shown in Fig. 2. (4.2 nmol/mg protein). The analysis of the samples with flow-cytometry showed a clear dose effect. At increasing concentrations of AAA, the Analysis of Schiff bases percentage of bacterium suffering oxidative stress at 24 h rise from 0.8% in control group to 1.8%, 2.1%, and 5.3% In the present study, the formation of Schiff bases is shown in bacteria exposed to 1, 5 and 10 mM AAA, respectively. in Fig. 3c and a clear dose effect of AAA was observed. No Specially, at the two final sampling times, the differences significant differences were found between AAA concentra- between groups were found to be higher than in the previ- tions during the first three sampling times. Nevertheless, ous ones. at the final sampling time (24 h) an increase was observed when the bacterium was exposed to the highest concentra- Analysis of thiobarbituric‑reactive substances tion (10 mM), which is coincident with carbonyls deple- tion found in the same group of samples at the end of the In Fig. 3a, the TBARS concentration in L. reuteri PL503 assay. At 24 h, the relative concentration of Schiff bases in during the assay is shown. In the presence of AAA, sig- L. reuteri followed the increasing order: control group (52 nificant changes occur at 20 h with 10 mM of AAA lower - fluorescent units) and bacterium exposed to 1, 5 and 10 mM ing TBARS content compared to control samples (0.89 mg AAA (80, 98, and 185 fluorescent units). Fig. 2 Percentage of Lactoba- cillus reuteri PL503 suffering from oxidative stress (positive to Cell Rox dye) when grown in the presence of increasing con- centrations (0, 1, 5 and 10 mM) of α-aminoadipic acid (AAA) for up to 24 h. Different letters on top of bars denote significant differences (p ≤ 0.05) between a AAA concentrations within the same sampling time c AAA concentra on (mM) 01 5100 15 10 01 5100 15 10 Incuba on me (h) 12 16 20 24 1 3 % C+ e l Rox An in vitro assay of the effect of lysine oxidation end‑product, α‑aminoadipic acid, on the redox… 669 (a) mM mM (b) Fig. 4 Concentration of free thiols (means ± standard deviation) in mM Lactobacillus reuteri PL503 grown in MRS broth with increasing concentrations (0, 1, 5, and 10  mM) of α-aminoadipic acid (AAA) during an incubation period for up to 24  h. Different letters at the same sampling time denote significant differences between AAA con- centrations (p ≤ 0.05) 24 h concentrations of 13.3 μmol/mg protein and 11.8 μmol/ mg protein, respectively. Conversely, the concentration of thiols in bacteria exposed to the highest AAA concentra- tion (10 mM) significantly decreased from the first sampling (10 μmol/mg protein) until the end of the assay (7.9 μmol/ (c) mg protein). mM Discussion Regulation of the uspA and dhaT genes by L. reuteri in response to AAA L. reuteri counts remained stable with the increasing applied doses of AAA (1 mM, 5 mM, and 10 mM) during the entire experimental assay (37 °C/24 h), so the survival was not jeopardized (data not shown). Yet, the challenge with this oxidized amino acid led to impairments of the bacterium’s Fig. 3 Concentration of thiobarbituric-reactive substances (TBARS) (a), allysine (b) and Schiff bases (c) (means ± standard deviation) in physiology. This finding reflects the ability of L. reuteri to Lactobacillus reuteri PL503 grown in MRS broth in the presence of activate mechanisms to neutralize the potential harmful increasing concentrations (0, 1, 5 and 10 mM) of α-aminoadipic acid effects of the sub-lethal concentrations of the added oxidized (AAA) during an incubation period for up to 24 h. Different letters at amino acid. In the present study, these mechanisms were the same sampling time denote significant differences between AAA concentrations (p ≤ 0.05) firstly assessed by the analysis of the relative expression of stress-related genes. The universal stress protein A (UspA) superfamily includes an ancient and conserved group of proteins found Analysis of protein thiols in assorted microorganisms, insects, and plants. The precise roles of Usp proteins in biological systems remain unclear; The concentration of free thiols in proteins from L. reu- teri PL503 during the incubation assay is shown in Fig. 4. yet, they seem to be involved in the defense against DNA- damaging agents and respiratory uncouplers (Kvint et al. Significant differences were observed in the first 12  h between the control group and the bacterium challenged 2003). Due to the defined function of the gene uspA, an upregulation was expected, which was only observed at with increasing AAA concentrations. From 16 h sampling time onwards, a significant increase of free thiols in samples 24 h and in the presence of the highest AAA concentra- tion (Fig. 1a). Yet, the downregulation observed at earlier exposed to 1 and 5 mM of AAA was detected, peaking at 1 3 670 P. Padilla et al. samplings and lower concentrations is consistent with data elemental mechanisms by which L. reuteri may seek to pro- reported by Oberg et al. (2015), who found a significant tect against AAA-induced biological damage through the downregulation of the uspA gene expression in Bifidobacte- activation of the 3-HPA pathway should be subjected to rium longum exposed to a hydroxyl-radical generating sys- scrutiny. As previously reported by Talarico et al. (1988), tem. Similar results were reported by Arcanjo et al. (2019) the 3-HPA pathway requires glycerol, commonly added working on the same bacterium and strain from the present as growth promoter in Lactobacillus cultures. In the pre- study. In that study, exposing L. reuteri to 0.5 mM of hydro- sent study, L. reuteri had no access to such precursor, and, gen peroxide led to a significant decrease of the uspA gene therefore, the 3-HPA pathway is unlikely to have occurred. expression. It is worth noting that both aforementioned stud- Considering the absence of glycerol, it seems reasonable to ies found the occurrence of oxidative stress and molecular consider that 1,3-PDO may have other substrates and that damage in the exposed bacteria. The fact that AAA exposure its cellular activity may be related to protection against a led to a similar effect on L. reuteri indicates that this oxi- potential pro-oxidative threat. To similar conclusions came dized amino acid is identified by the bacterium as a chemical Arcanjo et al. (2019) who found an increased expression threat. In fact, two recent studies agree in describing noxious of the dhaT gene in L. reuteri challenged with hydrogen effects of food-compatible AAA concentrations (200  μM) peroxide in simulated colonic conditions where glycerol on human intestinal (Díaz-Velasco et al. 2020) and human was, again, absent. The authors hypothesized whether the acinar pancreatic cells (Estaras et al. 2020). According to NAD + -dependent activity of the 1,3-PDO may be able these authors, the harmful effect of AAA involved the induc- to detoxify hydrogen peroxide in the presence of NADH. tion of pro-oxidative conditions within cells. Probiotic bac- Since no hydrogen peroxide was included in the present teria like L. reuteri may also be susceptible to this chemical assay, the implication of 1,3-PDO in balancing the redox species and, according to these results, the downregulation state of the cell seems to be a pertinent defense mechanism of the uspA gene seems to be related to a cellular signal of a against pro-oxidative threats. It is, still unknown how AAA pro-oxidative threat that both, the radical generating systems may impair the redox status of L reuteri but it is proven that (i.e., hydrogen peroxide) and oxidized amino acids such as AAA exposure to human eukaryotic cells cause oxidative AAA, may be able to induce. stress via mitochondrial disturbance and ROS generation It is