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Pseudomonas aeruginosa utilizes a type 3 secretion system to intoxicate host cells with the nucleotidyl cyclase ExoY. After activation by its host cell cofactor, filamentous actin, ExoY produces purine and pyrimidine cyclic nucleotides, including cAMP, cGMP, and cUMP. ExoY-generated cyclic nucleotides promote interendothelial gap formation, impair motility, and arrest cell growth. The disruptive activities of cAMP and cGMP during the P. aeruginosa infection are established; however, little is known about the function of cUMP. Here, we tested the hypothesis that cUMP contributes to endothelial cell barrier disruption during P. aeruginosa infection. Using a membrane permeable cUMP analog, cUMP-AM, we revealed that during infection with catalytically inactive ExoY, cUMP promotes interendothelial gap formation in cultured pulmonary microvascular endothelial cells (PMVECs) and contributes to increased filtration coefficient in the isolated perfused lung. These findings indicate that cUMP contributes to endothelial permeability during P. aeruginosa lung infection. NEW & NOTEWORTHY: During pneumonia, bacteria utilize a virulence arsenal to communicate with host cells. The Pseudomonas aeruginosa T3SS directly introduces virulence molecules into the host cell cytoplasm. These molecules are enzymes that trigger interkingdom communication. One of the exoenzymes is a nucleotidyl cyclase that produces noncanonical cyclic nucleotides like cUMP. Little is known about how cUMP acts in the cell. Here we found that cUMP instigates pulmonary edema during Pseudomonas aeruginosa infection of the lung. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) synthesis in Gram‐negative bacteria is completed at the outer leaflet of the inner membrane (IM). Following synthesis, seven LPS transport (Lpt) proteins facilitate the movement of LPS to the outer membrane (OM), an essential process that if disrupted at any stage has lethal effects on bacterial viability. LptB2FG, the IM component of the Lpt bridge system, is a type VI ABC transporter that provides the driving force for LPS extraction from the IM and subsequent transport across a stable protein bridge to the outer leaflet of the OM. LptC is a periplasmic protein anchored to the IM by a single transmembrane (TM) helix intercalating within the lateral gate formed by LptF TM5 and LptG TM1. LptC facilitates the hand‐off of LPS from LptB2FG to the periplasmic protein LptA and has been shown to regulate the ATPase activity of LptB2FG. Here, using an engineered chromosomal knockout system in Escherichia coli to assess the effects of LptC mutations in vivo, we identified six partial loss of function LptC mutations in the first unbiased alanine screen of this essential protein. To investigate the functional effects of these mutations, nanoDSF (differential scanning fluorimetry) and site‐directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy in combination with an in vitro ATPase assay show that specific residues in the TM helix of LptC destabilize the LptB2FGC complex and regulate the ATPase activity of LptB. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that utilizes a type IH secretion system to subvert host innate immunity. Of the 4 known effector proteins injected into eukaryotic cells, ExoS and ExoU are cytotoxic. The cytotoxic phenotype of ExoU depends on the enzymatic activity of the patatin-like phospholipase A2 domain localized to the N-terminal half of the protein. Amino acid residues located within the C-terminal region of ExoU are postulated to be required for trafficking or localization to the plasma membrane of eukaryotic cells. This report describes the characterization of a transposon-based linker insertion library in ExoU. Utilizing an unbiased screening approach and sensitive methods for measuring enzymatic activity, we identified regions of ExoU that are critical for activation of the phospholipase activity by the only known cofactor, SOD1. Insertions at D572 and L618 reduced the rate of substrate cleavage. Enzymatic activity could be restored to almost parental levels when SOD1 concentrations were increased, suggesting that the linker insertion disrupted the interaction between ExoU and SOD1. An enzyme-linked immunosorbent assay (ELISA)-based binding test was developed to measure ExoU-SOD1 binding. These experiments suggest that ExoU activation by SOD1 is hampered by linker insertion. ExoU derivatives harboring minimal phospholipase activity retained biological activity in tissue culture assays. These proteins affected primarily cellular architecture in a manner similar to that of ExoT. Our studies suggest that conformational changes in ExoU are facilitated by SOD1. Importantly, the level of phospholipase activity influences the biological outcome of ExoU intoxication. doi: 10.1128/JB.00904-09

