Copper Efflux System Required in Murine Lung Infection by Haemophilus influenzae Composed of a Canonical ATPase Gene and Tandem Chaperone Gene Copies

Copper is an essential micronutrient but is toxic at high concentrations. In Haemophilus influenzae mechanisms of copper resistance and its role in pathogenesis are unknown; however, our previous genetic screen by transposon insertion-site sequencing implicated a putative cation transporting ATPase ( ) in survival in a mouse lung infection model. Here, we demonstrate that H. influenzae (HI0290) is responsible for copper homeostasis involving the -type regulator, , as well as six tandem copies of the metallochaperone gene, . Deletion of the ATPase and metallochaperone genes resulted in increased sensitivity to copper but not to cobalt, zinc, or manganese. Nontypeable H. influenzae (NTHi) clinical isolate NT127 has the same locus organization but with three copies of . We showed that expression of the NTHi operon is activated by copper under the regulatory control of CueR. NTHi single and mutants and, especially, the double deletion mutant exhibited decreased copper tolerance, and the Δ mutant accumulated 97% more copper than the wild type when grown in the presence of 0.5 mM copper sulfate. Mutants of NT127 deleted of the ATPase ( ) alone and deleted of both the ATPase and chaperones ( ) were 4-fold and 20-fold underrepresented compared to the parent strain during mixed-infection lung challenge, respectively. Complementation of locus deletion mutations restored copper resistance and virulence properties. NTHi likely encounters copper as a host defense mechanism during lung infection, and our results indicate that the system encodes an important countermeasure to alleviate copper toxicity.