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• W4290703693 abstract "Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN. Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN. Streptococcus pneumoniae is a Gram-positive bacterium that causes pneumonia, otitis media, sepsis, and meningitis (1Weiser J.N. Ferreira D.M. Paton J.C. Streptococcus pneumoniae: transmission, colonization and invasion.Nat. Rev. Microbiol. 2018; 16: 355-367Crossref PubMed Scopus (326) Google Scholar, 2Yesilkaya H. Andisi V.F. Andrew P.W. Bijlsma J.J. Streptococcus pneumoniae and reactive oxygen species: an unusual approach to living with radicals.Trends Microbiol. 2013; 21: 187-195Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This bacterium generates hydrogen peroxide (H2O2) during metabolism (3Pericone C.D. Overweg K. Hermans P.W. Weiser J.N. Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract.Infect. Immun. 2000; 68: 3990-3997Crossref PubMed Scopus (261) Google Scholar), but it does not have H2O2-degrading enzymes (4Tettelin H. Nelson K.E. Paulsen I.T. Eisen J.A. Read T.D. Peterson S. et al.Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.Science. 2001; 293: 498-506Crossref PubMed Scopus (1108) Google Scholar). Research has been conducted to examine how S. pneumoniae survives H2O2 (5Pericone C.D. Park S. Imlay J.A. Weiser J.N. Factors contributing to hydrogen peroxide resistance in Streptococcus pneumoniae include pyruvate oxidase (SpxB) and avoidance of the toxic effects of the Fenton reaction.J. Bacteriol. 2003; 185: 6815-6825Crossref PubMed Scopus (191) Google Scholar, 6Hajaj B. Yesilkaya H. Benisty R. David M. Andrew P.W. Porat N. Thiol peroxidase is an important component of Streptococcus pneumoniae in oxygenated environments.Infect. Immun. 2012; 80: 4333-4343Crossref PubMed Scopus (39) Google Scholar, 7Hajaj B. Yesilkaya H. Shafeeq S. Zhi X. Benisty R. Tchalah S. et al.CodY regulates thiol peroxidase expression as part of the pneumococcal defense mechanism against H2O2 stress.Front. Cell. Infect. Microbiol. 2017; 7: 210Crossref PubMed Scopus (15) Google Scholar); however, in the host, this H2O2 will be consumed by host peroxidases. The heme peroxidase enzyme lactoperoxidase (LPO) is present in saliva, nasal, and airway lining fluid (8Wijkstrom-Frei C. El-Chemaly S. Ali-Rachedi R. Gerson C. Cobas M.A. Forteza R. et al.Lactoperoxidase and human airway host defense.Am. J. Respir. Cell Mol. Biol. 2003; 29: 206-212Crossref PubMed Scopus (172) Google Scholar, 9Chandler J.D. Day B.J. Thiocyanate: a potentially useful therapeutic agent with host defense and antioxidant properties.Biochem. Pharmacol. 2012; 84: 1381-1387Crossref PubMed Scopus (109) Google Scholar, 10Seidel A. Parker H. Turner R. Dickerhof N. Khalilova I.S. Wilbanks S.M. et al.Uric acid and thiocyanate as competing substrates of lactoperoxidase.J. Biol. Chem. 2014; 289: 21937-21949Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 11Thomas E.L. Jefferson M.M. Joyner R.E. Cook G.S. King C.C. Leukocyte myeloperoxidase and salivary lactoperoxidase: identification and quantitation in human mixed saliva.J. Dent. Res. 1994; 73: 544-555Crossref PubMed Scopus (85) Google Scholar, 12Vilja P. Lumikari M. Tenovuo J. Sievers G. Tuohimaa P. Sensitive immunometric assays for secretory peroxidase and myeloperoxidase in human saliva.J. Immunol. Methods. 1991; 141: 277-284Crossref PubMed Scopus (29) Google Scholar). This enzyme, and myeloperoxidase (MPO) and eosinophil peroxidase, present in inflammatory environments, catalyze the reaction of H2O2 and thiocyanate (SCN−) to generate hypothiocyanous acid (HOSCN) (13Aune T.M. Thomas E.L. Accumulation of hypothiocyanite ion during peroxidase-catalyzed oxidation of thiocyanate ion.Eur. J. Biochem. 