- Email: [email protected]
Helicobacter pylori, Its Urease and Carbonic Anhydrases, and Macrophage Nitric Oxide Synthase
TIMI 1459 No. of Pages 2
Letter
Helicobacter pylori, Its Urease and Carbonic Anhydrases, and Macrophage Nitric Oxide Synthase Dimitrios Tsikas,1,* Erik Hanff,1 and Gorig Brunner1
to form ONOO (ONOOH, pKa 6.8). ONOO and its acid, ONOOH, are very short-lived species in aqueous solutions of neutral and acidic pH values and decompose spontaneously to NO3 , NO2 , and O2 (Figure 1). Most notably, ONOO /ONOOH oxidize glutathione (GSH) to its disulfide (GSSG), and L-tyrosine to 3-nitro-L-tyrosine [3]. The formation of ONOOCO2 from ONOO and CO2 may occur in H. pylori, provided ONOO /ONOOH produced in macrophages can reach the bacterium [4,5]. In H. pylori, ONOOCO2 will spontaneously decompose to NO3 , NO2 , and O2. Yet, because of its peroxy group, ONOOCO2 is highly reactive. In other words, CO2 can influence the reactions of ONOO with biomolecules and thus attenuate or potentiate their intrinsic activities (Figure 1) [3,6,7]. First, CO2 can
accelerate and potentiate the ONOO induced oxidation of thiols [3,6,7] such as of GSH, cysteine residues in macromolecules, or thioredoxin [3,6], which are present in H. pylori. Second, CO2 can also potentiate the ONOO -induced nitration of tyrosine and tyrosine residues in enzymes, thus inhibiting their enzymatic activity [3,7]. Third, homolytic cleavage of the peroxy group of ONOOCO2 generates the highly toxic radicals NO2, OCO2 [60_TD$IF], and HO [3] (Figure 1). On the other hand, CO2 can decrease the ONOO -induced formation of the signaling molecules S-nitrosothiols [3]. Thus, suppression of ONOO –induced formation of S-nitrosothiols by CO2 may attenuate signaling and be potentially hazardous to H. pylori. Summing up, the reaction of ONOO with CO2 to form ONOOCO2 does not facilitate the
In their recent review article entitled “The immune battle against Helicobacter pylori infection: NO offense”, Gobert and Wilson [1] provided an excellent review on the most recent literature reporting on interactions between Helicobacter pylori infection and nitric oxide ( NO) synthesis in host cells and on potential underlying mechanisms. Emphasis was given to the O=N•=O Damage involvement of urease in the detoxification • O-C(=O)O– CO32-, NO2HO• of peroxynitrite (ONOO [58_TD$IF]), the reaction product of the radicals [59_TD$IF]NO and superox+ RSH Signaling ide anion (O2 –) [1]. In their discussion, NO2- NO3Gobert and Wilson considered the findO=N-O-O-C(=O)O– ings of Kuwahara et al. [2] that H. pylori + Carbonic suppresses the bactericidal activity of RSH anhydrase Urease ONOO by the urease product CO2. It Carbonic RSNO + HONO CO Urea H 2 anhydrasee is well known that ONOO reacts with H2O CO2 to form the labile nitrosoperoxycar+ bonate (ONOOCO2 ). Kuwahara et al. [2] and Gobert and Wilson [1] suggested that NO2- NO3O=N-O-O– O=N• O2•– the ONOOCO2 formation represents a TyrH RSH detoxification route in H. pylori, as CO2 _ + + ONOOCO2 decomposes to the chemiNO2- RSSR TyrNO O2 cally and biologically inactive NO3 . In our Damage Signaling RSNO H2O2 opinion, ONOOCO2 formation cannot represent a detoxification route in H. pylori. We wish to comment on this issue and to provide supportive results from the Figure 1. Helicobacter pylori Urease-Carbonic Anhydrase Modulation of Macrophages’ Peroxynitrite. Nitric literature. – As rightly stated by Gobert and Wilson [1], macrophages express a high-output inducible NO synthase (iNOS, NOS2). NOS2 can concomitantly produce NO and O2 –[59_TD$IF] which combine instantaneously
oxide ( NO) and superoxide anion (O2 ) from surrounding macrophages combine to form peroxynitrite (ONOO ). In the H. pylori cell, ONOO reacts with CO2 to form ONOOCO2 . ONOO and ONOOCO2 are strong oxidants and are harmful to the bacterium. Thiols (RSH) and tyrosine (TyrH) are oxidized to disulfides (RSSR) and 3-nitrotyrosine (TyrNO2), respectively. CO2 potentiates these reactions. ONOO nitrosates RSH to form S-nitrosothiols (RSNO). CO2 inhibits RSNO formation. NO2 from decomposed ONOO and ONOOCO2 is activated by carbonic anhydrase-derived H+ to nitrous acid (HONO) which, in turn, nitrosates RSH to the signaling molecules RSNO. The symbols + and indicate activation/increase and inactivation/ decrease, respectively.
