We used plants as an in vivo pathogenesis model for the identification of virulence factors of the human opportunistic pathogen Pseudomonas aeruginosa. Nine of nine TnphoA mutant derivatives of P. aeruginosa strain UCBPP-PA14 that were identified in a plant leaf assay for less pathogenic mutants also exhibited significantly reduced pathogenicity in a burned mouse pathogenicity model, suggesting that P. aeruginosa utilizes common strategies to infect both hosts. Seven of these nine mutants contain TnphoA insertions in previously unknown genes. These results demonstrate that an alternative nonvertebrate host of a human bacterial pathogen can be used in an in vivo high throughput screen to identify novel bacterial virulence factors involved in mammalian pathogenesis.Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium isolated from soil, water, and plants (1), and is an opportunistic human pathogen that infects patients who are immunodeficient or otherwise compromised. A variety of P. aeruginosa virulence factors have been described, and the majority of these, such as exotoxin A, elastase, and phospholipase C, were first detected biochemically on the basis of their cytotoxic activity (2). Subsequently, the genes corresponding to these factors or genes that regulate the expression of these factors were identified. In general, most pathogenicity-related genes in mammalian bacterial pathogens were first detected by using a biochemical assay. In contrast to mammalian pathogens, simple systematic genetic strategies have been routinely employed to identify pathogenicity-related genes in plant pathogens. Following random transposon-mediated mutagenesis, thousands of mutant clones of the phytopathogen are inoculated separately into individual plants to determine if they contain a mutation that affects the pathogenic interaction with the host (3-8). Comparable experiments with wholeanimal mammalian pathogenicity models are not feasible because of the vast number of animals that must be subjected to pathogenic attack.Reports indicating similarities between plant and animal pathogens (9, 10) prompted us to search for a strain of P. aeruginosa that was capable of eliciting disease in both a well-defined plant pathogenesis model and a well-defined animal pathogenesis model. Recently, we described (11) a clinical isolate of P. aeruginosa, UCBPP-PA14, that is infectious in both an Arabidopsis thaliana leaf infiltration model and in a mouse full-thickness skin thermal burn model. We showed that mutations in three P. aeruginosa pathogenicity-related genes, toxA, plcS, and gacA, caused significant decrease in pathogenicity in both models (11). The utilization of common virulence-related genes by P. aeruginosa for infecting animals and plants led us to hypothesize that previously unknown virulence determinants required for P. aeruginosa pathogenesis in animals could be identified by screening randomly mutagenized UCBPP-PA14 clones for ones that exhibited decreased virulence in plants. A subset of these mutants would...

Enterobacteriaceae produce amyloid proteins called curli that are the major proteinaceous component of biofilms. Amyloids are also produced by humans and are associated with diseases such as Alzheimer's. During the multistep process of amyloid formation, monomeric subunits form oligomers, protofibrils, and finally mature fibrils. Amyloid ␤ oligomers are more cytotoxic to cells than the mature amyloid fibrils. Oligomeric intermediates of curli had not been previously detected. We determined that turbulence inhibited biofilm formation and that, intriguingly, curli aggregates purified from cultures grown under high-turbulence conditions were structurally smaller and contained less DNA than curli preparations from cultures grown with less turbulence. Using flow cytometry analysis, we demonstrated that CsgA was expressed in cultures exposed to higher turbulence but that these cultures had lower levels of cell death than less-turbulent cultures. Our data suggest that the DNA released during cell death drives the formation of larger fibrillar structures. Consistent with this idea, addition of exogenous genomic DNA increased the size of the curli intermediates and led to binding to thioflavin T at levels observed with mature aggregates. Similar to the intermediate oligomers of amyloid ␤, intermediate curli aggregates were more cytotoxic than the mature curli fibrils when incubated with bone marrow-derived macrophages. The discovery of cytotoxic curli intermediates will enable research into the roles of amyloid intermediates in the pathogenesis of Salmonella and other bacteria that cause enteric infections. IMPORTANCE Amyloid proteins are the major proteinaceous components of biofilms, which are associated with up to 65% of human bacterial infections. Amyloids produced by human cells are also associated with diseases such as Alzheimer's. The amyloid monomeric subunits self-associate to form oligomers, protofibrils, and finally mature fibrils. Amyloid ␤ oligomers are more cytotoxic to cells than the mature amyloid fibrils. Here we detected oligomeric intermediates of curli for the first time. Like the oligomers of amyloid ␤, intermediate curli fibrils were more cytotoxic than the mature curli fibrillar aggregates when incubated with bone marrow-derived macrophages. The discovery of cytotoxic curli intermediates will enable research into the roles of amyloid intermediates in the pathogenesis of Salmonella and other bacteria that cause enteric infections.

The mechanisms by which the gut luminal environment is disturbed by the immune system to foster pathogenic bacterial growth and survival remain incompletely understood. Here, we show that STAT2 dependent type I IFN signaling contributes to the inflammatory environment by disrupting hypoxia enabling the pathogenic S . Typhimurium to outgrow the microbiota. Stat2 -/- mice infected with S . Typhimurium exhibited impaired type I IFN induced transcriptional responses in cecal tissue and reduced bacterial burden in the intestinal lumen compared to infected wild-type mice. Although inflammatory pathology was similar between wild-type and Stat2 -/- mice, we observed decreased hypoxia in the gut tissue of Stat2 -/- mice. Neutrophil numbers were similar in wild-type and Stat2 -/- mice, yet Stat2 -/- mice showed reduced levels of myeloperoxidase activity. In vitro , the neutrophils from Stat2 -/- mice produced lower levels of superoxide anion upon stimulation with the bacterial ligand N -formylmethionyl-leucyl-phenylalanine (fMLP) in the presence of IFNα compared to neutrophils from wild-type mice, indicating that the neutrophils were less functional in Stat2 -/- mice. Cytochrome bd- II oxidase-mediated respiration enhances S . Typhimurium fitness in wild-type mice, while in Stat2 -/- deficiency, this respiratory pathway did not provide a fitness advantage. Furthermore, luminal expansion of S . Typhimurium in wild-type mice was blunted in Stat2 -/- mice. Compared to wild-type mice which exhibited a significant perturbation in Bacteroidetes abundance, Stat2 -/- mice exhibited significantly less perturbation and higher levels of Bacteroidetes upon S . Typhimurium infection. Our results highlight STAT2 dependent type I IFN mediated inflammation in the gut as a novel mechanism promoting luminal expansion of S . Typhimurium.

Background:The structure and function of GPR35 are not understood. Results: Using a GPR35 activated state molecular model, we identified crucial amino acid residues required for ligand activation using ␤-arrestin trafficking, ERK1/2 activation, and calcium imaging. Conclusion: Arginines in TMH3-4-5-6 affected agonist signaling. Significance: Identification of residues for GPR35 agonist signaling is critical for the design of ligands with improved potency.