Chemico-Biological Interactions 238 (2015) 40–41
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Letter to the Editor Response to Cheluvappa and Eri: Conjugate products of pyocyanin–glutathione reactions
Pyocyanin is a major redox active virulence factor produced by the human respiratory pathogen Pseudomonas aeruginosa. Knowledge of the mechanisms associated with the inactivation of pyocyanin is important as this cytotoxin is considered to play a key role in the pathogenesis of pseudomonal infections, particularly in cystic fibrosis (CF). Previous reports have demonstrated a protective effect by glutathione (GSH) against pyocyanin toxicity [1,2] and this may be of clinical value as GSH is depleted in CF patients [3]. In our recent article we identified and characterised a novel pyocyanin–glutathionyl conjugate that, while retaining redox activity, lacked the cytotoxicity of the parent compound [4]. We proceeded to draw comparisons between our compound and one described in an earlier publication [5] in which an identical structure was proposed but with markedly different physico-chemical properties. We then pointed out a number of critical discrepancies between our respective studies. We now take this opportunity to address several concerns raised by the report of Cheluvappa et al. [5] and the correspondence of Cheluvappa and Eri [6]. We agree with one comment of Cheluvappa and Eri in that no structural data were presented in Fig. 6, Fig. 5 was intended, as stated elsewhere in the text, and we regret any confusion this may have caused. The initial difficulty with the study of Cheluvappa et al. [5] is an absence of data demonstrating the purity of the compound(s) submitted for 1H NMR analysis. Our structural analysis was conducted by tandem-MS on a single green-coloured compound initially prepared by liquid/liquid partitioning, then C18 solidphase extraction followed by high-performance thin layer chromatography. In contrast, Cheluvappa et al. used a single solid-phase extraction procedure to produce ‘‘red-brown products’’ for their analysis. It was not stated how many products their reaction produced, if further purification steps were performed or if the unfractionated mixture was subjected to 1H NMR analysis. Consequently, it remains uncertain what the chemical entity their 1 H NMR spectrum represents. It is further possible that the presence of multiple chemical species may have had a confounding influence on their spectral shift analysis. Clearly, the structure presented in Fig. 7 [5] is not based on a definitive structural analysis. The associated text to their Fig. 5 [5] states quite specifically the data are an ‘‘indication’’ of proton assignments and others ‘‘maybe’’ characteristic or ‘‘similar’’ to the predicted structure. Moreover, there was no attempt to verify the molecular weight of their compound against the predicted value. We maintain our view that Cheluvappa et al. [5] did not present data comparable to ours in support of their proposed structure and consider the ‘‘patently untrue’’ comment of Cheluvappa and Eri [6] as inappropriate.
http://dx.doi.org/10.1016/j.cbi.2015.06.003 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
The second major difficulty is the mechanisms associated with the formation of the respective products. We found GSH reacted with pyocyanin over the course of a few hours in a straightforward Michael-type addition reaction without the need for oxygen or hydrogen peroxide. In contrast, Cheluvappa et al. [5] found their red-brown products formed slowly over the course of several days under aerobic conditions and at a lesser rate under ‘‘oxygendeprived’’ conditions. They described the reaction as ‘‘O2-dependent’’ and the presence of hydrogen peroxide as ‘‘necessary for this reaction’’. Moreover, the presence of catalase completely inhibited the reaction supporting the view that hydrogen peroxide played a critical role in formation of their reaction products. If oxygen was necessary for the reaction it follows that their ‘‘oxygen-deprived’’ conditions were not anaerobic. Interestingly, Cheluvappa and Eri state that ‘‘no mechanistic overview was postulated’’ for the reaction mechanism [6] yet their legend to Fig. 7 [5] present specific mechanistic details. However, at no point was oxygen or the formation or involvement of hydrogen peroxide in the synthesis of their red-brown products implicated or even mentioned. We suggested that the hydrogen peroxide arose from the reduction of molecular oxygen due to autoxidation of GSH [4] yet in their current correspondence they described this suggestion as ‘‘totally speculative’’. It has been well established for several decades that thiols, including GSH, are unstable in aqueous aerobic media and generate reactive oxygen species, including hydrogen peroxide, during autoxidation [7], which is why we performed our studies under anaerobic conditions. In their correspondence, Cheluvappa and Eri concede autoxidation of GSH ‘‘may be partially true’’. Therefore, the possibility remains that much of their work was based on an experimental artefact. We found the rationale for the prolonged incubation times (up to 4 days) adopted by Cheluvappa et al. curious. They appear to argue that because periods of pseudomonal exacerbation in CF occur over a several days this is somehow relevant to the reaction time between pyocyanin and GSH. They state the ‘‘reaction rates and constants were deliberately not calculated owing to the obvious slow reaction time’’ [5]. Our own data [4] indicate the half-life of pyocyanin in the presence of GSH (0.1 mM) can be measured in hours, not days and it is difficult to understand why a time-course with more realistic time increments (e.g., hours) was not performed, if only to confirm that no significant reaction occurred at earlier time intervals. Finally, Cheluvappa and Eri suggest our article suffers from hyperbole. Challenging published methods or conclusions is not an exercise in hyperbole but an important element of the scientific process. We consider the comments presented in our report [5] were reasonable, consistent with current literature and our conclusions moderate and adequately supported by the available data.