Trends in Microbiology, Month Year, Vol. xx, No. yy
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TIMI 1459 No. of Pages 2
inactivation of ONOO , but may have serious effects on, and even kill, H. pylori. The extent of cellular damage will certainly depend upon the rate of formation of ONOO in the surrounding macrophages, and the urease and carbonic anhydrase activity, as well as the efficiency of cellular antioxidant systems in the bacterium. Kuwahara et al. [2] reported that CO2 inhibited 3-nitro-tyrosine formation in H. pylori treated with ONOO (see Figure 3 in [2]). This finding contradicts the well established 3-nitro-tyrosine-promoting effect of CO2 [3,7]. Kuwahara et al. [2] reported that CO2 itself increased the survival of H. pylori cells. This finding suggests that CO2 on its own is beneficial to H. pylori most likely not because it enhances the removal of ONOO via ONOOCO2 , but rather because CO2 may potentiate other not yet known mechanisms. These other mechanisms could include elevation of expression or activity of urease in the cytoplasm and its pH-gated transporter UreI in the inner membrane, as well as of a-carbonic anhydrase in the periplasm and of b-carbonic anhydrase in the cytoplasm of H. pylori [8]. This may be of particular importance because urease and carbonic anhydrase are essentially needed for pH acclimation and colonization of H. pylori and act synergistically [5].
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Experimental results obtained from the use of ONOO cannot be discussed without consideration of the extremely challenging handling of ONOO . Home-made ONOO usually contains excess NO2 [61_TD$IF] and H2O2 the starting materials of ONOO [9]. In addition, such ONOO preparations usually contain manganese ions (Mn2+) produced from MnO2 (used for removal of excess and unreacted H2O2). Urease and carbonic anhydrases are metalloenzymes [5], and their expression and activity in H. pylori may be influenced by contaminating Ni2+[62_TD$IF], Mn2+, and Zn2+ [8,10]. Furthermore, ONOO is stored and used in diluted NaOH or KOH and may also influence the outcome of experimental studies on H. pylori due to pH manipulation. In closing, we wish to mention that carbonic anhydrases possess additional activities, namely, the conversion of NO2 to ONOH and N2O3, and, in the presence of thiols, to S-nitrosothiols and [57_TD$IF]NO [11] (Figure 1). The occurrence and significance of this particular carbonic anhydrase activity in H. pylori, and its potential synergism with urease, remain to be studied. Conflict of Interest and Financial Support The authors declare no conflict of interest in association with this work and no financial support.
1 Centre of Pharmacology and Toxicology, Core Unit Proteomics, Hannover Medical School, Hannover, Germany
*Correspondence: [email protected] (D. Tsikas). http://dx.doi.org/10.1016/j.tim.2017.05.002 References 1. Gobert, A.P. and Wilson, K.T. (2016) The immune battle against Helicobacter pylori infection: NO offense. Trends Microbiol. 24, 366–376 2. Kuwahara, H. et al. (2000) Helicobacter pylori urease suppresses bactericidial activity of peroxynitrite via carbon dioxide production. Infect. Immun. 68, 4378–4383 3. Tsikas, D. and Duncan, M.W. (2014) Mass spectrometry and 3-nitrotyrosine: strategies, controversies, and our current perspective. Mass Spectrom. Rev. 33, 237–276 4. Sachs, G. et al. (2005) Acid acclimation by Helicobacter pylori. Physiology 20, 429–438 5. Nishimori, I. et al. (2008) The alpha and beta classes carbonic anhydrases from Helicobacter pylori as novel drug targets. Curr. Pharm. Des. 14, 622–630 6. Uppu, R.M. et al. (1996) Acceleration of peroxynitrite oxidations by carbon dioxide. Arch. Biochem. Biophys. 327, 335–343 7. Ducrocq, C. et al. (1999) Peroxynitrite: an endogenous oxidizing and nitrating agent. Cell. Mol. Life Sci. 55, 1068–1077 8. Scott, D.R. et al. (2010) Cytoplasmic histidine kinase (HP0244)-regulated assembly of urease with UreI, a channel for urea and its metabolites, CO2, NH3, and NH4(+), is necessary for acid survival of Helicobacter pylori. J. Bacteriol. 192, 94–103 9. Beckman, J.S. et al. (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U. S. A. 87, 1620–1624 10. Dosanjh, N.S. et al. (2007) Characterization of the Helicobacter pylori NikR-P(ureA) DNA interaction: metal ion requirements and sequence specificity. Biochemistry 46, 2520–2529 11. Hanff, E. et al. (2016) Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets. Amino Acids 48, 1695–1706
