ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system - PubMed (original) (raw)

ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system

T L Yahr et al. Proc Natl Acad Sci U S A. 1998.

Abstract

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.

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Figures

Figure 1

Figure 1

(A) Schematic representation of the ExoY, CyaA, and EF adenylate cyclases. The positions of conserved regions I, II, and III are shown. Solid boxes represent the calmodulin-binding domains of CyaA and EF. The glycine-rich repeats of CyaA (hatched), signal sequence (ss), and protective antigen-binding site of EF are labeled. (B)

pileup

alignment of conserved regions I and II. Residues within CyaA and EF that are homologous to ExoY are shaded. The position of ATP-binding site motif A and its consensus sequence are shown. Residues of ExoY altered by site-directed mutagenesis are indicated by arrows.

Figure 2

Figure 2

Reverse-phase HPLC analysis of cAMP production. The elution profile of ATP, AMP, and cAMP (30 nmol) is shown in A, standard reactions lacking enzyme in B, reactions containing a control extract from BL21pLysS in C, and 1.0 μM rExoY in D. ATP and cAMP (30 nmol) treated with 3′:5′-cyclic nucleotide phosphodiesterase is shown in E. A reaction identical to that seen in D treated with 5 μg of 3′:5′-cyclic nucleotide phosphodiesterase for 10 min at 30°C is shown in F.

Figure 3

Figure 3

A eukaryotic factor stimulates rExoY adenylate cyclase activity. rExoY (1.0 μM) was assayed for adenylate cyclase activity in the presence or absence of PNE from CHO cells for 30 min under standard conditions. ∗ indicates that PNE was heated to 100°C for 5 min prior to addition to the assay mixture. Calmodulin (CaM, 10 μM) or 50 μg/ml BSA did not stimulate rExoY adenylate cyclase activity.

Figure 4

Figure 4

Extracellular protein profiles of ExoY-expressing strains. P. aeruginosa strains (as in Table 1; wt, PA103; exsA, PA103_exsA_∷Ω; exoU/T, PA103Δ_exoUexoT_∷Tc; popD, PA103_popD_∷Ω) were grown to an _OD_540 between 4.0 and 5.0, and extracellular fractions were prepared. A is a Coomassie-stained gel of concentrated supernatants. Molecular-mass markers (in kDa) are labeled on the left of A. B_–_E are immunoblots of gels identical to A probed with antisera against ExoU, ExoS/T, ExoY, and PopD, respectively. For ExoY expression in strain PA103, ExoY was amplified with its native promoter and cloned into pUCP18. ∗ denotes the expression of a catalytically (K81M) inactive form of ExoY.

Figure 5

Figure 5

Kinetics of cAMP accumulation in infected CHO cells. CHO cells were infected with PA103Δ_exoUexoT_∷Tc expressing either native ExoY or catalytically inactive ExoYK81M. At the indicated times, lysate fractions were prepared and assayed for cAMP. Values are the average of three wells and reported as pmols of cAMP per 144 μg of cellular protein per well.

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