Achromobacter xylosoxidans modulates Pseudomonas aeruginosa virulence through a multi-target mechanism of competition

The colonization and persistence of Pseudomonas aeruginosa in chronically diseased lungs are driven by the production of various virulence factors. However, pulmonary infections in cystic fibrosis (CF) patients are predominantly polymicrobial. While Achromobacter xylosoxidans is an opportunistic pathogen in these patients, its impact on P. aeruginosa virulence during co-infection remains largely unknown. This study investigated the interaction between P. aeruginosa and two clonally related A. xylosoxidans strains, Ax 198 and Ax 200, co-isolated from a CF patient sputum. We found that the interaction between P. aeruginosa and co-isolated A. xylosoxidans was strain-dependent, with the Ax 200 strain significantly reducing P. aeruginosa virulence in a zebrafish model, providing the first in vivo evidence of interaction between these two species during co-infection. Proteomic analysis revealed that the P. aeruginosa proteome was differently impacted by the two A. xylosoxidans strains, with Ax 200 altering proteins involved in biofilm formation, swimming motility, iron acquisition, and secretion systems. These proteomic findings were further validated by phenotypic assays, which confirmed that A. xylosoxidans affected major P. aeruginosa virulence phenotypes, including biofilm formation, swimming motility, and siderophore production. Genetic analysis also confirmed that distinct regulatory mechanisms, including mechanisms involved in the iron cycle, may account for the strain-dependent interaction effects of A. xylosoxidans with P. aeruginosa. These findings reveal a novel multi-target competitive mechanism through which A. xylosoxidans significantly disrupts P. aeruginosa virulence. IMPORTANCEPseudomonas aeruginosa, a major human pathogen, is a leading cause of mortality in cystic fibrosis (CF) patients. Understanding its virulence mechanisms is critical for developing effective infection management strategies. Given the polymicrobial nature of CF infections, it is essential to investigate interspecies interactions that may influence bacterial virulence. While A. xylosoxidans is recognized as an opportunistic pathogen in CF, its impact on P. aeruginosa virulence during co-infection remains largely unexplored. Deciphering the molecular basis of P. aeruginosa virulence in polymicrobial settings could reveal novel and specific therapeutic targets to improve treatment strategies.