Letter to the Editor / Chemico-Biological Interactions 238 (2015) 40–41
Conflict of Interest The authors declare no conflict of interest. Transparency Document The Transparency document associated with this article can be found in the online version. References [1] M. Muller, Premature cellular senescence induced by pyocyanin, a redox active Pseudomonas aeruginosa toxin, Free Radic. Biol. Med. 41 (2006) 1670–1677. [2] M. Muller, Glutathione modulates the toxicity of, but is not a biologically relevant reductant for, the Pseudomonas aeruginosa redox toxin pyocyanin, Free Radic. Biol. Med. 50 (2011) 971–977. [3] J.H. Roum, R. Buhl, N.G. McElvaney, Z. Borok, R.G. Crystal, Systemic deficiency of glutathione in cystic fibrosis, J. Appl. Physiol. 75 (1993) 2419–2424. [4] M. Muller, N.D. Merrett, Mechanism for glutathione-mediated protection against the Pseudomonas aeruginosa redox toxin, pyocyanin, Chem. Biol. Interact. 232 (2015) 30–37.
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[5] R. Cheluvappa, R. Shimmon, M. Dawson, S.N. Hilmer, D.G. Le Couteur, Reactions of Pseudomonas aeruginosa pyocyanin with reduced glutathione, Acta Biochim. Pol. 55 (2008) 571–580. [6] R. Cheluvappa, R. Eri, Conjugate products of pyocyanin–glutathione reactions, Chem. Biol. Interact. (2015). [7] H.P. Misra, Generation of superoxide free radical during the autoxidation of thiols, J. Biol. Chem. 249 (1974) 2151–2155.
⇑
Michael Muller Neil D. Merrett Department of Surgery, School of Medicine, University of Western Sydney, Building 30, Goldsmith Avenue, Campbelltown, NSW 2560, Australia ⇑ Tel.: +61 2 4620 3739; fax: +61 2 4620 3890. E-mail address: [email protected] (M. Muller) Available online 6 June 2015
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Letter to the Editor Response to Cheluvappa and Eri: Conjugate products of pyocyanin–glutathione reactions
Pyocyanin is a major redox active virulence factor produced by the human respiratory pathogen Pseudomonas aeruginosa. Knowledge of the mechanisms associated with the inactivation of pyocyanin is important as this cytotoxin is considered to play a key role in the pathogenesis of pseudomonal infections, particularly in cystic fibrosis (CF). Previous reports have demonstrated a protective effect by glutathione (GSH) against pyocyanin toxicity [1,2] and this may be of clinical value as GSH is depleted in CF patients [3]. In our recent article we identified and characterised a novel pyocyanin–glutathionyl conjugate that, while retaining redox activity, lacked the cytotoxicity of the parent compound [4]. We proceeded to draw comparisons between our compound and one described in an earlier publication [5] in which an identical structure was proposed but with markedly different physico-chemical properties. We then pointed out a number of critical discrepancies between our respective studies. We now take this opportunity to address several concerns raised by the report of Cheluvappa et al. [5] and the correspondence of Cheluvappa and Eri [6]. We agree with one comment of Cheluvappa and Eri in that no structural data were presented in Fig. 6, Fig. 5 was intended, as stated elsewhere in the text, and we regret any confusion this may have caused. The initial difficulty with the study of Cheluvappa et al. [5] is an absence of data demonstrating the purity of the compound(s) submitted for 1H NMR analysis. Our structural analysis was conducted by tandem-MS on a single green-coloured compound initially prepared by liquid/liquid partitioning, then C18 solidphase extraction followed by high-performance thin layer chromatography. In contrast, Cheluvappa et al. used a single solid-phase extraction procedure to produce ‘‘red-brown products’’ for their analysis. It was not stated how many products their reaction produced, if further purification steps were performed or if the unfractionated mixture was subjected to 1H NMR analysis. Consequently, it remains uncertain what the chemical entity their 1 H NMR spectrum represents. It is further possible that the presence of multiple chemical species may have had a confounding influence on their spectral shift analysis. Clearly, the structure presented in Fig. 7 [5] is not based on a definitive structural analysis. The associated text to their Fig. 5 [5] states quite specifically the data are an ‘‘indication’’ of proton assignments and others ‘‘maybe’’ characteristic or ‘‘similar’’ to the predicted structure. Moreover, there was no attempt to verify the molecular weight of their compound against the predicted value. We maintain our view that Cheluvappa et al. [5] did not present data comparable to ours in support of their proposed structure and consider the ‘‘patently untrue’’ comment of Cheluvappa and Eri [6] as inappropriate.
