Abstract
Pseudomonas aeruginosa is an opportunistic human pathogen with a complex respiratory chain. The bacterium is predicted to express three NADH:ubiquinone oxidoreductases (NDH-1, NDH-2 and Nqr). We created deletions strains of the predicted NADH:ubiquinone oxidoreductases alone, and in combination to determine the respective roles of the NADH dehydrogenases in growth and virulence. NDH-1 and NDH-2 were largely redundant under aerobic conditions. Aerobic NADH dehydrogenase enzymatic activity assay was lost with deletion of both NDH-1 and NDH-2. Under anaerobic conditions, NDH-1 was required for robust growth, and overexpression of NDH-2 rescued the NDH-1 anaerobic growth defect in rich media. There was not compensatory upregulation of NDH-2 under anaerobic conditions in NDH-1 deletion strains. To test which genes were required for in vivo virulence, we used both an insect and plant disease model. In the Galleria mellonella model, neither deletion of NDH-1 nor NDH-2 led to a change in median lethal dose, although death occurred more slowly in the NDH-1 deletion infections. In a lettuce model of virulence, loss of NDH-1 caused a decrease in recovered viable bacteria and a decrease in visual tissue damage. The compound deletion of NDH-1/NDH-2 causes a severe growth defect, both under aerobic and anaerobic conditions, and was avirulent in a lettuce model. Together, these results demonstrate the redundancy of the P. aeruginosa respiratory chain at the NADH dehydrogenase level in aerobic growth and virulence.
Highlights
Pseudomonas aeruginosa is an opportunistic pathogen that causes pneumonia, chronic airway infections, and urinary tract infections (Magill et al, 2014; Crull et al, 2018)
The nuoIJ deletion was encountered while using gentamicin as a selection agent, and because the nuo operon has previously been reported to be under genetic pressure after exposure to aminoglycosides, we tested if deletion of nuoIJ resulted in an increase in gentamicin Minimum Inhibitory Concentration (MIC) using agar dilution (Yen and Papin, 2017)
We observed that of the NADH dehydrogenases in P. aeruginosa, NDH-1 is required for anaerobic growth in rich broth and glucose, as well as full virulence in plant and insect pathogenesis models
Summary
Pseudomonas aeruginosa is an opportunistic pathogen that causes pneumonia, chronic airway infections, and urinary tract infections (Magill et al, 2014; Crull et al, 2018). The bacterium is ubiquitous in both soil and water environments. To subsist in this wide range of environments, P. aeruginosa is able to metabolize a wide range of carbon sources. There are up to 17 predicted dehydrogenases that may be coupled to the quinone pool, though the physiological role of these enzymes remains poorly understood (Williams et al, 2007). The P. aeruginosa genome encodes at least three bioinformatically predicted NADH dehydrogenases (NADH:quinone oxidoreductases). NDH-1, encoded by the nuoA-N operon (PA2637-2649), is homologous to the mitochondrial complex I, and has a fused nuoCD subunit (Spero et al, 2016). NDH-1 enzymes both translocate protons and oxidize NADH to NAD+ (Williams et al, 2007). Mutations within the nuo operon are found in association with aminoglycoside resistance, presumably due to the energy dependency of aminoglycoside uptake (Amini et al, 2011; Ezraty et al, 2013; Yen and Papin, 2017)
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