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

Abstract

This study was designed to gain information about the underlying mechanisms of the effects of a food-occurring free oxidized 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 downregulation 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: Oxidative stress; Oxidized amino acids; Probiotic bacterium; Protein oxidation; Transcripts.

Fig. 1

Relative expression (2-^ΔΔC_T) of the uspA (a) and dhaT (b) genes in Lactobacillus reuteri PL503 grown in the presence of increasing concentrations (0, 1, 5, and 10 mM) of α-aminoadipic acid (AAA) for up to 24 h. Black line at 2-^ΔΔC_T = 1 denotes standardized expression rate for CONTROL group (0 mM) at each sampling time (calibrator). 2-^ΔΔC_T < 1 denotes suppression of the expression of the target gene; 2-^ΔΔC_T > 1 denotes activation of the expression of the target gene. Asterisks on top of bars denote significant differences between such treatment and the control within a particular sampling time (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

Fig. 2

Percentage of Lactobacillus reuteri PL503 suffering from oxidative stress (positive to Cell Rox dye) when grown in the presence of increasing concentrations (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 AAA concentrations within the same sampling time

Fig. 3

Concentration of thiobarbituric-reactive substances (TBARS) (a), allysine (b) and Schiff bases (c) (means ± standard deviation) in Lactobacillus reuteri PL503 grown in MRS broth in the presence of 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 concentrations (p ≤ 0.05)

Fig. 4

Concentration of free thiols (means ± standard deviation) in 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 concentrations (p ≤ 0.05)