(553) Determinants of LasR mutant Pseudomonas aeruginosa phagocytosis resistance

Background: Pseudomonas aeruginosa (Pa) LasR mutants are found at high prevalence in CF lungs, and have been associated with accelerated decline in lung function. Reasons for LasR mutant selection have been postulated to include selective growth advantage and resistance to phagocytosis by CF macrophages, among others. How LasR mutants resist phagocytosis however is not understood.

Methods: We utilized LasR mutant Pa from both clinical isolates and laboratory strains (PA14 background) to investigate determinants of phagocytosis resistance by CF Monocyte Derived Macrophages (MDM) purified from peripheral blood of F508del homozygous donors. We measured phagocytosis by a gentamicin protection assay in the presence or absence of CFTR modulators. We measured potential off-target effects of CFTR modulators on Pa growth and motility. We then supplemented these studies with assays using model THP-1 macrophages and measured phagocytosis of LasR mutants on anr, RhlR, and phenazine knockout backgrounds.

Results: While CFTR modulators augment phagocytosis of both WT and LasR- Pa by both CF and nonCF MDM, LasR mutants continue to resist phagocytosis. CFTR modulators had no discernable off-target effects on Pa growth or motility. In the model THP-1 system, LasR mutants continued to resist phagocytosis on an anr- background. RhlR or phenazine Pa mutants, however, displayed increased phagocytosis at baseline and differences with LasR mutants on these backgrounds were no longer evident.

Conclusions: LasR mutation appears to have broad effects on phagocytosis efficiency by CF MDM and THP-1 macrophages. These differences are not explained by previously described upregulation of anr regulated genes, as the LasR/anr double mutation had no effect. RhlR or phenazine mutants however were more efficiently phagocytosed compared with wild-type PA14, whereas LasR double mutants behaved similarly to single mutants. This may help to explain the common presence of LasR mutants in the CF lung but rarity of RhlR mutants. Work is ongoing to identify specific expression differences between these strains which may underlie phagocytosis resistance, providing a target to enhance Pa clearance from the lung.

Acknowledgements: CFF ARIDGI20L0 to DA; NIH/NHLBI R01HL122372 and CFF ASHARE15A0 to AA. This work was supported by DartCF (NIH P30-DK117469) and CFF RDP STANTO19R0.

References: