ABSTRACTPseudomonas aeruginosais a Gram-negative pathogen commonly associated with nosocomial infections such as hospital-acquired pneumonia. It uses a type III secretion system to deliver effector proteins directly into the cytosol of host cells. Type III secretion inP. aeruginosahas been linked to severe disease and worse clinical outcomes in animal and human studies. The majority ofP. aeruginosastrains secrete ExoS, a bifunctional toxin with GTPase-activating protein and ADP-ribosyltransferase activities. Numerousin vitrostudies have investigated the targets and cellular effects of ExoS, linking both its enzymatic activities with inhibition of bacterial internalization. However, little is known about how this toxin facilitates the progression of infectionin vivo. In this study, we used a mouse model to investigate the role of ExoS in inhibiting phagocytosis during pneumonia. We first confirmed previous findings that the ADP-ribosyltransferase activity of ExoS, but not the GTPase-activating protein activity, was responsible for bacterial persistence and decreased host survival in this model. We then used two distinct assays to demonstrate that ExoS inhibited phagocytosis during pneumonia. In contrast to the findings of severalin vitrostudies, thisin vivoinhibition was also dependent on the ADP-ribosyltransferase activity, but not the GTPase-activating protein activity, of ExoS. These results demonstrate for the first time the antiphagocytic function of ExoS in the context of an actual infection and indicate that blocking the ADP-ribosyltransferase activity of ExoS may have potential therapeutic benefit.IMPORTANCEPseudomonas aeruginosais a major cause of hospital-acquired infections. To cause severe disease, this bacterium uses a type III secretion system that delivers four effector proteins, ExoS, ExoT, ExoU, and ExoY, into host cells. The majority ofP. aeruginosastrains secrete ExoS, a bifunctional toxin with GTPase-activating protein and ADP-ribosyltransferase activities. In cell culture models, both enzymatic activities have been associated with decreased bacterial internalization. However, our study is the first to examine a role for ExoS in blocking phagocytosis in an animal model. We report that ExoS does inhibit phagocytosis during pneumonia. The ADP-ribosyltransferase activity, but not the GTPase-activating protein activity, of ExoS is necessary for this effect. Our findings highlight the ability ofP. aeruginosato manipulate the inflammatory response during pneumonia to facilitate bacterial survival.