worth clarifying that the higher AAA concentrations (Díaz-Velasco et al. 2020; Estaras et al. 2020). tested in the present study (1–10 mM) are plausibly compat- It is worth noting that the effect of AAA exposure on the ible with a physiological situation as explained as follows. expression of the dhaT gene at early stages of the assay (12 While AAA concentration in foods has been found to reach and 16 h) was opposite to that observed at advanced stages. up to 200 μM, it is also known that dietary proteins are fur- As discussed in due course, the activation of the gene at ther oxidized during digestion, increasing significantly the advanced stages of oxidative stress and oxidative damage final concentration of oxidized amino acids in the gut. For could have triggered defense mechanisms, in which the dhaT instance, in a study by Van-Hecke et al. (2019), the concen- gene may be implicated. At early stages, the underexpres- tration of protein oxidation products increased between 2 sion of this gene could respond to indefinite initial responses and fivefold times in assorted foods after simulated gastro- of the bacteria to the AAA exposure, in which the protein intestinal digestion. The same authors found in a more recent encoded by this gene was not found as essential. In line study (Goethals et al. 2020) sixfold times higher concentra- with this downregulation, a recent study by Díaz-Velasco tions of protein oxidation products in pork digests than in et al. (unpublished data) observed that AAA exposure to the original (undigested) pork product. CACO-2 cells led to an overall downregulation of gene The dhaT gene encodes the enzyme 1,3-PDO oxidore- expression due to the impairment of protein kinase A and C ductase which is known to play a relevant role in stressful (PKA and PKC, respectively) signaling pathways. Yet, the situations involving energetic demand. This enzyme enables mechanisms implicated in the downregulation of dhaT gene the main carbohydrate fermentation pathway (6-phosphoglu- at early stages of exposure to AAA in this bacterium remain conate/phosphoketolase; 6-PG/PK) through the production indefinite and require further elucidation. of NAD (required for glucose fermentation) from NADH in the conversion of 3-hydroxypropionaldehyde (3-HPA) ROS generation in L. reuteri by AAA (its substrate) into 1,3-PDO under anaerobic conditions. Additionally, 3-HPA, also known as reuterin, is excreted by The increased production of ROS in L. reuteri by the pres- L. reuteri strains under stressful situations (Schaefer et al. ence of AAA has no precedent in literature (Fig. 2). It is, 2010). The overexpression of this gene observed in bacteria however, consistent with results reported by Díaz-Velasco exposed to 5 and 1 mM AAA for 20 and 24 h, respectively, et al. (2020) in CACO-2 cells and Estaras et al. (2020) in could respond to an attempt of the bacteria to protect against pancreatic cells when the exposure to AAA led to impair- the oxidative threat caused by this oxidized amino acid. The ment of the oxidative status of the cell, ROS generation, 1 3 An in vitro assay of the effect of lysine oxidation end‑product, α‑aminoadipic acid, on the redox… 671 apoptosis, and necrosis. In addition, it is in accordance to also react with amino groups from neighboring amino acids Da Silva et al. (2017) who studied the effect of AAA on (e.g., lysine) to form an azomethine structure, also known as brain function of adolescent rats, and showed an induction Schiff bases (Estévez 2011). The dramatic drop of allysine of ROS generation and alteration of the cellular redox sta- concentration during the last 4 h of the assay in the bacte- tus via mitochondrial impairment. While the percentage rium exposed to the highest concentration of AAA (10 mM) of CelRox positive bacteria was found to be relatively low, is consistent with the sudden increase of Schiff bases in that previous studies using hydrogen peroxide and malondial- period of time (Fig. 3c). These results suggest that such fluo- dehyde (MDA) as inductors of oxidative stress in L. reuteri rescent structures were, at least, partially formed in bacteria reported similar percentages (Arcanjo et al. 2019; Padilla exposed to 10 mM as a result of allysine addition to other et al. 2021). The oxidative damage caused in bacterial lipids protein amines. The formation of Schiff bases in bacteria and proteins, explained in due course, denote severe oxida- exposed to intermediate AAA doses (1 and 5 mM), was not tive stress. The precise mechanisms by which AAA is able to so intense to reflect a decline of the reactant (allysine). Both, induce ROS generation in L. reuteri are indefinite. It is worth carbonylation and formation of non-reducible protein cross- noting that such mechanisms differ from those reported by links (i.e., Schiff bases), are irreversible protein modifica- the aforementioned authors since the bacterium lacks mito- tions with negative biological consequences (Davies 2005; chondria. Interestingly, Lactobacillus spp. have also been Ezraty et al. 2017; Estévez and Xiong 2019). Carbonylated found to be able to produce hydrogen peroxide and other proteins can be dysfunctional and may be labeled to removal ROS via implication of NAD(P)H oxidoreductases (Hertz- due to its accumulation causes impaired homeostasis that berger et al. 2014) which provides a plausible and coherent leads to chronic dysfunction and apoptosis (Shacter 2003). connection between AAA exposure, dhaT overexpression However, carbonylated proteins can also act as signaling and ROS generation. The molecular mechanisms underlying molecules, which may activate specific pathways, to pre- the interconnection between all these elements need to be serve homeostasis control senescence (Shacter 2003). precisely described. Both situations could be applied to the present experi- ment. The increase in carbonyls above 10 nmol/mg proteins Oxidative damage to L. reuteri by AAA in the bacterium challenged with 5 and 1 mM of AAA was coincident with the activation of the dhaT gene at sampling In the present work, the oxidative damage to bacterium times of 20 h and 24 h, respectively, and plausibly, the cor- caused by AAA-induced oxidative stress was assessed by responding synthesis of the NADH-dependent oxidoreduc- means of TBARS (lipid oxidation) and allysine (protein oxi- tase decoded by this gene. Given the proposed role of this dation). The basal TBARS concentration in control cultures, enzyme in detoxifying pro-oxidant species (Arcanjo et al. (~ 1 mg/L) may correspond to the occurrence of lipid peroxi- 2019), a relatively mild pro-oxidative threat, exhibited in a dation in the bacterium under physiological conditions and significant accretion of protein carbonyls, could have led to did not change significantly during the assay within groups the activation of an antioxidant response mediated, among (Fig. 3a). AAA did not significantly affect the extent of lipid others, by the activation of the dhaT gene. On the other hand, oxidation in L. reuteri. a severe oxidative damage caused by a more intense pro- On the other hand, AAA exposure had a significant oxidative environment, such as that observed in L. reuteri impact on the oxidative damage to bacterial proteins. A challenged with the highest concentration of AAA (10 mM) relatively low but significant increase in allysine, the main led to a sudden formation of advanced oxidation products protein carbonyl in biological systems (Stadtman and Levine (Schiff bases) and no dhaT gene-mediated response against 2000; Estévez and Luna 2016), was observed in the control the oxidative insult. These mechanisms were not present in group of L. reuteri (Fig. 3b). The present results show that the bacterium