# An effective response to respiratory inhibition by a Pseudomonas aeruginosa excreted quinoline promotes Staphylococcus aureus fitness and survival in co-culture

## TL;DR

Pseudomonas aeruginosa produces a toxin that inhibits Staphylococcus aureus respiration, but S. aureus adapts by switching to fermentation, improving its survival in co-culture.

## Contribution

The study identifies how S. aureus uses regulatory systems to adapt to P. aeruginosa toxins, promoting survival through fermentation.

## Key findings

- P. aeruginosa's HQNO inhibits S. aureus respiration and reduces SrrAB transcriptional output.
- S. aureus uses SrrAB and Rex to sense redox changes and increase fermentative pathways for survival.
- Strains with poor redox homeostasis show reduced fitness and survival when challenged with P. aeruginosa.

## Abstract

Pseudomonas aeruginosa and Staphylococcus aureus are primary bacterial pathogens frequently isolated from the airways of cystic fibrosis patients. P. aeruginosa produces secondary metabolites that negatively impact the fitness of S. aureus, allowing P. aeruginosa to become the most prominent bacterium when the species are co-cultured. Some of these metabolites inhibit S. aureus respiration. SrrAB is a staphylococcal two-component regulatory system (TCRS) that responds to alterations in respiratory status to help S. aureus transition between fermentative and respiratory metabolisms. Using P. aeruginosa mutant strains and chemical genetics, we established that P. aeruginosa secondary metabolites, 2-heptyl-4-quinolone N-oxide (HQNO) in particular, inhibit S. aureus respiration, resulting in decreased SrrAB transcriptional output. Metabolomic analyses demonstrated that the ratio of NAD+ to NADH increased upon prolonged culture with HQNO. Consistent with this, the activity of the Rex transcriptional regulator, which senses and responds to alterations in the NAD+/NADH ratio, repressed srrAB promoter activity upon HQNO treatment. The presence of SrrAB increased fitness when cultured with HQNO and enhanced survival when challenged with P. aeruginosa. S. aureus strains with reduced ability to maintain redox homeostasis via fermentation had decreased fitness when challenged with HQNO and lower survival when challenged with P. aeruginosa. These findings led to a model wherein P. aeruginosa secreted HQNO inhibits S. aureus respiration, resulting in manipulation of the redox status in both the membrane and cytoplasm, altering the transcriptional activities of SrrAB and Rex, which promote fitness and survival by increasing carbon flux through fermentative pathways to maintain redox homeostasis.

Cystic fibrosis is a hereditary respiratory disease that predisposes patients to bacterial infections, often caused by Staphylococcus aureus and Pseudomonas aeruginosa. Secondary metabolites excreted by P. aeruginosa decrease S. aureus fitness during co-infection, ultimately eliminating it. The regulatory systems and mechanisms that S. aureus uses to detect and respond to these metabolites are unknown. The data presented demonstrate that two regulatory systems that are stimulated by alterations in membrane or cytosolic redox status respond to the P. aeruginosa-produced respiratory toxin 2-heptyl-4-quinolone N-oxide (HQNO) by increasing transcription of genes utilized for fermentation, thereby promoting fitness. This study describes interactions between these two bacterial pathogens that could be exploited to decrease pathogen burden in individuals living with cystic fibrosis.