Controlling Pseudomonas aeruginosa Persister Cells by Human Granulocyte Macrophage Colony-Stimulating Factor

Bacteria are well known to cause chronic infections in humans by entering dormancy and by developing biofilms. These mechanisms allow bacteria to exhibit antibiotic tolerance and relapse to an active virulent state when the antibiotic treatments are discontinued. During bacterial invasions, the host immune cells secrete special signaling proteins, known as cytokines which orchestrate events leading to human immune response and elimination of bacterial pathogens. However, compared to the well documented activities of cytokines in immune reaction, little is known about the direct effects of cytokines on bacterial cells. In this study, we focused on granulocyte macrophage colony-stimulating factor (GM-CSF), a cytokine produced by macrophages, T-cells, endothelial cells, and fibroblasts. We chose Pseudomonas aeruginosa as a model bacterium. It is an opportunistic pathogenic bacterium and a major cause of nosocomial infections in individuals with compromised immune systems and cystic fibrosis (CF) patients. We show for the first time that GM-CSF can sensitize the persister cells of P. aeruginosa PAO1 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. The mucoid variant, P. aeruginosa PDO300 was also sensitized by GM-CSF to tobramycin in the presence of alginate lyase. In addition, GM-CSF sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in presence of biofilm matrix degrading enzymes DNase I and alginate lyase, respectively. In comparison, the normal cells of P. aeruginosa and the non-pathogenic Escherichia coli K12 persister cells were not affected by GM-CSF. DNA microarray and qPCR analyses revealed that GM-CSF induced flagella and pyocin associated genes in persister cells of P. aeruginosa PAO1, while the same genes in normal cells did not show significant change. Using co-immunoprecipitation (co-IP) and cross-linking, GM-CSF was found to interact with the protein FliC (flagellin). Deletion of fliC gene abolished the effects of GM-CSF on P. aeruginosa persister cells, which was restored by complementation of the fliC gene. Overall, the findings from this study suggest that cytokines have a direct interaction with bacterial cells and disturb their persistence. The results are helpful for understanding bacterial physiology and for developing new persistence control methods