ExoU is a potent Pseudomonas aeruginosa cytotoxin translocated into host cells by the type III secretion system. A comparison of genomes of various P. aeruginosa strains showed that that the ExoU determinant is found in the same polymorphic region of the chromosome near a [tRNA.sup.Lys] gene, suggesting that exoU is a horizontally acquired virulence determinant. We used yeast recombinational cloning to characterize four distinct ExoU-encoding DNA segments. We then sequenced and annotated three of these four genomic regions. The sequence of the largest DNA segment, named ExoU island A, revealed many plasmid- and genomic island-associated genes, most of which have been conserved across a broad set of [beta]- and [gamma]-Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 [tRNA.sup.Lys]-linked genomic island, the pathogenicity islands of strain PA14, and pKLC102 of clone C strains allowed us to propose a mechanism for the origin and transmission of the ExoU determinant. The evolutionary history very likely involved transposition of the ExoU determinant onto a transmissible plasmid, followed by transfer of the plasmid into different P. aeruginosa strains. The plasmid subsequently integrated into a [tRNA.sup.Lys] gene in the chromosome of each recipient, where it acquired insertion sequences and underwent deletions and rearrangements. We have also applied yeast recombinational cloning to facilitate a targeted mutagenesis of ExoU island A, further demonstrating the utility of the specific features of the yeast capture vector for functional analyses of genes on large horizontally acquired genetic elements.

Recombinant ExoU (rExoU) and yeast extract were used to optimize an in vitro phospholipase assay as a basis for identifying the mechanism for enzyme activation and substrate specificity. Our results support a model in which a eukaryotic protein cofactor or complex facilitates the interaction of rExoU with phospholipid substrates.

Acute cytotoxicity of epithelial cells and macrophage cell lines is caused by the effector protein ExoU of Pseudomonas aeruginosa. Molecular analysis reveal that N- and C-terminal regions are necessary for toxicity and a middle region of the protein reduces its activity. Data also indicate that ExoU does not oligomerize.

Research was conducted to examine the genetic characterization of the exoU locus in the Pseudomonas aeruginosa. A deletion analysis was performed to determine the minimal region of the ExoU locus required for expression and cytotoxicity. Results indicate that histidine-tagged recombinant SpcU and ExoU associate in a noncovalent complex that requires an amino-terminal domain within ExoU.

A comparative study was conducted on extracellular protein profiles from wild-type and regulatory or secretory isogenic mutants of the Pseudomonas aeruginosa exoenzyme S regulon to identify proteins coordinately secreted with ExoS. P aeruginosa homologs to proteins of Yersinia spp that act as regulators of the low calcium response, regulators of secretion, and mediators of the type III translocation mechanism were identified.

The mouse pathogen Pseudomonas aeruginosa AC869 was employed as a model to determine whether it could be attenuated without affecting its capacity to degrade 3,5-dichlorobenzoate, to grow in common laboratory media and to survive in 3,5-dichlorobenzoate-contaminated soil. A less virulent mutant, AC869-11, was isolated by utilizing bacteriophage E79. AC869-11 produced substantially less O antigen on its LPS than AC869 when grown in mouse lung and cecal mucus. Despite attenuation, AC869-11 grew as well as AC869 with 3,5-dichlorobenzoate as the only carbon and energy source.

Translational fusion, complementation and T7-directed protein expression analyses in Escherichia coli hosts were utilized to determine the contributions of ExsC and ExsB in the regulation of exoenzyme S synthesis. Functional analysis of the Pseudomonas aeruginosa transcriptional activators indicated the role of ExsA in promoting maximal exoenzyme S expression. On the other hand, ExsC influenced exoenzyme S expression in the posttranscriptional level.

The biochemical defects of the 53-kilodalton (Exo53) form of exoenzyme S in Pseudomonas aeruginosa were analyzed by utilizing recombinant deletion protein containing the catalytic domain of the exoenzyme S. Analysis of the amino acid sequence of the Exo53 form of the exoenzyme S indicated the absence of one or more amino acids that were required for the expression of adenosine 5'-diphosphate (ADP)-ribosyltransferase activity. Furthermore, the low specific activity of Exo53 for ADP-ribosylation was intrinsic to the protein and was not caused by steric inhibition from its amino terminus.