incubated with the lowest doses of AAA (1 allysine, formed in bacteria, as in eukaryotes, remarkably and 5 mM). Previous considerations made by Ezraty et al. contributes to protein carbonylation and may be used as a (2017) and Arcanjo et al. (2019) support the hypothesis that reliable indicator of oxidative stress. The results obtained are the dhaT gene could have been activated by pro-oxidant spe- in accordance with Ezraty et al. (2017) who proposed that cies and/or the effect of the former on protein carbonylation. protein carbonylation could be a reflection of bacterial senes- The evolution of protein thiols during the assay (Fig. 4) cence as oxidized proteins accumulate in non-proliferating provides additional strength to the aforementioned hypoth- bacteria. Allysine is typically formed in proteins because of eses. The oxidation of sulfur-containing amino acids, such the attack of ROS to lysine residues. This is plausibly the as cysteine (Cys) and methionine (Met), is a typical feature mechanism taking place in the present assay as the signifi- in biological systems attacked by ROS (Estévez et al. 2020). cant production of ROS in L. reuteri exposed to AAA expo- While the oxidation of thiols in proteins may lead to dys- sure could have caused the oxidation of lysine residues and function, irrelevant sulfur-containing amino acids are known hence, the accretion of allysine. Once formed, allysine may to act as antioxidants offering a sacrificial loss to ROS and 1 3 672 P. Padilla et al. the project AGL2017-84586R as well as by the Government of Extrem- protecting other amino acids with relevant significance, such adura and FEDER (grants GR18056 and GR15108). P. Padilla was as lysine (Davies, 2005; Estévez et al. 2020). This dual role employed through the contract PEJ2014-P-0057. of thiols was examined in the present experiment. Taking into account that these moieties can act as redox-active com- Declarations pounds and elements of antioxidant protection in biological systems, the coincidence of thiol accretion with the increase Conflict of interest The authors declare no conflict of interest. of carbonylation in those samples may respond to a strat- egy to keep a balanced redox status in cells in danger. The Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- incubation of L. reuteri with AAA caused an increase of tion, distribution and reproduction in any medium or format, as long thiol concentration since 12 h incubation onwards. The pro- as you give appropriate credit to the original author(s) and the source, oxidant changes induced by AAA, including the formation provide a link to the Creative Commons licence, and indicate if changes of protein carbonyls, possibly triggered the accumulation were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated of thiol groups by the novo synthesis of sulfur-containing otherwise in a credit line to the material. If material is not included in proteins/peptides with the purpose of protecting the bac- the article's Creative Commons licence and your intended use is not terium against this pro-oxidant threat. Thiol accumulation permitted by statutory regulation or exceeds the permitted use, you will is considered as an endogenous mechanism of antioxidant need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . defense owing to the recognized redox-active properties (Davies 2005). These moieties have been typically regarded as elements of antioxidant protection in eukaryotes and in lactic acid bacteria (Schaefer et al. 2010; Xiao et al. 2011). 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An in vitro assay of the effect of lysine oxidation end-product, α-aminoadipic acid, on the redox status and gene expression in probiotic Lactobacillus reuteri PL503

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Abstract

This study was designed to gain information about the underlying mechanisms of the effects of a food-occurring free oxi- dized amino acid, α-aminoadipic acid (AAA), on the probiotic Lactobacillus reuteri PL503. This bacterium was incubated in colonic-simulated conditions (37 °C for 24 h in microaerophilic conditions) and exposed to three food-compatible AAA concentrations, namely, 1 mM, 5 mM, and 10 mM. A control group with no AAA exposure was also considered. Each of the four experimental conditions was replicated three times and samplings were collected at 12, 16, 20, and 24 h. The down- regulation of the uspA gene by AAA (0.5-fold decrease as compared to control) suggests that AAA is identified as a potential chemical threat. The dhaT gene, implicated in the antioxidant defense, was found to be upregulated in bacteria treated with 1 and 5 mM AAA (up to twofold increase, as compared to control), which suggest the ability of the oxidized amino acid to impair the redox status of the bacterium. In fact, AAA caused an increased production of reactive oxygen species (ROS) and the accretion of post-translational changes (protein carbonylation) in L. reuteri (up to 13 nmol allysine/mg protein vs 1.8 nmol allysine/mg protein in control). These results suggest that probiotic bacteria identify oxidized amino acids as harmful species and activate mechanisms that may protect themselves and the host against their noxious effects. Keywords Oxidized amino acids · Oxidative stress · Probiotic bacterium · Protein oxidation · Transcripts Abbreviations 1,3-PDO Propane-1,3-diol 3-HPA 3-Hydroxypropionaldehyde Handling editor: D. Tsikas. AAA α-Aminoadipic acid AAS α-Aminoadipic semialdehyde * Mario Estévez ANOVA Analyses of variance mariovet@unex.es DNA Deoxyribonucleic acid Patricia Padilla ABA Aminobenzoic acid patriciapt@unex.es DPS 4,4′-Dipyridyldisulphide María J. Andrade DTPA Diethylenetriaminepentaacetic acid mjandrad@unex.es FLD Fluorescence detector Fernando J. Peña FMO Fluorescence minus one fjuanpvega@unex.es HPLC High-performance liquid chromatography Alicia Rodríguez MDA Malondialdehyde aliciarj@unex.es MES 2-(N-morpholino)ethanesulfonic acid Food Technology, IPROCAR Research Institute, University MRS Man Rogosa and Sharpe of Extremadura, 10003 Cáceres, Spain NADH Nicotinamide adenine dinucleotide Faculty of Veterinary Science, IPROCAR Research Institute, PBS Phosphate buffered saline solution Food Hygiene and Safety, University of Extremadura, PCR Polymerase chain reaction 10003 Cáceres, Spain ROS Reactive oxygen species Laboratory of Equine Reproduction and Equine RNA Ribonucleic acid Spermatology, University of Extremadura, 10003 Cáceres, TBARS Thiobarbituric-reactive substances Spain Vol.:(0123456789) 1 3 664 P. Padilla et al. TCA Trichloroacetic acid molecular mechanisms implicated in the responses of this TEP Tetraethoxypropane probiotic bacterium under specific pro-oxidant conditions UspA U niversal stress protein A are not well understood. According to some previous reports, the expression of the uspA and dhaT genes in L. reuteri is affected by the Introduction oxidative threat caused by reactive oxygen species (ROS) (Arcanjo et al. 2019). Usp proteins seem to be implicated in Protein oxidation is a post-translational modification induced the defense against DNA-damaging agents while the dhaT by reactive oxygen species (ROS) and other pro-oxidative gene encodes for a propane-1,3-diol (1,3-PDO) oxidoreduc- compounds, and plays an essential role in the pathogen- tase, which is involved in the protection of L. reuteri against esis of relevant degenerative diseases (Davies 2005). The oxidative stress (Arcanjo et al. 2019). In a preceding study, oxidative damage to proteins leads to depletion of original we investigated the molecular responses of this bacterium to amino acids and the formation, in its place of specific oxida- a major lipid oxidation