Letter
Helicobacter pylori, Its Urease and Carbonic Anhydrases, and Macrophage Nitric Oxide Synthase Dimitrios Tsikas,1,* Erik Hanff,1 and Gorig Brunner1
to form ONOO (ONOOH, pKa 6.8). ONOO and its acid, ONOOH, are very short-lived species in aqueous solutions of neutral and acidic pH values and decompose spontaneously to NO3 , NO2 , and O2 (Figure 1). Most notably, ONOO /ONOOH oxidize glutathione (GSH) to its disulfide (GSSG), and L-tyrosine to 3-nitro-L-tyrosine [3]. The formation of ONOOCO2 from ONOO and CO2 may occur in H. pylori, provided ONOO /ONOOH produced in macrophages can reach the bacterium [4,5]. In H. pylori, ONOOCO2 will spontaneously decompose to NO3 , NO2 , and O2. Yet, because of its peroxy group, ONOOCO2 is highly reactive. In other words, CO2 can influence the reactions of ONOO with biomolecules and thus attenuate or potentiate their intrinsic activities (Figure 1) [3,6,7]. First, CO2 can
accelerate and potentiate the ONOO induced oxidation of thiols [3,6,7] such as of GSH, cysteine residues in macromolecules, or thioredoxin [3,6], which are present in H. pylori. Second, CO2 can also potentiate the ONOO -induced nitration of tyrosine and tyrosine residues in enzymes, thus inhibiting their enzymatic activity [3,7]. Third, homolytic cleavage of the peroxy group of ONOOCO2 generates the highly toxic radicals NO2, OCO2 [60_TD$IF], and HO [3] (Figure 1). On the other hand, CO2 can decrease the ONOO -induced formation of the signaling molecules S-nitrosothiols [3]. Thus, suppression of ONOO –induced formation of S-nitrosothiols by CO2 may attenuate signaling and be potentially hazardous to H. pylori. Summing up, the reaction of ONOO with CO2 to form ONOOCO2 does not facilitate the
In their recent review article entitled “The immune battle against Helicobacter pylori infection: NO offense”, Gobert and Wilson [1] provided an excellent review on the most recent literature reporting on interactions between Helicobacter pylori infection and nitric oxide ( NO) synthesis in host cells and on potential underlying mechanisms. Emphasis was given to the O=N•=O Damage involvement of urease in the detoxification • O-C(=O)O– CO32-, NO2HO• of peroxynitrite (ONOO [58_TD$IF]), the reaction product of the radicals [59_TD$IF]NO and superox+ RSH Signaling ide anion (O2 –) [1]. In their discussion, NO2- NO3Gobert and Wilson considered the findO=N-O-O-C(=O)O– ings of Kuwahara et al. [2] that H. pylori + Carbonic suppresses the bactericidal activity of RSH anhydrase Urease ONOO by the urease product CO2. It Carbonic RSNO + HONO CO Urea H 2 anhydrasee is well known that ONOO reacts with H2O CO2 to form the labile nitrosoperoxycar+ bonate (ONOOCO2 ). Kuwahara et al. [2] and Gobert and Wilson [1] suggested that NO2- NO3O=N-O-O– O=N• O2•– the ONOOCO2 formation represents a TyrH RSH detoxification route in H. pylori, as CO2 _ + + ONOOCO2 decomposes to the chemiNO2- RSSR TyrNO O2 cally and biologically inactive NO3 . In our Damage Signaling RSNO H2O2 opinion, ONOOCO2 formation cannot represent a detoxification route in H. pylori. We wish to comment on this issue and to provide supportive results from the Figure 1. Helicobacter pylori Urease-Carbonic Anhydrase Modulation of Macrophages’ Peroxynitrite. Nitric literature. – As rightly stated by Gobert and Wilson [1], macrophages express a high-output inducible NO synthase (iNOS, NOS2). NOS2 can concomitantly produce NO and O2 –[59_TD$IF] which combine instantaneously
oxide ( NO) and superoxide anion (O2 ) from surrounding macrophages combine to form peroxynitrite (ONOO ). In the H. pylori cell, ONOO reacts with CO2 to form ONOOCO2 . ONOO and ONOOCO2 are strong oxidants and are harmful to the bacterium. Thiols (RSH) and tyrosine (TyrH) are oxidized to disulfides (RSSR) and 3-nitrotyrosine (TyrNO2), respectively. CO2 potentiates these reactions. ONOO nitrosates RSH to form S-nitrosothiols (RSNO). CO2 inhibits RSNO formation. NO2 from decomposed ONOO and ONOOCO2 is activated by carbonic anhydrase-derived H+ to nitrous acid (HONO) which, in turn, nitrosates RSH to the signaling molecules RSNO. The symbols + and indicate activation/increase and inactivation/ decrease, respectively.
