Pyoverdine and proteases affect the response of pseudomonas aeruginosa to gallium in human serum

Gallium is an iron mimetic which has recently been repurposed as an antibacterial agent due to its capability to disrupt bacterial iron metabolism. In this study, the antibacterial activity of gallium nitrate [Ga(NO<inf>3</inf> )<inf>3</inf> ] was investigated in complement-free human serum (HS) on 55 Pseudomonas aeruginosa clinical isolates from cystic fibrosis and non-cystic fibrosis patients. The susceptibility of P. aeruginosa to Ga(NO<inf>3</inf>)<inf>3</inf> in HS was dependent on the bacterial ability to acquire iron from serum binding proteins (i.e., transferrin). The extent of serum protein degradation correlated well with P. aeruginosa growth in HS, while pyoverdine production did not. However, pyoverdine-deficient P. aeruginosa strains were unable to grow in HS and overcome iron restriction, albeit capable of releasing proteases. Predigestion of HS with proteinase K promoted the growth of all strains, irrespective of their ability to produce proteases and/or pyoverdine. The MICs of Ga(NO<inf>3</inf>)<inf>3</inf> were higher in HS than in an iron-poor Casamino Acids medium, where proteolysis does not affect iron availability. Coherently, strains displaying high proteolytic activity were less susceptible to Ga(NO<inf>3</inf>)<inf>3</inf> in HS. Our data support a model in which both pyoverdine and proteases affect the response of P. aeruginosa to Ga(NO<inf>3</inf>)<inf>3</inf> in HS. The relatively high Ga(NO<inf>3</inf>)<inf>3</inf> concentration required to inhibit the growth of highly proteolytic P. aeruginosa isolates in HS poses a limitation to the potential of Ga(NO<inf>3</inf>)<inf>3</inf> in the treatment of P. aeruginosa bloodstream infections.