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Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause recurring bacterial infection in CF patients' lungs. However, the severity of CF lung disease correlates poorly with genotype. Antibiotic treatment helps dramatically prolong patients' life. The lung disease generally determines prognosis and causes most morbidity and mortality; early control of infections is thus critical. Staphylococcus aureus is a main cause of early infection in CF lungs. It secretes sphingomyelinase (SMase) C that can suppress CFTR activity. SMase C also inhibits voltage-gated K(+) channels in lymphocytes; inhibition of these channels causes immunosuppression. SMase C's pathogenicity is further illustrated by the demonstration that once Bacillus anthracis is engineered to express high levels of SMase C, the resulting mutant can evade the host immunity elicited by a live vaccine because additional pathogenic mechanisms are created. By screening a chemical library, we find that the natural product tannic acid is an SMase C antidote.
Figure 1. SMase C suppression of heretologously expressed and native CFTR currents.(A) Sphingomyelin hydrolysis reactions catalyzed by SMases C and D. (B) and (C) Currents of an oocyte injected with cRNA encoding CFTR before or after activation with 50 µM forskolin and 1 mM IBMX in the bath solution (B) and activated CFTR currents pre- or post-application of SMase C from S.aureus (SaSMase C: 0.4 ng/μl) (C), elicited by stepping membrane voltage from the â30 mV holding potential to â80 mV and then to 50 mV. (D) Time course of normalized CFTR currents at 50 mV where the arrows indicate addition of forskolin plus IBMX (arrow 1) or SaSMase C (arrow 2) (mean ± s.e.m, n = 6). (E) Normalized current amplitude before or after activation by forskolin plus IBMX and post-SMase C treatment (mean ± s.e.m, n = 6). (F) and (G) Native currents of a Calu-3 cell before or after activation with 50 µM forskolin and 1 mM IBMX in the bath solution (F) and activated CFTR currents before and after addition of SaSMase C (G), elicited by stepping membrane voltage from the 0 mV holding potential to â80 mV and then to 80 mV. (H) Time course of normalized CFTR currents at 80 mV where the arrows indicate addition of forskolin plus IBMX (arrow 1) or SaSMase C (0.5 ng/μl; arrow 2) (mean ± s.e.m, n = 7). (I) Normalized current amplitude before or after activation by forskolin plus IBMX and post-SMase C treatment (mean ± s.e.m, n = 7).DOI:
http://dx.doi.org/10.7554/eLife.03683.003
Figure 2. Effects of isoforms and mutations on SaSMase C suppression of CFTR current.(A) N-terminal sequences of three SaSMase C isoforms. (B) Percentage of SaSMase C suppression of CFTR current by individual isoforms at 1, 48 and 96 hr after final purification of SMase C (mean ± s.e.m, n = 5), where the final concentration was 0.4 ng/µl for isoform 1 and 40 ng/µl for isoforms 2 and 3. (C) Percentage of SaSMase C suppression of wild-type and mutant CFTR currents activated by expressing cRNA encoding the PKA catalytic subunit (mean ± s.e.m, n = 7â10).DOI:
http://dx.doi.org/10.7554/eLife.03683.004
Figure 3. Tannic acid (TA) counteracts SaSMase C and SMase D suppression of CFTR current.(A) Correlation plot of Z scores (raw scores minus the mean of the population, divided by the standard deviations of individual compounds) of individual compounds tested in two repeated screens; 27 compounds with higher Z scores are colored orange. (B) Percentage SaSMase C suppression of CFTR currents in the presence of 27 tested compounds, each at 10 µM. (C) Time course of normalized CFTR currents at 50 mV without or with 10 µM tannic acid (TA) present in the bath; arrow indicates addition of SaSMase C (0.6 ng/µl). (D) and (E) Time courses of normalized CFTR currents at 50 mV without and with 100 or 200 nM (D) or 300 nM (E) tannic acid present in the bath; arrow indicates addition of SaSMase C (D) and SMase D (E) (mean ± s.e.m, n = 3â5). (F) Apparent activities of SaSMase C (1 and 2) or SMase D (0.6 ng/μl) (3) plotted against the concentration of tannic acid, where the activities are expressed as half-time of the time course of sphingomyelin hydrolysis (1) or CFTR inhibition (2 and 3), which were obtained by the Amplex red assay (1) or by electrophysiological recordings (2 and 3). The half-time values were normalized to the corresponding ones obtained in the presence of relevant enzymes and the absence of tannic acid.DOI:
http://dx.doi.org/10.7554/eLife.03683.005
Figure 4. Tannic acid antagonizes suppression of CFTR and Kv1.3 currents by SMase C of B.anthracis (BaSMase C).(A) and (B) Currents of an oocyte injected with cRNA encoding CFTR, which was activated as in Figure 1, before (pre) and after (post) addition of BaSMase C (0.6 ng/μl) without (A) and with (B) 200 nM TA present. (C) Time course of normalized CFTR currents at 50 mV without or with 200 nM TA present in the bath; arrow indicates addition of BaSMase C (mean ± s.e.m, n = 10). (D) and (E) Currents of an oocyte injected with cRNA encoding Kv1.3 before (pre) and after (post) addition of BaSMase C without (D) and with (E) 200 nM TA, elicited by stepping membrane voltage from the â80 mV holding potential to 20 mV and then back to â80 mV. (F) Time course of normalized Kv1.3 currents at 20 mV without or with 200 nM TA in the bath; arrow indicates addition of BaSMase C (mean ± s.e.m, n = 4).DOI:
http://dx.doi.org/10.7554/eLife.03683.006
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