Four Superoxide Dismutases Contribute to Bacillus anthracis Virulence and Provide Spores with Redundant Protection from Oxidative Stress

Abstract

The Bacillus anthracis genome encodes four superoxide dismutases (SODs), enzymes capable of detoxifying oxygen radicals. That two of these SODs, SOD15 and SODA1, are present in the outermost layers of the B. anthracis spore is indicated by previous proteomic analyses of the exosporium. Given the requirement that spores must survive interactions with reactive oxygen species generated by cells such as macrophages during infection, we hypothesized that SOD15 and SODA1 protect the spore from oxidative stress and contribute to the pathogenicity of B. anthracis. To test these theories, we constructed a double-knockout (⌬sod15 ⌬sodA1) mutant of B. anthracis Sterne strain 34F2 and assessed its lethality in an A/J mouse intranasal infection model. The 50% lethal dose of the ⌬sod15 ⌬sodA1 strain was similar to that of the wild type (34F2), but surprisingly, measurable whole-spore SOD activity was greater than that in 34F2. A quadruple-knockout strain (⌬sod15 ⌬sodA1 ⌬sodC ⌬sodA2) was then generated, and as anticipated, spore-associated SOD activity was diminished. Moreover, the quadruple-knockout strain, compared to the wild type, was attenuated more than 40-fold upon intranasal challenge of mice. Spore resistance to exogenously generated oxidative stress and to macrophagemediated killing correlated with virulence in A/J mice. Allelic exchange that restored sod15 and sodA1 to their wild-type state restored wild-type characteristics. We conclude that SOD molecules within the spore afford B. anthracis protection against oxidative stress and enhance the pathogenicity of B. anthracis in the lung. We also surmise that the presence of four SOD alleles within the genome provides functional redundancy for this key enzyme.

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…Superoxide dismutase molecules within the spore may afford B. anthracis protection against oxidative stress and enhance pathogenicity in animal models of infection (107). The presence of multiple superoxide dismutase genes may provide redundancy needed to ensure protection of germinating spores in animal hosts.…”

“…Its contributions to oxidative stress and virulence were examined by Cybulski et al (107). A strain lacking all four superoxide dismutases encoded in the B. anthracis genome was found to be attenuated for virulence upon intranasal challenge of mice (107).…”

Selection and Characterization of Single Domain Antibodies Specific for Bacillus anthracis Spore Proteins

“…Although the isolated sdAbs were specific for B. anthracis, EA1 has been shown to be a spore contaminant and not an integral component of the bacterial spore [17]. The two llamas were subjected to a second series of immunizations with a combination of recombinant spore-specific proteins: BclA, gerQ, SODA1, SOD15, BxpB, and p5303 [18,19]. Centavo received a mix of all six proteins; while Whisper was immunized with a cocktail that did not include the BclA protein in an attempt to allow a stronger immune response toward the other spore proteins that may not be as immunodominant as the BclA antigen.…”

Roles of Cellular Redox Factors in Pathogen and Toxin Entry in the Endocytic Pathways

“…Based on the above evidence and given the fact that anthrax toxin requires a redox environment that favors receptor disulfide bond formation for translocation, an intriguing hypothesis is that the bacterium and the toxin stimulate an oxidative burst within the host cells so as to ensure anthrax toxin translocation. Consistent with this hypothesis, B. anthracis has evolved mechanisms defending itself against oxidative stress, including superoxide dismutases, peroxidases, and catalases, all of which suppress the damaging Fenton reaction catalyzed by reactive oxygen or nitrogen species [4,110,111]. Moreover, as mentioned above, the unusual "cysteine-free" feature of anthrax toxin might have been selected through evolution permitting the toxin to be exempt from the damaging thiol-modifications caused by oxidative stress.…”