Cytochrome oxidase requirements in Bordetella reveal insights into evolution towards life in the mammalian respiratory tract

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B. pertussis , the causal agent of human whooping cough, and B. bronchiseptica , which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B. bronchiseptica , which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B. pertussis , which evolved from a B. bronchiseptica -like ancestor but now only survives only in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1, ctaCDFGE1 , and cyoABCD1 . To test the hypothesis that the three cytochrome oxidases encoded within the B. pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B. bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo . No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B. pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B. bronchiseptica producing only the three B. pertussis -conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also showed that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on respiration requirements for bacteria that colonize the respiratory tract, the environment at the surface of the ciliated epithelium, and the evolution of virulence in bacterial pathogens. AUTHOR SUMMARY Cytochrome oxidases, critical components for aerobic respiration, have been shown to be vital for pathogenesis and tissue tropism in several bacterial species. However, the majority of the research has focused on facultative anaerobes and infections of microoxic to anaerobic host environments, like the gut. We sought to understand the role of cytochrome oxidases during respiratory infection by Bordetella bronchiseptica , an obligate aerobe, performing the first analysis of cytochrome oxidases in an extracellular respiratory pathogen that we know of. By comparing B. bronchiseptica to the closely related B. pertussis , a strictly human-specific pathogen and the causative agent of whooping cough, we found three cytochrome oxidases that are important for growth and survival within the mammalian respiratory tract. We also found that a bo3 -type cytochrome oxidase, predicted to have a low affinity for oxygen and therefore best suited to ambient air levels of oxygen, was sufficient for both the establishment of infection and persistence in the respiratory tract in mice. Our findings reveal the importance of low affinity cytochrome oxidases in respiratory pathogens, and emphasize the need to study the physiology of diverse pathogens. ### Competing Interest Statement The authors have declared no competing interest.