Cystic fibrosis is an inherited disease that mainly affects the lungs and the intestine. It is caused by defective copies of a protein called CFTR, which normally allows chloride ions to flow in and out of cells through the cell membrane. The activity of the CFTR protein is important for transferring fluid in and out of cells, and the dysfunctional CTFR protein causes the cells lining the lungs and the intestine to produce thick mucus. This leads to problems with breathing and absorbing nutrients, and makes the lungs of people with cystic fibrosis more susceptible to infections.

Recurring lung infections aggravate the symptoms of cystic fibrosis and worsen the predicted outcome for sufferers. A common skin bacterium, called Staphylococcus aureus, is one of the first to colonize the lungs of young cystic fibrosis patients. This pathogen releases an enzyme that can further reduce any residual activity of the defective CTFR protein. The enzyme—called sphingomyelinase C (or SMase C for short)—also interferes with the function of another protein that allows potassium ions to flow out of immune cells. This effect in turn reduces the body's ability to fight the infection.

Now Ramu et al. have found, by using cells grown in a laboratory, that the enzyme released by the bacteria would remain active long after the bacteria had been killed. This finding suggests that a combination therapy of antibiotics that kill the bacteria and a drug that inhibits the enzyme function may greatly improve treatments for cystic fibrosis.

Ramu et al. then tested a collection of over 2000 edible naturally-occurring chemicals and drugs, which are already approved for use in humans, to see if any could counteract the activity of the enzyme. A single natural chemical called tannic acid was shown to prevent both the negative effects on CTFR and those on the potassium channel protein.

Other pathogenic bacteria also produce an SMase C enzyme and Ramu et al. showed that tannic acid can also interfere with the anthrax bacterium's enzyme. This suggests that treatment with tannic acid may improve the outcome in a number of bacterial infections. Further experimental work is now needed to establish whether tannic acid can alleviate the symptoms of infectious disease in animal models.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel is activated when its regulatory (R) domain is phosphorylated by cyclic AMP-dependent protein kinase A (PKA) (Tabcharani et al., 1991). Mutations of CFTR cause CF disease that involves multiple organs (Knowles et al., 1983; Kerem et al., 1989; Riordan et al., 1989; Rommens et al., 1989). In the lungs of CF patients, defective CFTR leads to production of thick mucus that obstructs airways and thus predisposes the patients to recurring bacterial infection. Although CF disease originates from genetically defective CFTR protein, the severity of CF lung disease is not well correlated with genotype. The CF lung pathology is not fundamentally different from those of many other types of chronic pulmonary infectious and inflammatory diseases. Aggressive antibiotic treatment and supportive measures have prolonged the median life span of patients from 5 to 37 years (The Cystic Fibrosis Foundation, 2004). These observations underscore the profound impact of infections on progression and severity of CF lung disease. Given that the lung disease generally determines CF patients' prognosis and causes ∼90% of their morbidity and mortality, it is extremely important to effectively control or mitigate lung infections in the early stages of CF disease.

Staphylococcus aureus is a main cause of early infection in the lungs of CF patients (The Cystic Fibrosis Foundation, 2008). Most S. aureus strains, including methicillin-resistant S. aureus (MRSA), secrete the pathogenic factor sphingomyelinase (SMase) C, which cleaves sphingomyelin into phosphocholine and ceramide (Doery et al., 1963) (upper panel, Figure 1A). Our group has previously discovered that SMase C hydrolysis of sphingomyelin surrounding CFTR protein profoundly suppresses CFTR activity (Ramu et al., 2007). Thus, bacteria can further diminish critical residual CFTR activity in CF patients, worsening the negative impact of mutations. By inhibiting CFTR, SMase C-producing bacteria can also create a temporary condition, analogous to CFTR deficiency, in non-CF patients with lung infection. Additionally, compounding this already very serious problem, SMase C strongly inhibits the activity of Kv1.3 voltage-gated K⁺ (Kv) channels of lymphocytes (Xu et al., 2008). Inhibition of these channels is well known to cause immunosuppression (Chandy et al., 2004).

