Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity

Abstract

In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89, Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri, L. johnsonii, L. acidophilus, and L. reuteri, respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O(2)(.-) and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.

FIG. 1.

Construction of pSodA plasmid. pSodX-1 containing the sodA gene and flanking sequence from S. thermophilus in pGEM-T Easy cloning vector (Promega) was digested with EcoRI. The 1.2-kb fragment was gel purified and ligated into the EcoRI-digested shuttle vector, pTRK563. The new plasmid construct was termed pSodA.

FIG. 2.

PCR confirmation of sodA in the transformants. Lactobacillus spp. were grown at 37°C in MRS medium supplemented with 2% glucose. S. thermophilus AO54 was grown at 42°C in M17 medium supplemented with 0.5% glucose. Primers STSODF and STSODR and cells from presumptive transformants or S. thermophilus were used for PCRs. Lanes: 1, 1-kb ladder; 2, pSodA; 3, negative control; 4, L. gasseri NC1501; 5, L. johnsonii NC1511; 6, L. acidophilus NC1521; 7, L. reuteri NC1531; 8, S. thermophilus AO54.

FIG. 3.

Transcription/translation of sodA and MnSOD activity in the recombinant Lactobacillus spp. (A) Lactobacillus spp. and SOD activity (units per milligram of protein). (B) Western blots (dot blots) showing the levels of SodA protein in 15 μg of protein of CFEs from S. thermophilus AO54 and Lactobacillus harboring pSodA. (C) Results from RTQ-PCR showing the levels of sodA mRNA and 16S mRNA and their ratios for the recombinant Lactobacillus spp. and S. thermophilus AO54.

FIG. 4.

Sensitivity to hydrogen peroxide. Exponentially growing cells from L. gasseri and L. acidophilus harboring pSodA (continuous lines, solid symbols) or pTRK563 (dotted lines, open symbols) were tested for growth in the presence of increasing concentrations of hydrogen peroxide. The entire procedure was carried out in APT broth under aerobic conditions at 37°C as described in Materials and Methods.

FIG. 5.

Protection against hydrogen peroxide toxicity by DIP. Exponentially growing cells of L. gasseri were tested for growth in the presence of increasing concentrations of H₂O₂ and DIP. The entire procedure was carried out in APT broth under aerobic conditions at 37°C as described in Materials and Methods.

FIG. 6.

A schematic presentation showing how SODs, hydroperoxidases, iron chelators, iron-binding proteins, protectors of [Fe-S] clusters, and/or DNA and cellular repair mechanisms could protect against H₂O₂ toxicity. Reaction 1 shows the oxidation of labile iron-sulfur clusters by O₂^·−, reaction 2 shows the generation of HȮ by Fenton chemistry, reaction 3 shows the regeneration of Fe(II) from Fe(III) by O₂^·− (the sum of reactions 2 and 3 is also known as the Haber-Weiss reaction), and reaction 4 shows the deleterious effects of HȮ and the generation of damaged DNA and damaged cellular components. Protective molecules/mechanisms are shown in boxes: SODs inhibit reactions 1 and 3, hydroperoxidases inhibit reaction 2, iron-binding proteins (Dpr and Dps) and iron chelators (DIP) inhibit reaction 2, protectors of [Fe-S] clusters (YggX and FeSII) inhibit reaction 1, and DNA repair and other cellular repair mechanisms repair the damage caused by reaction 4.