Inactivation of the Moraxella catarrhalis superoxide dismutase SodA induces constitutive expression of iron-repressible outer membrane proteins

Abstract

Many pathogens produce one or more superoxide dismutases (SODs), enzymes involved in the detoxification of endogenous and exogenous reactive oxygen species that are encountered during the infection process. One detectable cytoplasmic SOD was identified in the human mucosal pathogen Moraxella catarrhalis, and the gene responsible for the SOD activity, sodA, was isolated from a recent pediatric clinical isolate (strain 7169). Sequence analysis of the cloned M. catarrhalis 7169 DNA fragment revealed an open reading frame of 618 bp encoding a polypeptide of 205 amino acids with 48 to 67% identity to known bacterial manganese-cofactored SODs. An isogenic M. catarrhalis sodA mutant was constructed in strain 7169 by allelic exchange. In contrast to the wild-type 7169, the 7169::sodK20 mutant was severely attenuated for aerobic growth, even in rich medium containing supplemental amino acids, and exhibited extreme sensitivity to the redox-active agent methyl viologen. The ability of recombinant SodA to rescue the aerobic growth defects of E. coli QC774, a sodA sodB-deficient mutant, demonstrated the functional expression of SOD activity by cloned M. catarrhalis sodA. Indirect SOD detection assays were used to visualize both native and recombinant SodA activity in bacterial lysates. This study demonstrates that M. catarrhalis SodA plays a critical role in the detoxification of endogenous, metabolically produced oxygen radicals. In addition, the outer membrane protein (OMP) profile of 7169::sodK20 was consistent with iron starvation in spite of growth under iron-replete conditions. This novel observation indicates that M. catarrhalis strains lacking SodA constitutively express immunogenic OMPs previously described as iron repressible, and this potentially attenuated mutant strain may be an attractive vaccine candidate.

FIG. 1.

Native-PAGE gel stained to detect SOD activity from whole-cell extracts of M. catarrhalis 7169 (lane1) and the sodA isogenic mutant 7169::sodK20 (lane 2).

FIG. 2.

Growth of 7169 (open symbols) and 7169::sodK20 (closed symbols) in a minimal salts medium (GC, dotted line), rich medium (BHI, dashed line), and rich, supplemented medium (HD, solid line) was monitored spectrophotometrically at 1-h intervals.

FIG. 3.

Sensitivity of M. catarrhalis 7169 (A) and 7169::sodK20 (B) to the superoxide-generating agent MV. Final concentrations of MV (micromolar) are shown on the left.

FIG. 4.

Complementation of SOD-deficient E. coli (QC774) by the recombinant M. catarrhalis SodA. Growth in rich medium under aerobic conditions (A), in the presence of 50 μM MV (B), and in minimal medium (C) was measured at 1-h intervals. The sodA sodB mutant QC774 (solid lines) and the parental strain GC4468 (dashed lines) were transformed with pMCSODA (closed symbols) and the control vector psodKAN (open symbols).

FIG. 5.

Native PAGE of crude bacterial cell extracts from 7169 (lane 1), 7169::sodK20 (lane 2), GC4468/psodKAN (lane 3), QC774/psodKAN (lane 4), and QC774/pMCSODA (lane 5) stained to visualize SOD activity. Locations of the M. catarrhalis SodA and the E. coli MnSOD, FeSOD, and HySOD (Mn/Fe SOD hybrid) are indicated.

FIG. 6.

A sodium dodecyl sulfate-PAGE gel, stained with Coomassie blue, comparing the OMP profiles of parental strain 7169 (lane 1), the 7169 sodA isogenic mutant 7169::sodK20 (lane 2), and a 7169 sodA-complemented revertant (lane 3). The iron-regulated OMPs TbpB (▸) and CopB (✖) and molecular sizes, in kilodaltons, are indicated.