Figure 1

Download asset Open asset

SMase C's pathogenicity is further illustrated by the disturbing characteristics reported for a genetically engineered Bacillus anthracis mutant. Natural B. anthracis, against which the live STI-1 (Sanitary Technical Institute, USSR) vaccine provides effective protection, produces little SMase C due to a ‘defective’ regulatory gene. However, once B. anthracis is engineered to express high levels of SMase C, the resulting mutant not only remains lethal but also evades the host immunity elicited by the live vaccine because additional pathogenic mechanisms are generated (Pomerantsev et al., 1997). Thus, it is important to find effective means to counteract the SMase C action.

Experimentally, the CFTR channels are often activated by boosting the concentration of intracellular cAMP with a combination of the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (Csanady et al., 2000). Figure 1B shows current of CFTR heterologously expressed in a Xenopus oocyte bathed in a forskolin- and IBMX-containing solution. Following exposure to extracellular SMase C from S. aureus, the CFTR current was markedly diminished in a time-dependent manner (Figure 1C–E). To investigate whether SMase C also suppresses native CFTR current, we tested the enzyme's effect on CFTR current in Calu-3 cells (a lung epithelial cell line commonly used in CFTR research) after activating their CFTR channels with forskolin and IBMX (Figure 1F–H). As in oocytes, SMase C suppressed CFTR current in Calu-3 cells (Figure 1G–I), a result confirming that this SMase C effect on CFTR activity is not unique to the oocyte heterologous expression system.

Individual strains of a given bacterium species may be expected to secrete different isoforms of SMase C. We compared three isoforms from S. aureus to learn their behaviors (Figure 2A; only isoform #1 was used elsewhere). All three recombinant isoforms suppressed CFTR current albeit with differing specific activity (Figure 2B). Two isoforms lost activity over 96 hr, and one retained full activity; all three isoforms exhibited measurable activity for at least 48 hr. It follows that secreted SMase C can remain active for a considerable time following death of the bacteria. Thus, while antibiotics remain critical in combating the infection, an effective means to counteract SMase C action would be important in limiting the negative impact of the bacterial infection on the host.

Figure 2

Download asset Open asset

We previously found that at levels beyond that required to achieve maximal CFTR activation, PKA's catalytic subunit significantly lessens suppression of CFTR current by SMase C (Ramu et al., 2007). This observation suggests that SMase C modifying the head group chemistry of sphingomyelin molecules surrounding CFTR makes activation of CFTR more energetically difficult, and this SMase C action can be markedly overcome by ‘over-phosphorylating’ the channel's R-domain until all four sites are phosphorylated. This hypothesis implies that mutant CFTR channels with individual key phosphorylation sites ablated would suffer more pronounced suppression by SMase C at these ‘supersaturating’ levels of PKA's catalytic subunit. To test this prediction, we replaced each of the four phosphorylation sites, one at a time, with alanine and examined SMase C's effect on these single-mutant channels. As a control, we showed that in the presence of a supersaturating level of PKA's catalytic subunit, SMase C suppressed only 25% of the wild-type CFTR current (Figure 2C). In contrast and as predicted, under the same experimental condition, SMase C suppressed 40–65% of the mutants' currents. A second corollary of our hypothesis is that a β adrenergic receptor agonist should alleviate the SMase C-caused suppression of CFTR current by boosting intracellular cAMP. In fact, β receptor agonists are routinely used as bronchodilators in patients suffering from pulmonary infectious and inflammatory diseases. Thus, application of β receptor agonists would not be contraindicated in cases of infection by SMase C-positive bacteria and likely be beneficial. However, given that a β receptor agonist unlikely causes overphosphorylation of the R domain, it may only modestly counteract the SMase C-dependent suppression of CFTR current.

To develop a clinically suitable drug from scratch is difficult and costly. One shortcut is to find novel medical benefits in already approved drugs or edible natural products. As such, we screened a chemical library (Spectrum 2000) consisting of approved drugs and natural products, each at 10 µM concentration. We employed a fluorescence-based assay (Amplex Red) involving a series of coupled enzymatic reactions. Figure 3A is a correlation plot of the Z scores obtained from two repeated screens. A positive result suggests an antagonizing effect of the tested compound on SMase C or on one or more of the coupled reactions. The 27 compounds with higher Z scores were then examined electrophysiologically for their antagonistic effects on SMase C (Figure 3B). We found that, of those 27 compounds at 10 µM, only tannic acid exhibited a clear SMase C-antagonizing effect (Figure 3B,C). Tannic acid was just as effective at concentrations down to 200 nM (Figure 3D). Furthermore, we found that it also antagonized SMase C of B. anthracis (Figure 4A–C).

Figure 3

Download asset Open asset

Figure 4

Download asset Open asset

Tannic acid is known to interact with many other proteins with apparent K_d or EC₅₀ between 8 µM and 150 mM (Gabbott, 2008). Consequently, high concentrations of tannic acid (5–50 mM) are often used to fix cells in EM studies (Hayat, 1981; Afzelius, 1992; Bozzola and Russell, 1992). To our knowledge, a EC₅₀ range of 50–100 nM observed in our study is by far the lowest concentration range at which tannic acid is found to bind effectively to a protein and/or to produce a meaningful biological effect (Figure 3F). Although tannic acid has been reported to bind to the choline group of phosphatidylcholine (PC), it does not appear to antagonize SMase C activity by binding to PC and thereby preventing SMase C from accessing sphingomyelin because tannic acid binds to PC only at much higher concentrations than what is required to antagonize SMase C activity (Kalina and Pease, 1977; Simon et al., 1994). Furthermore, the EC₅₀ (∼50 nM) of tannic acid for antagonizing the SMase C-catalyzed sphingomyelin hydrolysis in a biochemical reaction (involving no PC) is comparable to its EC₅₀ (∼100 nM) for antagonizing SMase C suppression of CFTR activity in a biological membrane (Figure 3F).

To assess the relative specificity of tannic acid's antagonizing effect on SMase C, we tested tannic acid against bacterial SMase D. Like SMase C, SMase D specifically hydrolyzes sphingomyelin (bottom panel, Figure 1A). However, unlike SMase C, SMase D removes only the choline group rather than the entire phosphocholine head group from sphingomyelin, leaving ceramide-1-phosphate instead of ceramide behind in the membrane. Our group previously showed that SMase D also suppresses CFTR activity (Ramu et al., 2007). Here, we show that tannic acid at 300 nM slightly slowed down SMase D suppression of CFTR activity (Figure 3E). In contrast, as shown above, tannic acid at 200 nM essentially eliminated SMase C suppression of CFTR activity (Figure 3D). Thus, tannic acid antagonizes SMase C and SMase D with differing potency (Figure 3F).

In lymphocytes, Kv1.3 channels help maintain a sufficiently negative resting membrane potential to sustain an adequate driving force for Ca²⁺ entry which acts as a key signal to activate lymphocytes (DeCoursey et al., 1984; Matteson and Deutsch, 1984; Cahalan and Chandy, 2009). Consequently, inhibition of Kv1.3 channels causes immunosuppression, a finding that has stimulated the development of inhibitors of Kv1.3 as effective immunosuppressants to treat autoimmune diseases (Chandy et al., 2004). On this backdrop, we previously showed that SMase C, by removing the phosphoryl head group of sphingomyelin molecules around Kv1.3 channels, profoundly diminishes these channels' activity (Xu et al., 2008). The underlying mechanism is that removal of the negatively charged phosphoryl group makes it energetically more difficult for positively charged voltage sensors to transition to the activated state. Here, we find that tannic acid can also effectively counteract suppression of Kv1.3 activity by SMase C (Figure 4D–F).

As illustrated above, we have shown that tannic acid is an antidote to SMase C. It is a readily available and inexpensive natural product, widely and abundantly used in the food industry. The US Food and Drug Administration terms it Generally Recognized As Safe (GRAS) (FDA, 2013). The use of tannic acid to treat various diseases was reported as early as 1850 (Alison, 1850; Allegrini and Costantini, 2012), for example in the treatment of human diarrhea. Recently reported clinical doses are 0.5–1 g (Bian et al., 2009), giving an estimated concentration in human body fluids of 8–16 µM, or 40–80 times higher than required to antagonize SMase C action. We suggest that tannic acid be considered in attempts to protect against B. anthracis mutants engineered to over-express SMase C, in the unfortunate event of an outbreak, or to lessen the harm caused by natural SMase C-positive bacteria including MRSA, in both CF and non-CF patients.

