ExoU, a cytotoxin translocated into host cells via the type III
ExoU, a cytotoxin translocated into host cells via the type III secretion system of in the lung. almost 20% of all pneumonia cases in rigorous care units (ICU) and is usually the leading cause of ventilator-associated pneumonia (VAP) (30). Additionally, as a causative agent of VAP, has a higher attributable mortality rate than most other bacteria, making it a particularly dangerous pathogen (4). Of the many bacterial factors that contribute to the pathogenesis of isolates, the repertoire of effector-encoding genes is usually variable. Approximately 28% of strains obtained buy SU14813 double bond Z from acute infections encode a potent cytotoxin, ExoU (8), which is usually a marker of highly virulent strains isolated from patients with VAP (35). In animal models, ExoU secretion greatly augments the virulence of (1, 20, 21, 37). The contribution of ExoU to virulence is usually attributable to its phospholipase A2 activity (27, 34). Upon injection into host cells, ExoU is usually activated and targeted to the plasma membrane, where it cleaves membrane phospholipids, resulting in rapid and complete cell lysis (10, 14, 27-29, 34). In addition to ExoU, isolates can encode other effectors, including ExoS, ExoT, and ExoY in various combinations (8). ExoS and ExoT are bifunctional enzymes with 75% amino acid identity and comparable functional domains, and each of them has GTPase-activating protein (GAP) and ADP-ribosyltransferase (ADPRT) activities (2). ExoY is usually an adenylate cyclase (41). Interestingly, most strains contain either or strains are capable of persisting in the lungs of infected animals despite the fact that they elicit an exaggerated immune response consisting of infiltrating inflammatory cells. Recruited phagocytic cells were impaired by ExoU during acute lung contamination, resulting in a localized paucity of functional phagocytes within the airways, which allowed bacteria to persist and cause severe disease (6). However, whether phagocytes were directly injected with ExoU or were compromised indirectly following intoxication of other cell types was unclear. Recently, a fluorogenic -lactam substrate, CCF2-AM, has been used to detect translocation of bacterial proteins into host cells and (3, 11, 17, 24, 25). Upon diffusion into host cells, intact CCF2-AM exhibits fluorescence resonance energy transfer (Worry) resulting in green LRP2 fluorescence. Cleavage of this substrate by a -lactamase molecule disrupts Worry, resulting in a shift to blue fluorescence (3, 42). Fusion of a bacterial protein (in this study ExoU) with a -lactamase tag allows detection of protein translocation into cells by virtue of the change in fluorescence emission. Here we used buy SU14813 double bond Z CCF2-AM to identify cell types intoxicated with ExoU in a buy SU14813 double bond Z mouse model of acute pneumonia. We found that phagocytic cells were injected with ExoU in the lung. Resident alveolar macrophages were injected with ExoU in the earliest stages of contamination, but as neutrophils and monocytes were rapidly recruited to the lung, they became the predominant cell types injected with ExoU. In comparison, only a small number of lymphocytes and type II alveolar epithelial cells were injected in the lung and progression to severe disease. MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains, mutants, and plasmids used in this study are listed in Table ?Table1.1. PA99 is a clinical isolate that naturally contains the genes but lacks the gene (8). PA99null and PA99secr? (PA99 TOP10 cells (Invitrogen) were used for cloning. TABLE buy SU14813 double bond Z 1. strains and plasmids used in this study Bacterial strains were streaked from frozen cultures onto Luria-Bertani (LB) agar or Vogel-Bonner minimal (VBM) agar (39). For infection, overnight cultures of grown in 5 ml MINS medium (26) at 37C were diluted into fresh medium and regrown to exponential phase. Generation of effector-Bla fusions. The stop codon of was altered, and an AgeI site was engineered at the 3 end of by site-directed mutagenesis using plasmid mini-CTX(29) as the template to generate mini-CTXfragment was ligated into mini-CTXpromoter was generated by amplifying the open reading frame of glutathione was altered by site-directed mutagenesis. In a subsequent reaction, the fragment was buy SU14813 double bond Z ligated into AgeI-digested mini-CTXstrain S17.1 (38) and, following conjugation, was introduced into a neutral site in the PA99null chromosome via integrase-mediated recombination at the site using previously described approaches (16) to generate PA99null+ExoU-Bla, PA99null+ExoU(S142A)-Bla, and PA99null+GST-Bla, respectively. mini-CTXand mini-CTXwere also introduced into the chromosome of PA99secr? to create strains PA99secr?+ExoU-Bla and PA99secr?+ExoU(S142A)-Bla. Integration was confirmed by PCR amplification of each construct from intact bacterial colonies..