A number of bacterial pathogens utilize the type III secretion pathway to deliver effector proteins directly into the host cell cytoplasm. Certain strains of Pseudomonas aeruginosa associated with acute infections express a potent cytotoxin, exoenzyme U (ExoU), that is delivered via the type III secretion pathway directly into contacting host cells. Once inside the mammalian cell, ExoU rapidly lyses the intoxicated cells via its phospholipase A 2 (PLA 2 ) activity. A high-throughput cell-based assay was developed to screen libraries of compounds for those capable of protecting cells against the cytotoxic effects of ExoU. A number of compounds were identified in this screen, including one group that blocks the intracellular activity of ExoU. In addition, these compounds specifically inhibited the PLA 2 activity of ExoU in vitro, whereas eukaryotic secreted PLA 2 and cytosolic PLA 2 were not inhibited. This novel inhibitor of ExoU-specific PLA 2 activity, named pseudolipasin A, may provide a new lead for virulence factor-based therapeutic design.Pseudomonas aeruginosa is the leading cause of hospitalacquired infections by gram-negative bacteria and is responsible for chronic infections of individuals with cystic fibrosis (15). As P. aeruginosa and other bacterial pathogens become multidrug resistant (22), there is a continual need for the identification of compounds directed at novel targets that could be developed into therapeutic agents. The ability of P. aeruginosa to cause a number of distinct infections has been attributed to its large genome, which encodes a variety of virulence factors (35). Prominent among these is the type III secretion system (TTSS), which allows the bacteria to deliver proteins directly into the host cell cytoplasm (39). ExoU is one of the toxic effector proteins delivered by the type III secretion apparatus, and its expression is associated with strains that cause acute infections (11,12). ExoU is a member of the patatin family of phospholipase A 2 (PLA 2 ), and this activity is required for cytotoxicity toward eukaryotic cells (33,34). In contrast to eukaryotic phospholipases, which remodel cellular membranes and synthesize proinflammatory secondary messengers such as arachidonic acid and leukotrienes (2, 9), ExoU PLA 2 activity primarily causes disruption of the host cytoplasmic membrane, resulting in cell lysis (24,32,33).We sought to identify compounds that inhibit type III secretion-mediated cytotoxicity by protecting tissue culture cells from infection by P. aeruginosa strains that elaborate ExoU as a sole cytotoxic effector. From a synthetic small-molecule library, we have identified compounds that protected Chinese hamster ovary (CHO) cells from the cytotoxic activity of P. aeruginosa expressing ExoU. One of the most potent compounds is pseudolipasin A (Pseudomonas phospholipase inhibitor A). Pseudolipasin A does not interfere with type III secretion in general, suggesting that the protection observed occurs downstream of the delivery of ExoU. Pseudolipasin A not only protected...

In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively.

SUMMARY Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of anti-nucleic acid autoantibodies, high levels of circulating type I interferon (IFN-I), and an IFN-I-dependent elevated expression of activating FcγR. Increases in neutrophils and monocytes are often observed in clinical SLE, but how these contribute to autoantibody and IFN-I production is poorly understood. We are analyzing SLE pathogenesis in 564Igi mice, an SLE-model strain carrying gene-targeted heavy and light chain antibody genes encoding an anti-RNA autoantibody in a C57BL/6 background. Similar to human SLE patients, 564Igi mice produce anti-RNA autoantibodies and expanded neutrophil and monocyte populations. These myeloid cells produce IFN-I and exhibit increased FcγRIV expression induced via an IFN-I autocrine loop. A direct effect of IFN-I on 564Igi bone marrow B cells and neutrophils is supported by their up-regulation of “IFN-I signature genes”. In addition, 564Igi developing B cells show up-regulated TLR7 resulting in IgG2a/2b class switch recombination and autoantibody production. Our results indicate that the production of anti-RNA autoantibody is sufficient to induce an increase of bone marrow, blood and spleen IFN-I-producing neutrophils, and suggest a mechanism by which autoantibody and IFN-I contribute to SLE by activating B lymphocytes, neutrophils and monocyte effector cells in vivo.