Pseudomonas aeruginosa is a major cause of severe infections that lead to bacteremia and high patient mortality. P. aeruginosa has evolved numerous evasion and subversion mechanisms that work in concert to overcome immune recognition and effector functions in hospitalized and immunosuppressed individuals. Here, we have used multilaser spinning-disk intravital microscopy to monitor the blood-borne stage in a murine bacteremic model of P. aeruginosa infection. P. aeruginosa adhered avidly to lung vasculature, where patrolling neutrophils and other immune cells were virtually blind to the pathogen’s presence. This cloaking phenomenon was attributed to expression of Psl exopolysaccharide. Although an anti-Psl mAb activated complement and enhanced neutrophil recognition of P. aeruginosa, neutrophil-mediated clearance of the pathogen was suboptimal owing to a second subversion mechanism, namely the type 3 secretion (T3S) injectisome. Indeed, T3S prevented phagosome acidification and resisted killing inside these compartments. Antibody-mediated inhibition of the T3S protein PcrV did not enhance bacterial phagocytosis but did enhance killing of the few bacteria ingested by neutrophils. A bispecific mAb targeting both Psl and PcrV enhanced neutrophil uptake of P. aeruginosa and also greatly increased inhibition of T3S function, allowing for phagosome acidification and bacterial killing. These data highlight the need to block multiple evasion and subversion mechanisms in tandem to kill P. aeruginosa.
Ajitha Thanabalasuriar, Bas G.J. Surewaard, Michelle E. Willson, Arpan S. Neupane, Charles K. Stover, Paul Warrener, George Wilson, Ashley E. Keller, Bret R. Sellman, Antonio DiGiandomenico, Paul Kubes
P. aeruginosa is captured by liver Kupffer cells from the circulation.
(A) Quantification of P. aeruginosa phagocytosis by Kupffer cells in the livers of mice. The number of bacteria arrested on or inside a Kupffer cell per field of view (FOV). n = 4; 3 FOV per group were assessed. Error bars represent SEM. (B) Representative spinning-disk intravital microscopy (SD-IVM) image of liver 15 minutes after i.v. infection with P. aeruginosa (GFP) WT, ΔpslA, or ΔpcrV. Scale bars: 50 μm. (C) Quantification of SD-IVM images for number of Kupffer cells associated with P. aeruginosa–GFP was assessed at 15 minutes after i.v. infection with WT, ΔpslA, or ΔpcrV strains. n = 4; 3 FOV per group were assessed. (D) WT mice treated 24 hours before i.v. infection with 15 mg/kg of control IgG, anti-Psl, anti-PcrV, or MEDI3902. Quantification of SD-IVM images for number of Kupffer cells associated with P. aeruginosa–GFP assessed at 15 minutes after i.v. infection with WT P. aeruginosa. Error bars represent SEM. One-way ANOVA was performed to determine statistical differences between groups. All experiments were repeated 3 times unless otherwise indicated.