product, malondialdehyde (MDA), at tion products (Davies 2005). Chemical species such as the concentrations between 5 and 100 μM (Padilla et al. 2021). α-aminoadipic semialdehyde (AAS), also known as allysine, Yet, the underlying mechanisms of the potential impact of and its end-product, the α-aminoadipic acid (AAA), are gen- oxidized amino acids on probiotic bacteria are unknown. erated by the oxidation of lysine through metal-catalyzed The aim of this study was to understand the molecular reactions (Stadtman and Oliver 1991). While both species mechanisms activated in L. reuteri in response to the poten- occur as intermediates in lysine metabolism, the accretion of tial harmful effects of AAA. To fulfil this objective, the such species in biological samples, including food systems, redox status (ROS generation and lipid and protein oxidation respond to a radical-mediated oxidation mechanism (Davies markers), and the expression of the uspA and dhaT genes in 2005; Stadtman and Oliver 1991). The chemical struc- L. reuteri challenged by increasing concentrations of AAA, tures and formation mechanisms of these oxidized amino was investigated. acids can be found in detail elsewhere (Luna et al. 2021). AAA has been identified in meat products, such as raw and cooked patties, cooked sausages and fermented meats, at Materials and methods levels ranging 50–200 µM (Utrera and Estévez 2012; Utrera et al. 2012). Recently, Estévez and Xiong (2019) collected Chemicals and raw material information about the scientific evidences of the potential harmful effects of dietary oxidized proteins and amino Chemicals and reagents used in this study were of Ameri- acids. AAA, in particular, has been found to exert, at food- can Chemical Society analytical grade and purchased from compatible concentrations of AAA (200 µM), mitochon- Sigma Chemicals (Sigma–Aldrich, Germany), Scharlab drial disturbance, oxidative stress, apoptosis, and necrosis S.L. (Spain), Pronadisa (Conda Laboratory, Spain), Applied in human intestinal and mice pancreatic cells (Díaz-Velasco Biosystems (USA), Epicentre (USA), and Acros Organics et al. 2020; Estaras et al. 2020). (Spain). L. reuteri PL503 was isolated from pig faeces and Among the assorted pathophysiological conditions then identified using 16S rRNA gene sequencing by Ruiz- induced by the intake of oxidized proteins and amino acids, Moyano et al. (2008). the disturbance of the microbiota has been found in both in vitro (Arcanjo et al. 2019) and in vivo (Goethals et al. Experimental setting 2020) studies. The protective role of microbiota is gain- ing interest since luminal oxidative stress in humans can L. reuteri PL503 was stored at − 80 °C in Man Rogosa and be counteracted by microbiota (Spyropoulos et al. 2011). Sharpe (MRS) broth with 20% (v/v) glycerol. To prepare In this regard, Lactobacillus reuteri, a natural colonizer of the working cultures, L. reuteri PL503 was cultivated twice the gastrointestinal tract in humans and animals, has been at 37 °C for 24 h in MRS broth supplemented with 0.5% used as a dietary supplement to enhance human gut health acetic acid 10% (v/v). A volume of 100  µL of such cul- (Shornikova et al. 1997), and its oral administration reduces ture of L. reuteri PL503 was inoculated in tubes of 5 mL gastrointestinal disorders and infections and contributes to of MRS broth containing different concentrations of free a balanced colonic microbiota (Shornikova et  al. 1997). AAA. In particular, four groups were considered based on L. reuteri has been reported to protect against oxidative the added concentration of free AAA: Control (L. reuteri), stress and inhibits the accretion of oxidation products in the 1 mM (L. reuteri + 1 mM AAA), 5 mM (L. reuteri + 5 mM lumen, according to the mechanisms related to its probiotic AAA), and 10 mM (L. reuteri + 10 mM AAA). They were effects (Amaretti et al. 2013). While the benefits of L. reuteri incubated at 37 °C for up to 24 h in microaerophilic condi- against oxidative stress are documented (Petrella 2016), the tions to simulate physiological conditions in the colon. The 1 3 An in vitro assay of the effect of lysine oxidation end‑product, α‑aminoadipic acid, on the redox… 665 concentrations of free AAA are those expected to be found SYBR Premix Ex Taq™ (Takara Bio Inc.), 0.625 µL of in the colon after gastrointestinal digestion of a severely ROX™ Reference Dye (Takara Bio Inc.), and 300 nM of processed muscle food (Utrera and Estévez 2012; Utrera each primer pair (Table 1). The qPCR program consisted of et al. 2012; Goethals et al. 2020). For each treatment, three an initial denaturation step at 95 °C for 10 min; 40 cycles at replicates were performed. During the incubation period, a denaturation temperature of 95 °C for 15 s and annealing/ samples were taken at 12, 16, 20, and 24 h. For counting extension temperatures of 55 °C and 60 °C for the 16S and viable cells, 100 µL of L. reuteri PL503 of each treatment target genes, respectively, during 30 s. After the final qPCR and sampling time were inoculated on MRS agar at the same cycle, a melting curve was included by heating the product sampling time and conditions as the experimental tubes. For from 60 to 99 °C and continuous measurement of the fluo- protein analyses, to avoid possible contamination from the rescence was performed to verify the qPCR products. All culture medium, two washes with phosphate buffered saline samples were analyzed in triplicate, including control sam- solution (PBS, pH 7.4) were made. ples consisting of adding sterile ultrapure water instead of −ΔΔC cDNA. The expression ratio was calculated using the 2 Gene expression studies method reported by Livak and Schmittgen (2001). The cali- brator sample corresponded to the value of the expression RNA extraction of the experimental group Control at each sampling time. The RNA extraction of each experimental group and incuba- Analytical procedures tion time was performed using the MasterPure™ RNA puri- fication kit (Epicentre), which includes DNase treatment. Analysis of ROS by flow‑cytometry The obtained RNA was eluted in 35 μL TE buffer and kept at − 80 °C until further use. RNA quantity (ng/µL) and qual- Flow cytometry detection of ROS (e.g., hydroxyl and ity (A /A ratio) were spectrophotometrically determined superoxide radicals) in L. reuteri PL503 was performed as 260 280 using the Nanodrop 2000 (Thermo Scientific, USA). determined using protocols described by Díaz-Velasco et al. (2020) with some minor modifications. In brief, samples Reverse transcription reaction of L. reuteri PL503 (1 × 10  ufc/mL) of each experimen- tal group and incubation time, were extended in 1 mL of The cDNA was synthesized using about 500  ng of total PBS, and stained with CellRox Deep Red (5 μM; Ther- RNA, according to the PrimeScript™ RT Reagent kit moFisher, USA) (excitation and emission wavelengths, 644 (Takara Bio Inc., Japan). The cDNA was stored at − 20 °C and 645 nm, respectively) for detecting the bacterium pro- until being used for the PCR reactions. ducing ROS, and Hoechst 33,342 (0.5 μM; Sigma–Aldrich) (excitation and emission wavelengths, 345 nm and 488 nm, Real‑time PCR analysis of gene expression respectively) to identify the bacterium and remove debris from the analysis. After thorough mixing, the cell sus- The uspA and dhaT genes were selected for relative expres- pension was incubated at room temperature for 25 min in sion studies using real-time PCR (qPCR), being used the 16S the dark, washed in PBS and immediately run on the flow gene as reference gene. The amplification was performed in cytometer. The analyses were conducted using a Cytoflex MicroAmp optical 96-well plates sealed with optical adhe- flow cytometer (Beckman Coulter, USA) equipped