CrpH of Bordetella pertussis, a prototypic PepSYTM protein supporting heme-copper oxidoreductase function

In the living world, copper is both toxic in excess and necessary for the activity of specific oxidoreductases and electron transfer chains and as such is involved in the host-pathogen interface. Mammalian hosts deploy anti-microbial strategies of copper intoxication or starvation of invading microorganisms, collectively called nutritional immunity, and bacteria have developed both protection and acquisition systems in response. We recently described a TonB-dependent copper importer, CrtABp in the whooping cough agent Bordetella pertussis. Here we characterized another protein encoded in the same operon and similarly upregulated by copper starvation, CrpH. By combining in vitro and in vivo experiments with transcriptomics, we showed that CrpH contributes to bacterial fitness and enhances respiration by the heme-copper oxidoreductases (HCO) of B. pertussis. CrpH belongs to the PepSYTM superfamily of membrane-associated bacterial enzymes, whose known members catalyze heme-mediated ferrisiderophore reduction in the periplasm. The corresponding heme-binding motifs of CrpH are similarly required for function. Furthermore, we uncovered a synthetic growth phenotype of a double crpH-ccoG mutant, the latter gene encoding a putative copper reductase involved in HCO assembly. In silico analyses identified thousands of CrpH orthologs, leading us to define a new subfamily of PepSYTM proteins found in diverse bacterial species all harboring HCO genes. Collectively, our results indicate that CrpH of B. pertussis is a prototype of a family of proteins supporting HCO function.