Trends in Microbiology, Month Year, Vol. xx, No. yy
1
TIMI 1459 No. of Pages 2
inactivation of ONOO , but may have serious effects on, and even kill, H. pylori. The extent of cellular damage will certainly depend upon the rate of formation of ONOO in the surrounding macrophages, and the urease and carbonic anhydrase activity, as well as the efficiency of cellular antioxidant systems in the bacterium. Kuwahara et al. [2] reported that CO2 inhibited 3-nitro-tyrosine formation in H. pylori treated with ONOO (see Figure 3 in [2]). This finding contradicts the well established 3-nitro-tyrosine-promoting effect of CO2 [3,7]. Kuwahara et al. [2] reported that CO2 itself increased the survival of H. pylori cells. This finding suggests that CO2 on its own is beneficial to H. pylori most likely not because it enhances the removal of ONOO via ONOOCO2 , but rather because CO2 may potentiate other not yet known mechanisms. These other mechanisms could include elevation of expression or activity of urease in the cytoplasm and its pH-gated transporter UreI in the inner membrane, as well as of a-carbonic anhydrase in the periplasm and of b-carbonic anhydrase in the cytoplasm of H. pylori [8]. This may be of particular importance because urease and carbonic anhydrase are essentially needed for pH acclimation and colonization of H. pylori and act synergistically [5].
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Trends in Microbiology, Month Year, Vol. xx, No. yy
Experimental results obtained from the use of ONOO cannot be discussed without consideration of the extremely challenging handling of ONOO . Home-made ONOO usually contains excess NO2 [61_TD$IF] and H2O2 the starting materials of ONOO [9]. In addition, such ONOO preparations usually contain manganese ions (Mn2+) produced from MnO2 (used for removal of excess and unreacted H2O2). Urease and carbonic anhydrases are metalloenzymes [5], and their expression and activity in H. pylori may be influenced by contaminating Ni2+[62_TD$IF], Mn2+, and Zn2+ [8,10]. Furthermore, ONOO is stored and used in diluted NaOH or KOH and may also influence the outcome of experimental studies on H. pylori due to pH manipulation. In closing, we wish to mention that carbonic anhydrases possess additional activities, namely, the conversion of NO2 to ONOH and N2O3, and, in the presence of thiols, to S-nitrosothiols and [57_TD$IF]NO [11] (Figure 1). The occurrence and significance of this particular carbonic anhydrase activity in H. pylori, and its potential synergism with urease, remain to be studied. Conflict of Interest and Financial Support The authors declare no conflict of interest in association with this work and no financial support.
1 Centre of Pharmacology and Toxicology, Core Unit Proteomics, Hannover Medical School, Hannover, Germany
*Correspondence: [email protected] (D. Tsikas). http://dx.doi.org/10.1016/j.tim.2017.05.002 References 1. Gobert, A.P. and Wilson, K.T. (2016) The immune battle against Helicobacter pylori infection: NO offense. Trends Microbiol. 24, 366–376 2. Kuwahara, H. et al. (2000) Helicobacter pylori urease suppresses bactericidial activity of peroxynitrite via carbon dioxide production. Infect. Immun. 68, 4378–4383 3. Tsikas, D. and Duncan, M.W. (2014) Mass spectrometry and 3-nitrotyrosine: strategies, controversies, and our current perspective. Mass Spectrom. Rev. 33, 237–276 4. Sachs, G. et al. (2005) Acid acclimation by Helicobacter pylori. Physiology 20, 429–438 5. Nishimori, I. et al. (2008) The alpha and beta classes carbonic anhydrases from Helicobacter pylori as novel drug targets. Curr. Pharm. Des. 14, 622–630 6. Uppu, R.M. et al. (1996) Acceleration of peroxynitrite oxidations by carbon dioxide. Arch. Biochem. Biophys. 327, 335–343 7. Ducrocq, C. et al. (1999) Peroxynitrite: an endogenous oxidizing and nitrating agent. Cell. Mol. Life Sci. 55, 1068–1077 8. Scott, D.R. et al. (2010) Cytoplasmic histidine kinase (HP0244)-regulated assembly of urease with UreI, a channel for urea and its metabolites, CO2, NH3, and NH4(+), is necessary for acid survival of Helicobacter pylori. J. Bacteriol. 192, 94–103 9. Beckman, J.S. et al. (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U. S. A. 87, 1620–1624 10. Dosanjh, N.S. et al. (2007) Characterization of the Helicobacter pylori NikR-P(ureA) DNA interaction: metal ion requirements and sequence specificity. Biochemistry 46, 2520–2529 11. Hanff, E. et al. (2016) Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets. Amino Acids 48, 1695–1706