Heterologous expression of the Campylobacter jejuni globins: Roles of CGB and CTB in the nitrosative stress resistance in Escherichia coli

Abstract

Campylobacter jejuni, one of the main causal agents of human gastrointestinal disease worldwide, is exposed to high concentrations of nitric oxide (NO) produced by the host during transmission and pathogenesis. However, under nitrosative stress conditions, C. jejuni expresses two haemoglobins implicated in tolerance to this condition. One, a single-domain haemoglobin (Cgb) detoxifies NO apparently by its conversion to nitrate via a dioxygenase or denitrosylase activity. The other, (the truncated haemoglobin Ctb) has been linked to oxygen chemistry rather than protection from NO toxicity. Details about the molecular mechanisms of these globins remain obscure, since both lack the reductase domain for restoration of the ferrous state required for ligand-binding activity in the most studied bacterial globin, the Escherichia coli flavohaemoglobin Hmp. In the present work, over-expression of Cgb and Ctb in an hmp mutant of E. coli demonstrated that Cgb but not Ctb complements the NO resistance phenotype. Both Cgb- and Ctb-expressing cell suspensions moderately resist to respiratory inhibition by NO under aerobic conditions. However, only Cgb consumes NO endogenously produced by E. coli in anaerobic conditions. Spectroscopic studies of these proteins reveal that the haem is converted to the ferrous state in the presence of E. coli or C. jejuni soluble extracts. Re-reduction of the haem groups after oxidation by NO suggests the presence of a redox partner or reductant environment in the actual and the heterologous host. Unraveling the molecular mechanisms of these globins constitutes a key step in the understanding of the NO resistance ability of C. jejuni.