Non-typeable Haemophilus influenzae (NTHi) is a host-adapted human pathogen that causes diseases of the respiratory tract. During host colonization, NTHi encounter reactive oxygen and chlorine species (ROS, RCS) generated mostly by human immune cells. In NTHi, while intracellular stress defence mechanisms are well studied, oxidative stress defense mechanisms that protect the NTHi cell envelope from ROS and RCS released by host cells during bacterial infection are not well studied.
We have recently identified three periplasmic enzymes, the two molybdenum-containing S-/N-oxide reductases, DmsA & MtsZ and a peptide methionine sulfoxide reductase (MsrAB) which play significant roles in enhancing NTHi's survival during infection and are induced by ROS and RCS. We found that MsrAB repairs MetSO residues in essential H. influenzae proteins and is required for survival in the presence of RCS while NTHi MtsZ and DmsABC can convert various S- and N-oxides, but not protein-bound MetSO. Mutations in MtsZ and DmsABC strongly attenuated NTHi strains during infection. As the S-oxide repair system appears to have some redundancy, we created double mutant strains, that each contain a single functional S-oxide reductase. Interestingly, under microaerobic conditions, growth of all strains remained comparable to the wild-type; however, under aerobic conditions, strains lacking the msrAB gene exhibited significantly reduced growth rates. As expected, the absence of msrAB rendered strains sensitive to RCS killing, but unexpectedly DmsA and MtsZ also played a role in resistance to less reactive RCS such as N-chlorotaurine (NCT), compounds commonly encountered during bacterial infection. In infection assays all double mutants showed decreased attachment and invasion of human bronchial epithelial cells after 24 hours of co-culture, with decreases in intracellular populations being strongest in strains lacking dmsA. This collective evidence strongly suggests that MsrAB, DmsABC, and MtsZ are a new bacterial stress defence system that plays an essential role in NTHi in-host survival and pathogenesis by protecting the bacterial cell envelope. Similar defense systems may exist in a range of other, Gram-negative bacterial pathogens, in which homologues of these three enzymes are found. If confirmed, MsrAB and the molybdenum-containing S-oxide reductases might serve as potential bacterial-specific drug targets with wide applicability.