RESUMEN
Here, we tested the hypothesis that a promiscuous bacterial cyclase synthesizes purine and pyrimidine cyclic nucleotides in the pulmonary endothelium. To test this hypothesis, pulmonary endothelial cells were infected with a strain of the Gram-negative bacterium Pseudomonas aeruginosa that introduces only exoenzyme Y (PA103 ΔexoUexoT::Tc pUCPexoY; ExoY(+)) via a type III secretion system. Purine and pyrimidine cyclic nucleotides were simultaneously detected using mass spectrometry. Pulmonary artery (PAECs) and pulmonary microvascular (PMVECs) endothelial cells both possess basal levels of four different cyclic nucleotides in the following rank order: cAMP > cUMP ≈ cGMP ≈ cCMP. Endothelial gap formation was induced in a time-dependent manner following ExoY(+) intoxication. In PAECs, intercellular gaps formed within 2 h and progressively increased in size up to 6 h, when the experiment was terminated. cGMP concentrations increased within 1 h postinfection, whereas cAMP and cUMP concentrations increased within 3 h, and cCMP concentrations increased within 4 h postinfection. In PMVECs, intercellular gaps did not form until 4 h postinfection. Only cGMP and cUMP concentrations increased at 3 and 6 h postinfection, respectively. PAECs generated higher cyclic nucleotide levels than PMVECs, and the cyclic nucleotide levels increased earlier in response to ExoY(+) intoxication. Heterogeneity of the cyclic nucleotide signature in response to P. aeruginosa infection exists between PAECs and PMVECs, suggesting the intracellular milieu in PAECs is more conducive to cNMP generation.
Asunto(s)
Células Endoteliales/metabolismo , Nucleótidos Cíclicos/fisiología , Pseudomonas aeruginosa/enzimología , Permeabilidad Capilar , Células Cultivadas , Células Endoteliales/microbiología , Interacciones Huésped-Patógeno , Microvasos/citología , Arteria Pulmonar/citologíaRESUMEN
[19F]-1H heteronuclear difference nuclear Overhauser effect experiments are performed on a sample of 5,5-difluorohexanoyl acyl carrier protein from Escherichia coli. Interaction of the fluorines at the 5-position of the acyl chain with protons on methyl groups of isoleucine 54 and alanine 59 is clearly indicated. The covalent attachment of the acyl chain via a prosthetic group to serine 36 and the known alpha-helix which exists from residues 36 to 51 greatly restrict the structural models which would allow acyl chain contact with residues 54 and 59.