The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.

Pseudomonas aeruginosa is a gram-negative pathogen causing life-threatening infections. Lung injury and the development of sepsis depend largely on the expression of type III secretion system (TTSS) virulence. TTSS functions as a molecular syringe to deliver toxins directly to the cytosol of cells, inhibit innate immune mechanisms, and prevent bacterial clearance. Polyclonal antibodies that bind to PcrV of P. aeruginosa inhibit the delivery of type III toxins and enhance the clearance of bacteria during acute lung infections. PcrV is a homologue of LcrV, a protective antigen in the Yersinia TTSS and an integral component of TTSS. In this study, a murine monoclonal antibody (MAb) to PcrV was generated: MAb 166, which is protective against P. aeruginosa when coinstilled with the bacterial inoculum or intraperitoneally transferred to mice. Fab fragments from MAb 166 prevent sepsis and death. The epitope bound by MAb 166 was mapped to the carboxyl-terminus of PcrV.

Summary Pseudomonas aeruginosa delivers exoenzyme S (ExoS

The presence of invasion-inhibitory activity that is regulated by the transcriptional activator ExsA of cytotoxic Pseudomonas aeruginosa has previously been proposed. The results of this study show that both ExoT and ExoS, known type III secreted effector proteins of P. aeruginosa that are regulated by ExsA, possess this activity. Invasion was reduced 94.4% by ExoT and 96.0% by ExoS. Invasion-inhibitory activity is not linked to ADP-ribosylation activity, at least for ExoS, since a noncatalytic mutant also inhibits uptake by an epithelial cell line (invasion was reduced 96.0% by ExoSE381A).

AMPD1 genotype, relative fiber type composition, training status, and gender were evaluated as contributing factors to the reported variation in AMP deaminase enzyme activity in healthy skeletal muscle. Multifactorial correlative analyses demonstrate that AMPD1 genotype has the greatest effect on enzyme activity. An AMPD1 mutant allele frequency of 13.7 and a 1.7% incidence of enzyme deficiency was found across 175 healthy subjects. Homozygotes for the AMPD1 normal allele have high enzyme activities, and heterozygotes display intermediate activities. When examined according to genotype, other factors were found to affect variability as follows: AMP deaminase activity in homozygotes for the normal allele exhibits a negative correlation with the relative percentage of type I fibers and training status. Conversely, residual AMP deaminase activity in homozygotes for the mutant allele displays a positive correlation with the relative percentage of type I fibers. Opposing correlations in different homozygous AMPD1 genotypes are likely due to relative fiber-type differences in the expression of AMPD1 and AMPD3 isoforms. Gender also contributes to variation in total skeletal muscle AMP deaminase activity, with normal homozygous and heterozygous women showing only 85-88% of the levels observed in genotype-matched men.

This study was initiated to characterize the regulation and secretion of ExoS by Pseudomonas aeruginosa during contact with eukaryotic cells. The production of ExoS was monitored by a sensitive ADP-ribosyltransferase activity assay, and specific activities were calculated for supernatant and cell-associated fractions. Time course analysis indicated that ExoS was produced after a lag period, suggesting that induction of the regulon is necessary for the expression of detectable amounts of enzyme activity. Under tissue culture growth conditions, ExoS was induced when P. aeruginosa was in contact with Chinese hamster ovary (CHO) cells or after growth in tissue culture medium with serum. The serum induction of ExoS appeared to result in generalized type III secretion, while induction by contact with CHO cells appeared to result in polarized type III secretion. Mutants in the type III secretory system that express a null phenotype for ExoS production in bacteriological medium produced but did not secrete the enzyme when P. aeruginosa was grown under inducing conditions in tissue culture medium. These results suggest that both induction and secretion of ExoS may differ when the bacteria are exposed to different growth environments. The putative type III translocation proteins and secretion apparatus of P. aeruginosa were required for translocation of bacterial factors that mediate changes in CHO cell morphology during infection.

SummaryExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N-terminus (residues 1±234) is a GTPase activating protein (GAP) for RhoGTPases, while the C-terminus (residues 232±453) encodes an ADP-ribosyltransferase. Utilizing a series of Nterminal deletion peptides of ExoS and an epitopetagged full-length ExoS, two independent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N-terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90±234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP-ribosyltransferase activities.

A strain of Pseudomonas aeruginosa that fails to express known type III-secreted effector proteins was constructed as an expression host. Individual effectors were expressed in trans, and their biological effects on CHO cells were assessed in an acute cellular infection model. Intoxication with ExoS, ExoT, or ExoY resulted in alterations in cell morphology. As shown in previous genetic studies, ExoU expression was linked to acute cytotoxicity.