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Summary Salmonellae survive and propagate in macrophages to cause serious systemic disease. Periplasmic superoxide dismutase plays a critical role in this survival by combating phagocytic superoxide. Salmonella Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Although both proteins are produced during infection, only SodCI is functional in the macrophage phagosome. We have previously shown that SodCI, relative to SodCII, is both protease resistant and tethered within the periplasm and that either of these properties is sufficient to allow a SodC to protect against phagocytic superoxide. Tethering is defined as remaining cell-associated after osmotic shock or treatment with cationic antimicrobial peptides. Here we show that SodCI non-covalently binds peptidoglycan. SodCI binds to Salmonella and Bacillus peptidoglycan, but not peptidoglycan from Staphylococcus. Moreover, binding can be inhibited by a diaminopimelic acid containing tripeptide, but not a lysine containing tripeptide, showing that the protein recognizes the peptide portion of the peptidoglycan. Replacing nine amino acids in SodCII with the corresponding residues from SodCI confers tethering, partially delineating an apparently novel peptidoglycan binding domain. These changes in sequence increase the affinity of SodCII for peptidoglycan fragments to match that of SodCI and allow the now tethered SodCII to function during infection.

Salmonella Typhimurium combats phagocytic superoxide by producing the periplasmic superoxide dismutase, SodCI. The homologous protein, SodCII, is also produced during infection, but does not contribute to virulence. The proteins physically differ in that SodCI is dimeric, protease resistant, and non-covalently tethered within the periplasm. Conversely, SodCII is a protease sensitive monomer that is released normally from the periplasm by osmotic shock. To identify which properties correlate with virulence, we constructed over 20 enzymatically functional hybrid SodC proteins and assayed them for protease susceptibility, release by osmotic shock, multimerization, and affinity for metal cofactors. Protease susceptibility maps to the C-terminus of SodCII, while SodCI residues 120–131 are required for tethering. A protease resistant SodCII hybrid was able to substitute for SodCI during infection. Interestingly, a tethered but protease sensitive SodCII hybrid was also able to confer protection. Thus, either tethering or protease resistance is sufficient for a SodC to function during infection. These results support our model that in the macrophage, the outer membrane of Salmonella is partially disrupted by antimicrobial peptides. Periplasmic proteins, including SodCII, are released and/or phagocytic proteases gain access. SodCI is both tethered within the periplasm and protease resistant, thereby surviving to detoxify superoxide.

Salmonellae survive and propagate in macrophages to cause serious systemic disease. Periplasmic superoxide dismutase plays a critical role in this survival by combating phagocytic superoxide. Salmonella Typhimurium strain 14028 produces two periplasmic superoxide dismutases, SodCI and SodCII. Although both proteins are produced during infection, only SodCI is functional in the macrophage phagosome. We have previously shown that SodCI, relative to SodCII, is both protease resistant and tethered within the periplasm, and that either of these properties is sufficient to allow a SodC to protect against phagocytic superoxide. Tethering is defined as remaining cell-associated after osmotic shock or treatment with cationic antimicrobial peptides. Here we show that SodCI non-covalently binds peptidoglycan. SodCI binds to Salmonella and Bacillus peptidoglycan, but not peptidoglycan from Staphylococcus. Moreover, binding can be inhibited by a diaminopimelic acid containing tripeptide, but not a lysine containing tripeptide, showing that the protein recognizes the peptide portion of the peptidoglycan. Replacing nine amino acids in SodCII with the corresponding residues from SodCI confers tethering, partially delineating an apparently novel peptidoglycan binding domain. These changes in sequence increase the affinity of SodCII for peptidoglycan fragments to match that of SodCI, and allow the now tethered SodCII to function during infection.