with vio- sive covers (Applied Biosystems) on a ViiA™ 7 Real-Time let, blue, and red lasers. The instrument was daily calibrated System (Applied Biosystems) using the SYBR Green tech- using specific calibration beads provided by the manufac- nology. Each well contained 2.5 µL of cDNA, 6.25 µL of turer. A compensation overlap was performed before each Table 1 Sequences of primers used for reverse transcription real-time PCR assays to conduct gene expression analyses Primers Gene Nucleotide sequence (5′-3′) Annealing tem- References perature uspALr-F1 uspA CTT GGG TAG CGT TCA CCA TT 60 °C Arcanjo et al. (2019) uspALr-R1 TGA AAA AGC GGT TGA CAC TG 60 °C Arcanjo et al. (2019) LS67 dhaT TGA CTG GAT CCT AAT TTG GTC CTG GTG TTA TTGC 60 °C Schaefer et al. (2010) LS68 TGA CTG AAT TCT TCC GGA TCT TAG GGT TAG G 60 °C Schaefer et al. (2010) Lr16S_F 16S rRNA CCG CTT AAA CTC TGT TGT TG 55 °C Arcanjo et al. (2019) Lr16S_R CGT GAC TTT CTG GTT GGA TA 55 °C Arcanjo et al. (2019) 1 3 666 P. Padilla et al. experiment; however, due to emission and excitation char- the pellet was treated with 6 M HCl and kept in an oven at acteristics of the combination of the used probes, spectral 110 °C for 18 h until completion of hydrolysis. The hydro- overlap was negligible. Files were exported as FCS files and lysates were dried in vacuo in a centrifugal evaporator. The analyzed using FlowJoV 10.5.3 Software for Mac OS (Ash- generated residue was reconstituted with 200 µL of milliQ land, USA). Unstained, single-stained, and Fluorescence water and then filtered through hydrophilic polypropylene Minus One (FMO) controls were used to determine com- GH Polypro (GHP) syringe filters (0.45  μm pore size, Pall pensations and positive and negative events, as well as to Corporation, USA) for HPLC analysis. set regions of interest. A Shimadzu ‘Prominence’ HPLC apparatus (Shimadzu Corporation, Japan), equipped with a quaternary solvent Synthesis of allysine standard compound delivery system (LC-20AD), a DGU-20AS on-line degasser, a SIL-20A auto-sampler, a RF-10A XL fluorescence detec- N-Acetyl-L-AAS (allysine) was synthesized from tor (FLD), and a CBM-20A system controller, was used. An Nα-acetyl-L-lysine using lysyl oxidase activity from egg aliquot (1 μL) from the reconstituted protein hydrolysates shell membrane following the procedure described by Aka- was injected and analyzed in the HPLC-FLD equipment. gawa et al. (2002). Briefly, 10 mM Nα -acetyl-L-lysine was Allysine–ABA was eluted in a Cosmosil 5C -AR-II RP- incubated at constant stirring with 5 g of egg shell mem- HPLC column (5 µm, 150 × 4.6 mm) equipped with a guard brane in 50 mL of 20 mM sodium phosphate buffer, pH 9.0 column (10 × 4.6 mm) packed with the same material (Phe- at 37 °C for 24 h. The egg shell membrane was then removed nomenex, PA, USA). The flow rate was kept at 1 mL/min by centrifugation and the pH of the solution adjusted to 6.0 and the temperature of the column was maintained constant using 1 M HCl. The resulting aldehydes were reductively at 30  °C. The eluate was monitored with excitation and aminated with 3 mM 4-aminobenzoic acid (ABA) in the emission wavelengths set at 283 and 350 nm, respectively. presence of 4.5 mM sodium cyanoborohydride (NaBH CN) Standards (0.1 μL) were run and analyzed under the same at 37 °C for 2 h with stirring. ABA derivatives were then conditions. Identification of both derivatized semialdehydes hydrolyzed by 50 mL of 12 M HCl at 110 °C for 10 h. The in the chromatograms was carried out by comparing their hydrolysates were evaporated at 40 °C in vacuo to dryness. retention times with those from the standard compounds. The resulting allysine–ABA was purified using silica gel The peak corresponding to allysine–ABA was manually column chromatography and ethyl acetate/acetic acid/water integrated from the FLD chromatograms and the resulting (20:2:1, v/v/v) as elution solvent. The purity of the resulting areas plotted against an ABA standard curve with known solution (70%) and authenticity of the standard compounds concentrations that ranged from 0.1 to 0.5 mM (Utrera et al. obtained following the aforementioned procedures were 2011). Results were expressed as nmol of allysine per mg checked using MS and H NMR (Estévez et al. 2009). of protein. Quantification of allysine Analysis of Schiff bases Allysine was quantified in bacterial protein as a marker of The formation of Schiff bases in bacterial protein was oxidation-induced post-translational modification, according assessed in each experimental group and incubation time to the method described by Utrera et al. (2011). Five hun- by fluorescence spectroscopy. Prior to the analysis, reac - dred μL of each experimental group and incubation time of tion mixtures were diluted (1:20) with 8 M urea in 100 mM culture were dispensed in 2 mL microtubes and treated with sodium phosphate buffer, pH 7. Diluted samples were dis- cold 10% (v/v) trichloroacetic acid (TCA) solution. Each pensed in spectrofluorometric cuvettes and excited at 350 nm microtube was vortexed and then subjected to centrifugation using a LS-55 Perkin–Elmer fluorescence spectrometer at 600 × g for 5 min at 4 °C. The supernatants were removed, (PerkinElmer, UK). The fluorescence emitted by Schiff and the pellets were incubated with the following freshly bases was recorded at 450 nm. The excitation and emission prepared solutions: 0.5  mL 250  mM 2-(N-morpholino) slit widths were set at 10 nm and the speed of data collection ethanesulfonic acid (MES) buffer pH 6.0 containing 1 mM while scanning was of 500 nm per min. The height of the diethylenetriaminepentaacetic acid (DTPA), 0.5 mL 50 mM peaks corresponding to Schiff bases spectra was recorded. ABA in 250 mM MES buffer pH 6.0 and 0.25 mL 100 mM After taking into consideration the applied dilutions, the NaBH CN in 250 mM MES buffer pH 6.0. After vortexing, results were expressed as fluorescence units. the tubes were incubated in a water bath at 37 °C for 90 min. The samples were stirred every 15 min. The samples were Analysis of protein thiols then treated with a cold 50% TCA (v/v) solution and cen- trifuged at 1200 × g for 10 min. The pellets were washed Thiols from sulfur-containing amino acids in bacterial pro- twice with 10% TCA and diethyl ether-ethanol (1:1). Finally, teins were quantified in accordance to the method reported 1 3 An in vitro assay of the effect of lysine oxidation end‑product, α‑aminoadipic acid, on the redox… 667 by Rysman et al. (2014). A volume of 250 µL of each L. a standard curve with tetraethoxypropane (TEP). The results reuteri PL503 experimental group and incubation time, was are expressed as mg TBARS per L of sample. washed twice with PBS and ethanol:ethyl acetate (1:1) to avoid possible contamination with thiols from the medium. Statistical analysis Upon centrifugation (600 × g/5 min), the pellet was resus- pended in 250 µL of guanidine hydrochloride, treated with True replicates (n = 3) were subjected to duplicate analyses 250 µL of 4,4′-dipyridyldisulphide (DPS) in 12 mM HCl and and data were collected and subjected to statistical analysis. dispensed in a spectrophotometric cuvette. Absorbance was Earlier, the data were analyzed for normality (Shapiro–Wilk measured at 324 nm against a blank sample in which DPS test) and homoscedasticity (Bartlett test). The effects of was replaced with an equivalent volume of guanidine hydro- AAA concentration and incubation times were studied using chloride. Quantification was made by preparing a standard analyses of variance (ANOVA) (SPSS v. 15.5). The effect of curve with cysteine. The results were expressed as µmol of AAA on the gene expression (ΔΔC values) was analyzed free thiol groups per mg of protein. using the