1. 1. Afzelius BA

   (1992) Section staining for electron microscopy using tannic acid as a mordant: a simple method for visualization of glycogen and collagen

   Microscopy Research and Technique 21:65–72.

   https://doi.org/10.1002/jemt.1070210110

   • Google Scholar
2. 1. Alison SS

   (1850) On the use and administration of tannic acid in various diseases

   London Journal of Medicine 2:1–9.

   https://doi.org/10.1136/bmj.s2-2.13.1

   • Google Scholar
3. 1. Allegrini A
   2. Costantini M

   (2012) Gelatine tannate for the treatment of acute diarrhoea in adults

   Journal of Gastrointestinal and Digestive System 2:110.

   https://doi.org/10.4172/2161-069X.1000110

   • Google Scholar
4. 1. Bian Y
   2. Masuda A
   3. Matsuura T
   4. Ito M
   5. Okushin K
   6. Engel AG
   7. Ohno K

   (2009) Tannic acid facilitates expression of the polypyrimidine tract binding protein and alleviates deleterious inclusion of CHRNA1 exon P3A due to an hnRNP H-disrupting mutation in congenital myasthenic syndrome

   Human Molecular Genetics 18:1229–1237.

   https://doi.org/10.1093/hmg/ddp023

   • Google Scholar
5. Book

   1. Bozzola JJ
   2. Russell LD

   (1992)

   Electron microscopy: principles and techniques for biologists

   Jones and Bartlett.

   • Google Scholar
6. 1. Cahalan MD
   2. Chandy KG

   (2009) The functional network of ion channels in T lymphocytes

   Immunological Reviews 231:59–87.

   https://doi.org/10.1111/j.1600-065X.2009.00816.x

   • Google Scholar
7. 1. Chandy KG
   2. Wulff H
   3. Beeton C
   4. Pennington M
   5. Gutman GA
   6. Cahalan MD

   (2004) K⁺ channels as targets for specific immunomodulation

   Trends in Pharmacological Sciences 25:280–289.

   https://doi.org/10.1016/j.tips.2004.03.010

   • Google Scholar
8. 1. Cheng SH
   2. Rich DP
   3. Marshall J
   4. Gregory RJ
   5. Welsh MJ
   6. Smith AE

   (1991) Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel

   Cell 66:1027–1036.

   https://doi.org/10.1016/0092-8674(91)90446-6

   • Google Scholar
9. 1. Csanady L
   2. Chan KW
   3. Seto-Young D
   4. Kopsco DC
   5. Nairn AC
   6. Gadsby DC

   (2000) Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains

   The Journal of General Physiology 116:477–500.

   https://doi.org/10.1085/jgp.116.3.477

   • Google Scholar
10. 1. DeCoursey TE
    2. Chandy KG
    3. Gupta S
    4. Cahalan MD

    (1984) Voltage-gated K⁺ channels in human T lymphocytes: a role in mitogenesis?

    Nature 307:465–468.

    https://doi.org/10.1038/307465a0

    • Google Scholar
11. 1. Doery HM
    2. Magnusson BJ
    3. Cheyne IM
    4. Sulasekharam J

    (1963) A phospholipase in staphylococcal toxin which hydrolyses sphingomyelin

    Nature 198:1091–1092.

    https://doi.org/10.1038/1981091a0

    • Google Scholar
12. 1. FDA

    (2013)

    Food and drugs

    21.

    • Google Scholar
13. Book

    1. Gabbott P

    (2008)

    Principles and applications of thermal analysis

    Blackwell Publication Limited.

    • Google Scholar
14. Book

    1. Hayat MA

    (1981)

    Fixation for electron microscopy

    London: Academic press.

    • Google Scholar
15. 1. Kalina M
    2. Pease DC

    (1977) The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

    The Journal of Cell Biology 74:726–741.

    https://doi.org/10.1083/jcb.74.3.726

    • Google Scholar
16. 1. Kerem B
    2. Rommens JM
    3. Buchanan JA
    4. Markiewicz D
    5. Cox TK
    6. Chakravarti A
    7. Buchwald M
    8. Tsui LC

    (1989) Identification of the cystic fibrosis gene: genetic analysis

    Science 245:1073–1080.

    https://doi.org/10.1126/science.2570460

    • Google Scholar
17. 1. Knowles MR
    2. Stutts MJ
    3. Spock A
    4. Fischer N
    5. Gatzy JT
    6. Boucher RC

    (1983) Abnormal ion permeation through cystic fibrosis respiratory epithelium

    Science 221:1067–1070.

    https://doi.org/10.1126/science.6308769

    • Google Scholar
18. 1. Liman ER
    2. Tytgat J
    3. Hess P

    (1992) Subunit stoichiometry of a mammalian K⁺ channel determined by construction of multimeric cDNAs

