cCMP and cUMP occur in vivo.

Mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. It is unknown whether these tentative new second messenger molecules occur in vivo. We used high performance liquid chromatography quadrupole tandem mass spectrometry to quantitate nucleoside 3',5'-cyclic monophosphates. cCMP was detected in all organs studied, most notably pancreas, spleen and the female reproductive system. cUMP was not detected in organs, probably due to the intrinsically low sensitivity of mass spectrometry to detect this molecule and organ matrix effects. Intratracheal infection of mice with recombinant Pseudomonas aeruginosa harboring the nucleotidyl cyclase toxin ExoY massively increased cUMP in lung. The identity of cCMP and cUMP in organs was confirmed by high performance liquid chromatography quadrupole time of flight mass spectrometry. cUMP also appeared in serum, urine and faeces following infection. Taken together, this report unequivocally shows for the first time that cCMP and cUMP occur in vivo.

The Role of Pseudomonas aeruginosa ExoY in an Acute Mouse Lung Infection Model.

The effector protein Exotoxin Y (ExoY) produced by Pseudomonas aeruginosa is injected via the type III secretion system (T3SS) into host cells. ExoY acts as nucleotidyl cyclase promoting the intracellular accumulation of cyclic nucleotides. To what extent nucleotidyl cyclase activity contributes to the pathogenicity of ExoY and which mechanisms participate in the manifestation of lung infection is still unclear. Here, we used an acute airway infection model in mice to address the role of ExoY in lung infection. In infected lungs, a dose-dependent phenotype of infection with bacteria-expressing ExoY was mirrored by haemorrhage, formation of interstitial oedema in alveolar septa, and infiltration of the perivascular space with erythrocytes and neutrophilic granulocytes. Analyses of the infection process on the cellular and organismal level comparing infections with Pseudomonas aeruginosa mutants expressing either nucleotidyl cyclase-active or -inactive ExoY revealed differential cytokine secretion, increased prevalence of apoptosis, and a break of lung barrier integrity in mice infected with cyclase-active ExoY. Notably, of all measured cyclic nucleotides, only the increase of cyclic UMP in infected mouse lungs coincides temporally with the observed early pathologic changes. In summary, our results suggest that the nucleotidyl cyclase activity of ExoY can contribute to P. aeruginosa acute pathogenicity.