paired Students’ t test (SPSS v. 15.5). The statisti- cal significance was set at p ≤ 0.05. Analysis of thiobarbituric‑reactive substances Results The quantification of MDA and other thiobarbituric-reactive substances (TBARS) in all experimental groups and incuba- Relative expression of the uspA gene tion times, was made in accordance to the method described by Ganhao et al. (2011). An aliquot of 200 µL of L. reu- The relative expression of the L. reuteri PL503 uspA gene teri PL503 experimental group was treated with 500 µL of during the incubation assay in the presence of different thiobarbituric acid (0.02 M) and 500 µL of TCA (10%) and concentrations of AAA is shown in Fig. 1a. A significant incubated during 20 min at 90 °C. After cooling, a 5 min downregulation of the uspA gene was particularly observed centrifugation at 600 × g was made and the supernatant was at 12 h of incubation in the presence of 5 and 10 mM of measured at 532 nm. Quantification was made by preparing AAA (0.22- and 0.40-fold decrease, respectively) as well Fig. 1 Relative expression (a) ΔΔC (2- ) of the uspA (a) and dhaT (b) genes in Lactobacil- lus reuteri PL503 grown in the presence of increasing concen- trations (0, 1, 5, and 10 mM) of α-aminoadipic acid (AAA) for up to 24 h. Black line at ΔΔC 2- = 1 denotes standardized expression rate for CONTROL group (0 mM) at each sampling ΔΔC time (calibrator). 2- < 1 denotes suppression of the expression of the target gene; ΔΔC 2- > 1 denotes activation of the expression of the target gene. Asterisks on top of bars (b) denote significant differences between such treatment and the control within a particu- lar sampling time (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) 1 3 668 P. Padilla et al. as at 16 h sampling with 10 mM (0.38-fold decrease). An TBARS/L vs. 1.10 mg TBARS/L). At 24 h, the concentra- upregulation of the gene (1.43-fold increase) was observed tion of TBARS in control samples (1.17 mg TBARS/L) was at 24 h when the bacterium was exposed to the highest AAA significantly higher than in the bacterium challenged with concentration. AAA (ranging from 1.05 to 1.10 mg TBARS/L). Relative expression of the dhaT gene Quantification of allysine The relative expression of the L. reuteri PL503 dhaT gene The changes of the concentration of allysine in L. reuteri during the incubation assay in the presence of different PL503 during the incubation period is shown in Fig.  3b. concentrations of AAA is shown in Fig. 1b. A significant Compared to control, the exposure to AAA caused a sig- downregulation of the gene was found at 12 h in the pres- nificant increase in the concentration of allysine in proteins ence of 5 mM (0.42-fold decrease) and at 16 h in the pres- from L. reuteri PL503 for 20 h. At that sampling point, the ence of both 1 mM and 10 mM of AAA (0.75- and 0.81- concentration of allysine in control samples (1.8 nmol/mg fold decreases, respectively). Nonetheless, in the two final protein) was significantly lower than in those treated with sampling times, an upregulation of the relative transcription 1, 5, and 10 mM AAA (11.7, 10.4, and 8.8 nmol/mg pro- of the dhaT gene was observed. In particular, significant tein, respectively). At 24 h sampling, the behavior varied changes were found in the presence of 5 mM of AAA at 20 h between groups. In L. reuteri challenged with 1 and 5 mM (1.98-fold increase) and 1 mM of AAA at 24 h (1.83-fold of AAA, the increase of allysine was constant during the increase). complete assay reaching the highest concentration at 24 h (12.0 and 13.5 nmol/mg protein, respectively). On the other ROS generation by flow‑cytometry analyses hand, when the bacterium was exposed to the highest AAA concentration (10 mM) allysine peaked at 20 h, after which The incubation of L. reuteri PL503 in the presence of AAA a decrease was observed at the end of the incubation period led to an increased production of ROS as shown in Fig. 2. (4.2 nmol/mg protein). The analysis of the samples with flow-cytometry showed a clear dose effect. At increasing concentrations of AAA, the Analysis of Schiff bases percentage of bacterium suffering oxidative stress at 24 h rise from 0.8% in control group to 1.8%, 2.1%, and 5.3% In the present study, the formation of Schiff bases is shown in bacteria exposed to 1, 5 and 10 mM AAA, respectively. in Fig. 3c and a clear dose effect of AAA was observed. No Specially, at the two final sampling times, the differences significant differences were found between AAA concentra- between groups were found to be higher than in the previ- tions during the first three sampling times. Nevertheless, ous ones. at the final sampling time (24 h) an increase was observed when the bacterium was exposed to the highest concentra- Analysis of thiobarbituric‑reactive substances tion (10 mM), which is coincident with carbonyls deple- tion found in the same group of samples at the end of the In Fig. 3a, the TBARS concentration in L. reuteri PL503 assay. At 24 h, the relative concentration of Schiff bases in during the assay is shown. In the presence of AAA, sig- L. reuteri followed the increasing order: control group (52 nificant changes occur at 20 h with 10 mM of AAA lower - fluorescent units) and bacterium exposed to 1, 5 and 10 mM ing TBARS content compared to control samples (0.89 mg AAA (80, 98, and 185 fluorescent units). Fig. 2 Percentage of Lactoba- cillus reuteri PL503 suffering from oxidative stress (positive to Cell Rox dye) when grown in the presence of increasing con- centrations (0, 1, 5 and 10 mM) of α-aminoadipic acid (AAA) for up to 24 h. Different letters on top of bars denote significant differences (p ≤ 0.05) between a AAA concentrations within the same sampling time c AAA concentra on (mM) 01 5100 15 10 01 5100 15 10 Incuba on me (h) 12 16 20 24 1 3 % C+ e l Rox An in vitro assay of the effect of lysine oxidation end‑product, α‑aminoadipic acid, on the redox… 669 (a) mM mM (b) Fig. 4 Concentration of free thiols (means ± standard deviation) in mM Lactobacillus reuteri PL503 grown in MRS broth with increasing concentrations (0, 1, 5, and 10  mM) of α-aminoadipic acid (AAA) during an incubation period for up to 24  h. Different letters at the same sampling time denote significant differences between AAA con- centrations (p ≤ 0.05) 24 h concentrations of 13.3 μmol/mg protein and 11.8 μmol/ mg protein, respectively. Conversely, the concentration of thiols in bacteria exposed to the highest AAA concentra- tion (10 mM) significantly decreased from the first sampling (10 μmol/mg protein) until the end of the assay (7.9 μmol/ (c) mg protein). mM Discussion Regulation of the uspA and dhaT genes by L. reuteri in response to AAA L. reuteri counts remained stable with the increasing applied doses of AAA (1 mM, 5 mM, and 10 mM) during the entire experimental assay (37 °C/24 h), so the survival was not jeopardized (data not shown). Yet, the challenge with this oxidized amino acid led to impairments of the bacterium’s Fig. 3 Concentration of thiobarbituric-reactive substances (TBARS) (a), allysine (b) and Schiff bases (c) (means ± standard deviation) in physiology. This finding reflects the ability of L. reuteri to Lactobacillus reuteri PL503 grown in MRS broth in the presence of activate mechanisms to neutralize the potential harmful increasing concentrations (0, 1, 5 and 10 mM) of α-aminoadipic acid effects of the sub-lethal concentrations of the added oxidized (AAA) during an incubation period for up to 24 h. Different letters at amino acid. In the present study, these mechanisms were the same sampling time denote significant differences between AAA concentrations (p ≤ 0.05) firstly assessed by the analysis of the relative expression of stress-related genes. The