Using a FRET-based reporter assay, we directly showed that phagocytic cells are extensively injected with ExoU during acute pneumonia caused by P. aeruginosa. Resident alveolar macrophages were injected early during infection, while recruited neutrophils and monocytes were injected slightly later as these cells entered the lungs in response to the invading microbes. To our knowledge, this is the first identification of cell types targeted by a P. aeruginosa type III effector in vivo and the first examination of injection into cells during pneumonia for any type III secretion system. Thus, our results provide important insights into the mechanisms by which bacteria cause pneumonia.
Using a noncytotoxic ExoU variant, we found that the types of cells injected with ExoU reflect the overall repertoire of immune cells in the lungs over the first 18 h of infection. Injection was observed as early as 3 h postinfection, at a time when few inflammatory cells were present in the lungs. Thus, resident alveolar macrophages comprised the majority of the cells initially injected in the lung. The subsequent influx of neutrophils and monocytes into the lung correlated with an increase in injection of these cell types, suggesting that cell type targeting of ExoU is to a large extent determined by the availability of cells rather than a specific preference of the type III secretion apparatus or P. aeruginosa
adhesins for particular cell types. However, a somewhat higher proportion of neutrophils than of other cell types in the lungs were injected with ExoU. This may have simply reflected increased numbers of P. aeruginosa
bacteria binding to neutrophils because of the large surface area and phagocytic nature of these cells or may have been because a larger proportion of neutrophils migrate into the airways and come into contact with bacteria. In any case, our results indicate that phagocytic cells are a primary target of ExoU injection in vivo
. Since most ExoU+
clinical isolates also secrete ExoT and many ExoU+
clinical isolates secrete ExoY, it may be that these effector proteins are similarly targeted. ExoS, however, is rarely secreted by ExoU+
isolates, and ExoS-secreting strains constitute a clonal group that is distinct from ExoU+
). For these reasons, it remains possible that ExoS-secreting strains differ in pathogenesis and that this effector protein is injected into distinct populations of host cells. Examination of the cell types injected with other P. aeruginosa
effectors during acute pneumonia is currently under way.
Targeting of phagocytic cells, including neutrophils, has previously been implicated in the pathogenesis of other Gram-negative bacterial pathogens. Geddes and colleagues showed that Salmonella enterica
targets splenic neutrophils for injection of type III effectors to promote intracellular survival in vivo
). Similarly, Marketon et al. (24
) and Koberle et al. (17
) observed injection into neutrophils, macrophages, and dendritic cells in the spleen during infection with Yersinia
spp. In these cases, targeting of splenic innate immune cells was shown to be a mechanism for evading or disrupting host immune responses. Our results demonstrate that neutrophils are also targets for type III secretion in the lung. Previous experimental observations suggested that impairment of recruited phagocytic cells by ExoU was important for P. aeruginosa
survival in the lung and progression to severe disease. In agreement with this functional evidence, we found that P. aeruginosa
injected ExoU into neutrophils and macrophages during pneumonia. Impairment or killing of these cells by direct injection of ExoU may prevent effective clearance from the lung and contribute to the enhanced disease observed with ExoU+
Interestingly, we did not observe significant injection into type II pneumocytes during infection. There are several possible explanations for this finding. Infiltrating inflammatory cells may bind P. aeruginosa
with increased affinity, thereby reducing the number of bacteria available to bind to epithelial cells. Alternatively, type II pneumocytes may be resistant to injection of ExoU. If this is the case, damage to the lung epithelium, previously observed during infection with ExoU+ P. aeruginosa
), may be an indirect effect caused by release of damaging inflammatory mediators from ExoU-lysed phagocytic cells rather than by direct killing by injection of ExoU into epithelial cells. Additional studies are necessary to understand the interaction between pulmonary epithelial cells and ExoU.
Injection of ExoU occurred disproportionately in the airspace compared to other compartments of the lung during early pneumonia. This finding is consistent with several previous observations. First, the majority of P. aeruginosa
bacteria reside in the airways and alveoli of the lungs during pneumonia (7
). Since type III secretion requires direct contact between the bacterium and the host cell, this implies that secretion occurs primarily in the airspace. Second, we previously observed a relative reduction in the number of viable neutrophils in the airways and alveoli compared to whole lung compartments of mice infected with ExoU+ P. aeruginosa
), suggesting that neutrophils are injected with ExoU upon transmigration across the respiratory epithelium. Thus, in early pneumonia, type III secretion may be employed most actively against immune cells as they enter the airways and alveoli.
We were unable to detect secretion of catalytically active ExoU-Bla into host cells in vivo
. This was likely due to the rapid cell lysis induced by this molecule since cells injected with ExoU(S142A)-Bla, a noncytotoxic ExoU variant, were detected at all time points examined. Previously, it has been shown that ExoU induces rapid and complete cell lysis of neutrophils in vitro
, which precludes detection of cells into which ExoU has been injected in vivo
). Similarly, other investigators have also been unable to detect cells containing catalytically active ExoU (9
). Our inability to detect cells injected with catalytically active ExoU further supports the idea that ExoU induces rapid lysis of phagocytic cells in vivo
. The use of a noncatalytic variant of ExoU rather than wild-type ExoU was one limitation of this study because the lack of cell killing by ExoU alters bacterial survival, disease progression, and the host immune response. We attempted to more accurately mimic infection with a strain producing wild-type ExoU by increasing the infecting dose to increase the overall number of bacteria in the lung and enhance recruitment of inflammatory cells to the lung. Nonetheless, it is possible that the dynamics of translocation may be somewhat different in the presence of wild-type ExoU. Another potential limitation is that throughout this study we utilized a strain which secreted ExoU(S142A)-Bla alone, without other effector proteins. Clinical isolates of P. aeruginosa
usually secrete combinations of type III effector proteins. However, this does not appear to be a major limitation since the presence of ExoS and ExoT did not dramatically alter the types of cells injected with ExoU and since the injected cell populations continued to closely mirror the total phagocytic cell populations present in the lung (Fig. ). This is consistent with reports indicating that cosecretion of ExoS or ExoT along with ExoU did not substantially alter the severity of pneumonia (36
). Nonetheless, cosecretion of other effector proteins could have subtle effects on the total numbers and proportions of host cells injected with ExoU. Interestingly, the repertoire of inflammatory cells recruited to the lung did change when ExoS or ExoS and ExoT were secreted along with ExoU(S142A), indicating that the effector proteins did dictate the nature of the inflammatory response.
Based on direct analysis of injection into cells in the lung during acute infection, the results of this study suggested a model for the mechanism of ExoU activity during early pneumonia. During the initial stage of infection, ExoU is injected into alveolar macrophages, which may incapacitate these sentinel cells that are crucial in the host defense during early P. aeruginosa
). Later injection of ExoU into phagocytes may allow P. aeruginosa
to evade phagocytosis by these immune cells, allowing large numbers of bacteria to persist in the lung. Thus, injection of ExoU creates an environment in which the bacteria can survive and cause the pathology observed during severe pneumonia.