Volume 11 Supplement 1

5th International Conference on cGMP: Generators, Effectors and Therapeutic Implications

Open Access

The effector protein ExoY secreted by Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for GTP

  • Ulrike Voigt1Email author,
  • Heike Burhenne1,
  • Lena Sonnow1,
  • Christine Wölfel1,
  • Corinna Spangler1,
  • Daniel Ladant2,
  • Dara W Frank3,
  • Volkhard Kaever1 and
  • Roland Seifert1
BMC Pharmacology201111(Suppl 1):P74

https://doi.org/10.1186/1471-2210-11-S1-P74

Published: 1 August 2011

Background

Pseudomonas aeruginosa is an important opportunistic pathogen causing serious pulmonary, urogenital and systemic infections. P. aeruginosa injects four effector proteins into host cells via the type III secretion system. ExoY is one of these proteins.

ExoY was originally classified as adenylyl cyclase with homology to the typical calmodulin-stimulated adenylyl cyclase exotoxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis, but the pathophysiologial function of ExoY has remained elusive [1, 2]. Recently, our group showed that CyaA and edema factor possess a rather broad base specifity (ATP >> CTP > UTP) [3, 4], raising the question of wether ExoY may also bind and metabolize nucleoside 5’- triphosphates other than ATP.

Methods

We determined cyclic nucleotide concentrations in cells transfected with ExoY plasmid or infected with P. aeruginosa with a highly sensitive HPLC-MS/MS method. Moreover, we determined the catalytic activity of purified ExoY.

Results

In mammalian cells transfected with ExoY plasmid and infected with ExoY-encoding P. aeruginosa, massive production of cGMP and cUMP was observed, with little production of cAMP. Purified ExoY was a highly effective nucleotidyl cyclase with the substrate preference GTP >> UTP ~ ATP > CTP. Fluorescence resonance energy transfer studies with methylanthraniloyl-substituted nucleotides corroborated the preference of ExoY for GTP. In contrast to ExoY, CyaA induced accumulation of cyclic nucleotides in the order cAMP > cCMP > cUMP in mammalian cells, and edema factor induced only cAMP- and cCMP accumulation.
Table 1

Comparison of ExoY with CyaA and EF

Parameter

ExoY

CyaA

EF

Bacterial source

Pseudomonas aeruginosa

Bordetella pertussis

Bacillus anthracis

Secretion type

III

I

II

Function as toxin

Unknown, “antitoxin?”

yes

yes

Activation mechanism

Cytosolic cofactor (mammalian cells and D. discoideum)

calmodulin

calmodulin

Substrate-specificity purified enzyme

GTP >> UTP ≥ ATP > CTP

ATP >> CTP > UTP > GTP

ATP >> CTP > UTP > GTP

Substrate-specificity intact cells

UTP ~ GTP > ATP > CTP

(except for B103 cells)

ATP >> CTP > UTP >>

GTP (ineffective)

ATP >> CTP >>

UTP and GTP(ineffective)

Conclusion

ExoY is a nucleotidyl cyclase with preference for GTP, and the substrate-specificity of ExoY is clearly different from that of CyaA and edema factor. Our data open the door for future studies aiming at the elucidation of the as yet unknown pathophysiological function of ExoY and which role cCMP, cGMP and cUMP play in this process.

Authors’ Affiliations

(1)
Institute of Pharmacology, Medical School of Hannover
(2)
Department of Structural Biology and Chemistry, Institut Pasteur
(3)
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin

References

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Copyright

© Voigt et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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