    Neuron 9:861–871.

    https://doi.org/10.1016/0896-6273(92)90239-A

    • Google Scholar
19. 1. Matteson DR
    2. Deutsch C

    (1984) K channels in T lymphocytes: a patch clamp study using monoclonal antibody adhesion

    Nature 307:468–471.

    https://doi.org/10.1038/307468a0

    • Google Scholar
20. 1. Pomerantsev AP
    2. Staritsin NA
    3. Mockov Y
    4. Marinin LI

    (1997) Expression of cereolysine AB genes in Bacillus anthracis vaccine strain ensures protection against experimental hemolytic anthrax infection

    Vaccine 15:1846–1850.

    https://doi.org/10.1016/S0264-410X(97)00132-1

    • Google Scholar
21. 1. Ramu Y
    2. Xu Y
    3. Lu Z

    (2006) Enzymatic activation of voltage-gated potassium channels

    Nature 442:696–699.

    https://doi.org/10.1038/nature04880

    • Google Scholar
22. 1. Ramu Y
    2. Xu Y
    3. Lu Z

    (2007) Inhibition of CFTR Cl^- channel function caused by enzymatic hydrolysis of sphingomyelin

    Proceedings of the National Academy of Sciences of USA 104:6448–6453.

    https://doi.org/10.1073/pnas.0701354104

    • Google Scholar
23. 1. Riordan JR
    2. Rommens JM
    3. Kerem B
    4. Alon N
    5. Rozmahel R
    6. Grzelczak Z
    7. Zielenski J
    8. Lok S
    9. Plavsic N
    10. Chou JL
    11. Drumm ML
    12. Iannuzzi MC
    13. Collins FS
    14. Tsui LC

    (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA

    Science 245:1066–1073.

    https://doi.org/10.1126/science.2475911

    • Google Scholar
24. 1. Rommens JM
    2. Iannuzzi MC
    3. Kerem B
    4. Drumm ML
    5. Melmer G
    6. Dean M
    7. Rozmahel R
    8. Cole JL
    9. Kennedy D
    10. Hidaka N
    11. Zsiga M
    12. Riordan JR
    13. Tsui LC
    14. Collins FS

    (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping

    Science 245:1059–1065.

    https://doi.org/10.1126/science.2772657

    • Google Scholar
25. 1. Simon SA
    2. Disalvo EA
    3. Gawrisch K
    4. Borovyagin V
    5. Toone E
    6. Schiffman SS
    7. Needham D
    8. McIntosh TJ

    (1994) Increased adhesion between neutral lipid bilayers: interbilayer bridges formed by tannic acid

    Biophysical Journal 66:1943–1958.

    https://doi.org/10.1016/S0006-3495(94)80988-9

    • Google Scholar
26. 1. Tabcharani JA
    2. Chang XB
    3. Riordan JR
    4. Hanrahan JW

    (1991) Phosphorylation-regulated Cl^- channel in CHO cells stably expressing the cystic fibrosis gene

    Nature 352:628–631.

    https://doi.org/10.1038/352628a0

    • Google Scholar
27. 1. The Cystic Fibrosis Foundation

    (2004)

    Patient registry annual data report

    Bethesda, M.D.

    • Google Scholar
28. 1. The Cystic Fibrosis Foundation

    (2008)

    Annual report

    Bethesda, M.D.

    • Google Scholar
29. 1. Xu Y
    2. Ramu Y
    3. Lu Z

    (2008) Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K⁺ channels

    Nature 451:826–829.

    https://doi.org/10.1038/nature06618

    • Google Scholar

Author details

1. Yajamana Ramu

   Department of Physiology, Perelman School of Medicine, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, United States

   Contribution

   YR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Yanping Xu (deceased)

   Department of Physiology, Perelman School of Medicine, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, United States

   Contribution

   YX, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Hyeon-Gyu Shin

   Department of Physiology, Perelman School of Medicine, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, United States

   Contribution

   H-GS, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Zhe Lu

   Department of Physiology, Perelman School of Medicine, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, United States

   Contribution

   ZL, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   zhelu@mail.med.upenn.edu

   Competing interests

   The authors declare that no competing interests exist.

• 1,291

  views

• 75

  downloads

• 4

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.