http://dx.doi.org/10.1016/j.cbi.2015.06.003 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
The second major difficulty is the mechanisms associated with the formation of the respective products. We found GSH reacted with pyocyanin over the course of a few hours in a straightforward Michael-type addition reaction without the need for oxygen or hydrogen peroxide. In contrast, Cheluvappa et al. [5] found their red-brown products formed slowly over the course of several days under aerobic conditions and at a lesser rate under ‘‘oxygendeprived’’ conditions. They described the reaction as ‘‘O2-dependent’’ and the presence of hydrogen peroxide as ‘‘necessary for this reaction’’. Moreover, the presence of catalase completely inhibited the reaction supporting the view that hydrogen peroxide played a critical role in formation of their reaction products. If oxygen was necessary for the reaction it follows that their ‘‘oxygen-deprived’’ conditions were not anaerobic. Interestingly, Cheluvappa and Eri state that ‘‘no mechanistic overview was postulated’’ for the reaction mechanism [6] yet their legend to Fig. 7 [5] present specific mechanistic details. However, at no point was oxygen or the formation or involvement of hydrogen peroxide in the synthesis of their red-brown products implicated or even mentioned. We suggested that the hydrogen peroxide arose from the reduction of molecular oxygen due to autoxidation of GSH [4] yet in their current correspondence they described this suggestion as ‘‘totally speculative’’. It has been well established for several decades that thiols, including GSH, are unstable in aqueous aerobic media and generate reactive oxygen species, including hydrogen peroxide, during autoxidation [7], which is why we performed our studies under anaerobic conditions. In their correspondence, Cheluvappa and Eri concede autoxidation of GSH ‘‘may be partially true’’. Therefore, the possibility remains that much of their work was based on an experimental artefact. We found the rationale for the prolonged incubation times (up to 4 days) adopted by Cheluvappa et al. curious. They appear to argue that because periods of pseudomonal exacerbation in CF occur over a several days this is somehow relevant to the reaction time between pyocyanin and GSH. They state the ‘‘reaction rates and constants were deliberately not calculated owing to the obvious slow reaction time’’ [5]. Our own data [4] indicate the half-life of pyocyanin in the presence of GSH (0.1 mM) can be measured in hours, not days and it is difficult to understand why a time-course with more realistic time increments (e.g., hours) was not performed, if only to confirm that no significant reaction occurred at earlier time intervals. Finally, Cheluvappa and Eri suggest our article suffers from hyperbole. Challenging published methods or conclusions is not an exercise in hyperbole but an important element of the scientific process. We consider the comments presented in our report [5] were reasonable, consistent with current literature and our conclusions moderate and adequately supported by the available data.
Letter to the Editor / Chemico-Biological Interactions 238 (2015) 40–41
Conflict of Interest The authors declare no conflict of interest. Transparency Document The Transparency document associated with this article can be found in the online version. References [1] M. Muller, Premature cellular senescence induced by pyocyanin, a redox active Pseudomonas aeruginosa toxin, Free Radic. Biol. Med. 41 (2006) 1670–1677. [2] M. Muller, Glutathione modulates the toxicity of, but is not a biologically relevant reductant for, the Pseudomonas aeruginosa redox toxin pyocyanin, Free Radic. Biol. Med. 50 (2011) 971–977. [3] J.H. Roum, R. Buhl, N.G. McElvaney, Z. Borok, R.G. Crystal, Systemic deficiency of glutathione in cystic fibrosis, J. Appl. Physiol. 75 (1993) 2419–2424. [4] M. Muller, N.D. Merrett, Mechanism for glutathione-mediated protection against the Pseudomonas aeruginosa redox toxin, pyocyanin, Chem. Biol. Interact. 232 (2015) 30–37.
41
[5] R. Cheluvappa, R. Shimmon, M. Dawson, S.N. Hilmer, D.G. Le Couteur, Reactions of Pseudomonas aeruginosa pyocyanin with reduced glutathione, Acta Biochim. Pol. 55 (2008) 571–580. [6] R. Cheluvappa, R. Eri, Conjugate products of pyocyanin–glutathione reactions, Chem. Biol. Interact. (2015). [7] H.P. Misra, Generation of superoxide free radical during the autoxidation of thiols, J. Biol. Chem. 249 (1974) 2151–2155.
⇑
Michael Muller Neil D. Merrett Department of Surgery, School of Medicine, University of Western Sydney, Building 30, Goldsmith Avenue, Campbelltown, NSW 2560, Australia ⇑ Tel.: +61 2 4620 3739; fax: +61 2 4620 3890. E-mail address: [email protected] (M. Muller) Available online 6 June 2015