universal stress protein A (UspA) superfamily includes an ancient and conserved group of proteins found Analysis of protein thiols in assorted microorganisms, insects, and plants. The precise roles of Usp proteins in biological systems remain unclear; The concentration of free thiols in proteins from L. reu- teri PL503 during the incubation assay is shown in Fig. 4. yet, they seem to be involved in the defense against DNA- damaging agents and respiratory uncouplers (Kvint et al. Significant differences were observed in the first 12  h between the control group and the bacterium challenged 2003). Due to the defined function of the gene uspA, an upregulation was expected, which was only observed at with increasing AAA concentrations. From 16 h sampling time onwards, a significant increase of free thiols in samples 24 h and in the presence of the highest AAA concentra- tion (Fig. 1a). Yet, the downregulation observed at earlier exposed to 1 and 5 mM of AAA was detected, peaking at 1 3 670 P. Padilla et al. samplings and lower concentrations is consistent with data elemental mechanisms by which L. reuteri may seek to pro- reported by Oberg et al. (2015), who found a significant tect against AAA-induced biological damage through the downregulation of the uspA gene expression in Bifidobacte- activation of the 3-HPA pathway should be subjected to rium longum exposed to a hydroxyl-radical generating sys- scrutiny. As previously reported by Talarico et al. (1988), tem. Similar results were reported by Arcanjo et al. (2019) the 3-HPA pathway requires glycerol, commonly added working on the same bacterium and strain from the present as growth promoter in Lactobacillus cultures. In the pre- study. In that study, exposing L. reuteri to 0.5 mM of hydro- sent study, L. reuteri had no access to such precursor, and, gen peroxide led to a significant decrease of the uspA gene therefore, the 3-HPA pathway is unlikely to have occurred. expression. It is worth noting that both aforementioned stud- Considering the absence of glycerol, it seems reasonable to ies found the occurrence of oxidative stress and molecular consider that 1,3-PDO may have other substrates and that damage in the exposed bacteria. The fact that AAA exposure its cellular activity may be related to protection against a led to a similar effect on L. reuteri indicates that this oxi- potential pro-oxidative threat. To similar conclusions came dized amino acid is identified by the bacterium as a chemical Arcanjo et al. (2019) who found an increased expression threat. In fact, two recent studies agree in describing noxious of the dhaT gene in L. reuteri challenged with hydrogen effects of food-compatible AAA concentrations (200  μM) peroxide in simulated colonic conditions where glycerol on human intestinal (Díaz-Velasco et al. 2020) and human was, again, absent. The authors hypothesized whether the acinar pancreatic cells (Estaras et al. 2020). According to NAD + -dependent activity of the 1,3-PDO may be able these authors, the harmful effect of AAA involved the induc- to detoxify hydrogen peroxide in the presence of NADH. tion of pro-oxidative conditions within cells. Probiotic bac- Since no hydrogen peroxide was included in the present teria like L. reuteri may also be susceptible to this chemical assay, the implication of 1,3-PDO in balancing the redox species and, according to these results, the downregulation state of the cell seems to be a pertinent defense mechanism of the uspA gene seems to be related to a cellular signal of a against pro-oxidative threats. It is, still unknown how AAA pro-oxidative threat that both, the radical generating systems may impair the redox status of L reuteri but it is proven that (i.e., hydrogen peroxide) and oxidized amino acids such as AAA exposure to human eukaryotic cells cause oxidative AAA, may be able to induce. stress via mitochondrial disturbance and ROS generation It is worth clarifying that the higher AAA concentrations (Díaz-Velasco et al. 2020; Estaras et al. 2020). tested in the present study (1–10 mM) are plausibly compat- It is worth noting that the effect of AAA exposure on the ible with a physiological situation as explained as follows. expression of the dhaT gene at early stages of the assay (12 While AAA concentration in foods has been found to reach and 16 h) was opposite to that observed at advanced stages. up to 200 μM, it is also known that dietary proteins are fur- As discussed in due course, the activation of the gene at ther oxidized during digestion, increasing significantly the advanced stages of oxidative stress and oxidative damage final concentration of oxidized amino acids in the gut. For could have triggered defense mechanisms, in which the dhaT instance, in a study by Van-Hecke et al. (2019), the concen- gene may be implicated. At early stages, the underexpres- tration of protein oxidation products increased between 2 sion of this gene could respond to indefinite initial responses and fivefold times in assorted foods after simulated gastro- of the bacteria to the AAA exposure, in which the protein intestinal digestion. The same authors found in a more recent encoded by this gene was not found as essential. In line study (Goethals et al. 2020) sixfold times higher concentra- with this downregulation, a recent study by Díaz-Velasco tions of protein oxidation products in pork digests than in et al. (unpublished data) observed that AAA exposure to the original (undigested) pork product. CACO-2 cells led to an overall downregulation of gene The dhaT gene encodes the enzyme 1,3-PDO oxidore- expression due to the impairment of protein kinase A and C ductase which is known to play a relevant role in stressful (PKA and PKC, respectively) signaling pathways. Yet, the situations involving energetic demand. This enzyme enables mechanisms implicated in the downregulation of dhaT gene the main carbohydrate fermentation pathway (6-phosphoglu- at early stages of exposure to AAA in this bacterium remain conate/phosphoketolase; 6-PG/PK) through the production indefinite and require further elucidation. of NAD (required for glucose fermentation) from NADH in the conversion of 3-hydroxypropionaldehyde (3-HPA) ROS generation in L. reuteri by AAA (its substrate) into 1,3-PDO under anaerobic conditions. Additionally, 3-HPA, also known as reuterin, is excreted by The increased production of ROS in L. reuteri by the pres- L. reuteri strains under stressful situations (Schaefer et al. ence of AAA has no precedent in literature (Fig. 2). It is, 2010). The overexpression of this gene observed in bacteria however, consistent with results reported by Díaz-Velasco exposed to 5 and 1 mM AAA for 20 and 24 h, respectively, et al. (2020) in CACO-2 cells and Estaras et al. (2020) in could respond to an attempt of the bacteria to protect against pancreatic cells when the exposure to AAA led to impair- the oxidative threat caused by this oxidized amino acid. The ment of the oxidative status of the cell, ROS generation, 1 3 An in vitro assay of the effect of lysine oxidation end‑product, α‑aminoadipic acid, on the redox… 671 apoptosis, and necrosis. In addition, it is in accordance to also react with amino groups from neighboring amino acids Da Silva et al. (2017) who studied the effect of AAA on (e.g., lysine) to form an azomethine structure, also known as brain function of adolescent rats, and showed an induction Schiff bases (Estévez 2011). The dramatic drop of allysine of ROS generation and alteration of the cellular redox sta- concentration during the last 4 h of the assay in the bacte- tus via mitochondrial impairment. While the percentage rium exposed to the highest concentration of AAA (10 mM) of CelRox positive bacteria was found