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.
Authors
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
(A) Still images of lung taken 20 minutes after i.v. infection by the specified pathogens using spinning-disk intravital imaging. Neutrophils, red; P. aeruginosa, S. aureus, S. pneumoniae, and E. coli, green; endothelial cells, blue. Scale bar: 50 μm. (B) Quantification of 10-minute intravital videos: percentage of neutrophils with bacteria per FOV was assessed in each mouse. n = 3; 3 fields were assessed per group. Error bars represent SEM. **P = 0.0006, ***P = 0.0002. (C) SD-IVM was used to assess the ability of pulmonary intravascular neutrophils to recognize the P. aeruginosa mutants ΔpslA and ΔpcrV in the lung capillaries after blood-borne infections. Images represent still captures of live videos 20 minutes after infection. Graphs represent quantification of intravital videos: number of neutrophils with bacteria per FOV was assessed in each mouse. n = 4; 3 fields were assessed per group; 10-minute videos. Error bars represent SEM. ****P < 0.0001. (D) Neutrophil phagocytosis of P. aeruginosa WT and mutants was assessed by flow cytometry. Percentage of Ly6G-positive cells containing P. aeruginosa–GFP. n = 3. **P = 0.0087. (E) SD-IVM was used to assess the ability of pulmonary intravascular neutrophils to recognize P. aeruginosa mutants noted on the figure in the lung capillaries after blood-borne infections of complement component 3–knockout mice (C3–/–). n = 3; 3 fields were assessed per group. Error bars represent SEM. Unless otherwise stated, 1-way ANOVA statistical analysis was performed on data points; multiple comparisons were performed between indicated columns. Graphs labeled “IVM” quantify intravital microscopy data, not flow cytometry data. All experiments were repeated 3 times unless otherwise indicated.