Molecular mechanisms of the disturbance caused by malondialdehyde on probiotic Lactobacillus reuteri PL503

Abstract

This study aimed to provide insight into the molecular and genetic mechanisms implicated in the responses of Lactobacillus reuteri against the oxidative stress induced by malondialdehyde (MDA) by analysing protein oxidation and assessing the uspA and the dhaT genes. Four experimental groups were evaluated depending on the concentration of MDA added in Man, Rogosa and Sharpe (MRS) broth: Control (L. reuteri), 5 µM (L. reuteri + 5 µM MDA), 25 µM (L. reuteri + 25 µM MDA) and 100 µM (L. reuteri + 100 µM MDA). Three replicates were incubated at 37 °C for 24 h in microaerophilic conditions and sampled at 12, 16, 20 and 24 h. The upregulation of the uspA gene by L. reuteri indicates the recognition of MDA as a potential DNA-damaging agent. The dhaT gene, encoding a NADH-dependent-oxidoreductase, was also upregulated at the highest MDA concentrations. This gene was proposed to play a role in the antioxidant response of L. reuteri. The incubation of L. reuteri with MDA increased the production of ROS and caused thiol depletion and protein carbonylation. L. reuteri is proposed to detoxify pro-oxidative species while the underlying mechanism requires further elucidation.

Fig. 1

Relative expression (2^‐ΔΔC _T) of the uspA (A) and dhaT (B) genes in Lactobacillus reuteri PL503 as affected by increasing concentrations of malondialdehyde (MDA) for up to 24 h. Means for each experimental group are calculated from analyses applied to three biological replicates and all analyses were technically duplicated. Black line at 2^‐ΔΔC _T = 1 denotes standardized expression rate for CONTROL group at each sampling time (calibrator). 2^‐ΔΔC _T < 1 denotes suppression of gene expression; 2^‐ΔΔC _T > 1 denotes activation of gene expression. Asterisks on top of bars denote significant diferences in paired Student’s t‐tests performed to compare each MDA concentration with CONTROL [MDA = 0]: *P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001.

Fig. 2

Production of reactive oxygen species (ROS) in Lactobacillus reuteri PL503 grown in MRS broth with increasing concentrations of malondialdehyde (MDA) for 24 h. A: DNA containing bacterium (debris and nonbacterium events are removed); B: Gate for bacterium; C‐F: X‐axis bacterium (DNA +) Y‐axis ROS; C: CONTROL; D: L. reuteri PL503 incubated with 5 µM MDA; E: L. reuteri PL503 incubated with 25 µM MDA; F: L. reuteri PL503 incubated with 100 µM MDA. Analyses were applied to three biological replicates and all analyses were technically duplicated.

Fig. 3

Concentration of TBARS (A); allysine (B) and Schiff bases (C) (means ± standard deviation) in Lactobacillus reuteri PL503 grown in MRS broth with increasing concentrations of malondialdehyde (MDA) during an incubation period for up to 24 h. Means for each experimental group are calculated from analyses applied to three biological replicates and all analyses were technically duplicated. Different letters at the same sampling time denote significant differences between means within the same sampling point in ANOVA (P ≤ 0.05).

Fig. 4

Concentration of allysine (A) and Schiff bases (B) (means ± standard deviation) in human serum albumin (HSA), human haemoglobin (HH) and β‐lactoglobulin (LAC) (5 mg ml⁻¹) after incubation with MDA (0.25 µM) at 37 °C (HSA, HH) and 80 °C (LAC) for 24 h. Means for each experimental group are calculated from analyses applied to three biological replicates and all analyses were technically duplicated. Different letters denote significant differences between means within the same sampling point in ANOVA (P ≤ 0.05).

Fig. 5

Concentration of free thiols (means ± standard deviation) in Lactobacillus reuteri PL503 grown in MRS broth with increasing concentrations of malondialdehyde (MDA) during an incubation period for up to 24 h. Means for each experimental group are calculated from analyses applied to three biological replicates and all analyses were technically duplicated. Different letters at the same sampling time denote significant differences between means within the same sampling point in ANOVA (P ≤ 0.05).

Fig. 6

General scheme of the proposed mechanisms whereby malondialdehyde (MDA) damages biomolecules in Lactobacillus reuteri PL503 (red lines) and mechanism by which the bacterium may protect against MDA‐induced oxidative stress (green lines).