to be relatively low, is consistent with the sudden increase of Schiff bases in that previous studies using hydrogen peroxide and malondial- period of time (Fig. 3c). These results suggest that such fluo- dehyde (MDA) as inductors of oxidative stress in L. reuteri rescent structures were, at least, partially formed in bacteria reported similar percentages (Arcanjo et al. 2019; Padilla exposed to 10 mM as a result of allysine addition to other et al. 2021). The oxidative damage caused in bacterial lipids protein amines. The formation of Schiff bases in bacteria and proteins, explained in due course, denote severe oxida- exposed to intermediate AAA doses (1 and 5 mM), was not tive stress. The precise mechanisms by which AAA is able to so intense to reflect a decline of the reactant (allysine). Both, induce ROS generation in L. reuteri are indefinite. It is worth carbonylation and formation of non-reducible protein cross- noting that such mechanisms differ from those reported by links (i.e., Schiff bases), are irreversible protein modifica- the aforementioned authors since the bacterium lacks mito- tions with negative biological consequences (Davies 2005; chondria. Interestingly, Lactobacillus spp. have also been Ezraty et al. 2017; Estévez and Xiong 2019). Carbonylated found to be able to produce hydrogen peroxide and other proteins can be dysfunctional and may be labeled to removal ROS via implication of NAD(P)H oxidoreductases (Hertz- due to its accumulation causes impaired homeostasis that berger et al. 2014) which provides a plausible and coherent leads to chronic dysfunction and apoptosis (Shacter 2003). connection between AAA exposure, dhaT overexpression However, carbonylated proteins can also act as signaling and ROS generation. The molecular mechanisms underlying molecules, which may activate specific pathways, to pre- the interconnection between all these elements need to be serve homeostasis control senescence (Shacter 2003). precisely described. Both situations could be applied to the present experi- ment. The increase in carbonyls above 10 nmol/mg proteins Oxidative damage to L. reuteri by AAA in the bacterium challenged with 5 and 1 mM of AAA was coincident with the activation of the dhaT gene at sampling In the present work, the oxidative damage to bacterium times of 20 h and 24 h, respectively, and plausibly, the cor- caused by AAA-induced oxidative stress was assessed by responding synthesis of the NADH-dependent oxidoreduc- means of TBARS (lipid oxidation) and allysine (protein oxi- tase decoded by this gene. Given the proposed role of this dation). The basal TBARS concentration in control cultures, enzyme in detoxifying pro-oxidant species (Arcanjo et al. (~ 1 mg/L) may correspond to the occurrence of lipid peroxi- 2019), a relatively mild pro-oxidative threat, exhibited in a dation in the bacterium under physiological conditions and significant accretion of protein carbonyls, could have led to did not change significantly during the assay within groups the activation of an antioxidant response mediated, among (Fig. 3a). AAA did not significantly affect the extent of lipid others, by the activation of the dhaT gene. On the other hand, oxidation in L. reuteri. a severe oxidative damage caused by a more intense pro- On the other hand, AAA exposure had a significant oxidative environment, such as that observed in L. reuteri impact on the oxidative damage to bacterial proteins. A challenged with the highest concentration of AAA (10 mM) relatively low but significant increase in allysine, the main led to a sudden formation of advanced oxidation products protein carbonyl in biological systems (Stadtman and Levine (Schiff bases) and no dhaT gene-mediated response against 2000; Estévez and Luna 2016), was observed in the control the oxidative insult. These mechanisms were not present in group of L. reuteri (Fig. 3b). The present results show that the bacterium incubated with the lowest doses of AAA (1 allysine, formed in bacteria, as in eukaryotes, remarkably and 5 mM). Previous considerations made by Ezraty et al. contributes to protein carbonylation and may be used as a (2017) and Arcanjo et al. (2019) support the hypothesis that reliable indicator of oxidative stress. The results obtained are the dhaT gene could have been activated by pro-oxidant spe- in accordance with Ezraty et al. (2017) who proposed that cies and/or the effect of the former on protein carbonylation. protein carbonylation could be a reflection of bacterial senes- The evolution of protein thiols during the assay (Fig. 4) cence as oxidized proteins accumulate in non-proliferating provides additional strength to the aforementioned hypoth- bacteria. Allysine is typically formed in proteins because of eses. The oxidation of sulfur-containing amino acids, such the attack of ROS to lysine residues. This is plausibly the as cysteine (Cys) and methionine (Met), is a typical feature mechanism taking place in the present assay as the signifi- in biological systems attacked by ROS (Estévez et al. 2020). cant production of ROS in L. reuteri exposed to AAA expo- While the oxidation of thiols in proteins may lead to dys- sure could have caused the oxidation of lysine residues and function, irrelevant sulfur-containing amino acids are known hence, the accretion of allysine. Once formed, allysine may to act as antioxidants offering a sacrificial loss to ROS and 1 3 672 P. Padilla et al. the project AGL2017-84586R as well as by the Government of Extrem- protecting other amino acids with relevant significance, such adura and FEDER (grants GR18056 and GR15108). P. Padilla was as lysine (Davies, 2005; Estévez et al. 2020). This dual role employed through the contract PEJ2014-P-0057. of thiols was examined in the present experiment. Taking into account that these moieties can act as redox-active com- Declarations pounds and elements of antioxidant protection in biological systems, the coincidence of thiol accretion with the increase Conflict of interest The authors declare no conflict of interest. of carbonylation in those samples may respond to a strat- egy to keep a balanced redox status in cells in danger. The Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- incubation of L. reuteri with AAA caused an increase of tion, distribution and reproduction in any medium or format, as long thiol concentration since 12 h incubation onwards. The pro- as you give appropriate credit to the original author(s) and the source, oxidant changes induced by AAA, including the formation provide a link to the Creative Commons licence, and indicate if changes of protein carbonyls, possibly triggered the accumulation were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated of thiol groups by the novo synthesis of sulfur-containing otherwise in a credit line to the material. If material is not included in proteins/peptides with the purpose of protecting the bac- the article's Creative Commons licence and your intended use is not terium against this pro-oxidant threat. Thiol accumulation permitted by statutory regulation or exceeds the permitted use, you will is considered as an endogenous mechanism of antioxidant need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . defense owing to the recognized redox-active properties (Davies 2005). These moieties have been typically regarded as elements of antioxidant protection in eukaryotes and in lactic acid bacteria (Schaefer et al. 2010; Xiao et al. 2011). 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Journal

Amino Acids – Springer Journals

Published: Apr 1, 2022

Keywords: Oxidized amino acids; Oxidative stress; Probiotic bacterium; Protein oxidation; Transcripts

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