Molecular and cellular analysis of the "adenylyl cyclase" exotoxins from Bacillus anthracis and Bordetella pertussis

The adenylyl cyclase (AC) toxins EF and CyaA contribute to the pathogenity and mortality of anthrax and whooping cough caused by Bacillus anthracis and Bordetella pertussis, respectively. Inhibitors of bacterial toxins would mitigate the course of disease and increase survival rates. A luminescence-based AC assay was developed as screening tool for AC toxin inhibitors. The assay prooved to be fast and inexpensive and no work-up steps like chromatography are needed. It is performed in 96-well plates, and therefore is suitable for high-throughput screening. The luminescent complex TbNflx responds to changes in analyte concentrations immediately, and hence allows monitoring of real-time enzyme kinetics. The full characterization of CyaA and EF in vitro and in vivo improves our knowledge of intracellular mechanisms upon infection and leads to a better understanding of disease courses. Thus, substrate-specificity of both toxins was explored in vitro. Cytidylyl cyclase activity was determined to be approximately 300 times lower than AC activity. Endogenous cCMP and cUMP was identified in HL-60 and J774 cells by a highly specific and sensitive HPLC-MS/MS method. Additionally, we demonstrated the accumulation of cCMP after toxin treatment. To identify a potential source of endogenous cCMP and cUMP in cells, mammalian ACs were analyzed for cytidylyl and uridylyl activity. Membrane-bound ACs indead produce cCMP in very small amounts, whereas soluble AC produces cCMP as well as cUMP. In microarray and RT-PCR experiments no significant change in mRNA expression upon addition of cCMP and the cell permeable derivative Bt2cCMP could be detected. However, the increase in mRNA expression levels by CyaA could not be explained by the action of cAMP alone. Therefore, a number of questions remain to be solved: Which effects does cCMP display? Are there target proteins for cCMP? Is there a specific CC or does endogenous cCMP amount from already known mACs? How is the action of cCMP terminated? In conclusion, this work opens a new field of research in an area troubled with artifacts and unconfirmed data

Nucleotidyl Cyclase Activity of Particulate Guanylyl Cyclase A: Comparison with Particulate Guanylyl Cyclases E and F, Soluble Guanylyl Cyclase and Bacterial Adenylyl Cyclases Cyaa and Edema Factor

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.</p

Nucleotidyl cyclase activity of particulate guanylyl cyclase A: comparison with particulate guanylyl cyclases E and F, soluble guanylyl cyclase and bacterial adenylyl cyclases CyaA and edema factor.

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides

Kinetic parameters of membrane preparations of HEK293 stably overexpressing pGC-A for various nucleotides.

<p>NC activities were analyzed as mentioned in Materials and Methods. Membranes from HEK293 cells overexpressing pGC-A (10–80 µg of protein per tube) were incubated with 2–7,500 µM (XTP/Me²⁺: 2–2,000 µM) NTP/Me²⁺ in the presence of 1 µM ANP at 37°C for 5–10 min depending on the analyzed NTP. Apparent s_0.5, V_max, and <i>n</i>_Hill represent the means ± SEM of six independent experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone-0070223-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070223#pone-0070223-g002" target="_blank">2</a> and are given in alphabetical order of NTPs. Curves were analyzed by nonlinear regression using Prism version 5.0. nd: not detected, nq: not quantified.</p>*<p>only detectable in the presence of 500 µM ATP/Mg²⁺.</p

Inhibition of GC activity by XTP in membrane preparations of HEK293 cells stably overexpressing pGC-A.

<p>Membranes (10 µg protein per tube) was stimulated with 200 µM GTP/Mn²⁺ and 1 µM ANP at 37°C for 5 min and increasing concentrations of XTP/Mn²⁺ (2–1,000 µM). Data were best fitted by using a competitive binding model at one binding site with an IC₅₀ of 145.3 ± 1.2 µM. Values based on the means ± SEM of six independent experiments.</p