Antimicrobial Effects of Reactive Nitrogen Species and Reactive Oxygen Species on Bordetella and a Role for Dermonecrotic Toxin in Bacterial Physiology

Bordetella species express virulence factors that enable host immune evasion and establishment of prolonged respiratory infection. Upon interaction with ciliated cells of the host upper airway, bordetellae are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS) as part of the host innate immune defense. I hypothesized that RNS and ROS have anti-Bordetella activity at physiologically relevant concentrations. In a novel in vitro assay, B. bronchiseptica exposure to prototypical redox active species revealed toxicity at physiologically relevant (nM-μM) concentrations. Antimicrobial synergy was observed when individual redox active species were applied in combination. Additionally, there was increased bacteriostatic activity of nitric oxide towards Bordetella relative to hydrogen peroxide. Analysis of B. bronchiseptica mutant strains unable to respond to physiological stimuli via the two-component virulence control system BvgAS identified a protective role for BvgAS in the Bordetella redox stress response. The observation that RNS and ROS produced by airway epithelial cells have anti-Bordetella activity at physiologically relevant concentrations encourages further analysis of how manipulation of redox active species in the airway can be utilized to combat airway infection. Species of the Bordetella genus express a conserved virulence factor, dermonecrotic toxin (DNT), that in purified form can target host cell cytoskeletal regulators. However, DNT is not released from cultured bacteria, and no mechanism for bacteria to host transfer has been described. I hypothesized that loss of DNT expression affects bacterial physiological processes. Comparison of wild type B. bronchiseptica and a DNT mutant strain revealed an altered proteome that included several proteins associated with bacterial metabolism. In vitro, loss of DNT expression resulted in increased sensitivity to physiological stress. Comparison of wild type and DNT mutant B. bronchiseptica interactions with primary cultured rabbit tracheal epithelial cells did not reveal a function for DNT in host cell actin cytoskeletal rearrangement during the B. bronchiseptica-host interaction. It is suggested that DNT is conserved across the Bordetella genus because of its effects on bacterial physiology. This is a novel perspective on a Bordetella factor under BvgAS control and may have implications for understanding similar necrotizing factors from other virulent bacteria