1977; 80: 209-214Crossref PubMed Scopus (221) Google Scholar, 14van Dalen C.J. Whitehouse M.W. Winterbourn C.C. Kettle A.J. Thiocyanate and chloride as competing substrates for myeloperoxidase.Biochem. J. 1997; 327: 487-492Crossref PubMed Scopus (343) Google Scholar, 15van Dalen C.J. Kettle A.J. Substrates and products of eosinophil peroxidase.Biochem. J. 2001; 358: 233-239Crossref PubMed Scopus (101) Google Scholar). In the lung and nasal airway fluid, the concentration of SCN− ranges from 30 to 800 μM, whereas in the saliva, the concentration can reach up to 3 mM (8Wijkstrom-Frei C. El-Chemaly S. Ali-Rachedi R. Gerson C. Cobas M.A. Forteza R. et al.Lactoperoxidase and human airway host defense.Am. J. Respir. Cell Mol. Biol. 2003; 29: 206-212Crossref PubMed Scopus (172) Google Scholar, 16Thomson E. Brennan S. Senthilmohan R. Gangell C.L. Chapman A.L. Sly P.D. et al.Identifying peroxidases and their oxidants in the early pathology of cystic fibrosis.Free Radic. Biol. Med. 2010; 49: 1354-1360Crossref PubMed Scopus (81) Google Scholar, 17Schultz C.P. Ahmed M.K. Dawes C. Mantsch H.H. Thiocyanate levels in human saliva: quantitation by Fourier transform infrared spectroscopy.Anal. Biochem. 1996; 240: 7-12Crossref PubMed Scopus (81) Google Scholar, 18van Haeringen N. Ensink F. Glasius E. The peroxidase-thiocyanate-hydrogenperoxide system in tear fluid and saliva of different species.Exp. Eye Res. 1979; 28: 343-347Crossref PubMed Scopus (39) Google Scholar, 19Lorentzen D. Durairaj L. Pezzulo A.A. Nakano Y. Launspach J. Stoltz D.A. et al.Concentration of the antibacterial precursor thiocyanate in cystic fibrosis airway secretions.Free Radic. Biol. Med. 2011; 50: 1144-1150Crossref PubMed Scopus (55) Google Scholar). Thus, HOSCN will be a major product generated at these sites. How S. pneumoniae survives this microbicidal secondary oxidant has been largely overlooked.We recently reported that S. pneumoniae is relatively tolerant of HOSCN, when compared with another respiratory microbe Pseudomonas aeruginosa (20Shearer H.L. Kaldor C.D. Hua H. Kettle A.J. Parker H.A. Hampton M.B. Resistance of Streptococcus pneumoniae to hypothiocyanous acid generated by host peroxidases.Infect. Immun. 2022; 90e0053021Crossref PubMed Scopus (2) Google Scholar). This raises the question as to what mechanisms are responsible for HOSCN tolerance. We first investigated the contribution of the low molecular weight thiol GSH in S. pneumoniae (21Shearer H.L. Paton J.C. Hampton M.B. Dickerhof N. Glutathione utilization protects Streptococcus pneumoniae against lactoperoxidase-derived hypothiocyanous acid.Free Radic. Biol. Med. 2022; 179: 24-33Crossref PubMed Scopus (2) Google Scholar). The genes required for the biosynthesis of GSH are absent in S. pneumoniae, so the bacteria rely on its import from their environment (22Potter A.J. Trappetti C. Paton J.C. Streptococcus pneumoniae uses glutathione to defend against oxidative stress and metal ion toxicity.J. Bacteriol. 2012; 194: 6248-6254Crossref PubMed Scopus (73) Google Scholar, 23Lanie J.A. Ng W.L. Kazmierczak K.M. Andrzejewski T.M. Davidsen T.M. Wayne K.J. et al.Genome sequence of Avery's virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6.J. Bacteriol. 2007; 189: 38-51Crossref PubMed Scopus (318) Google Scholar, 24Kumaresan K. Springhorn S.S. Lacks S.A. Lethal and mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine potentiated by oxidized glutathione, a seemingly harmless substance in the cellular environment.J. Bacteriol. 1995; 177: 3641-3646Crossref PubMed Scopus (27) Google Scholar). The ability to import and recycle GSH was shown to be important for the ability of S. pneumoniae to cope with HOSCN; however, even when they lacked GSH, the bacteria could still survive HOSCN exposure for up to 1 h. Also, they were able to grow, albeit more slowly, in the presence of LPO, where in the presence of SCN−, bacterially produced H2O2 is converted to HOSCN (21Shearer H.L. Paton J.C. Hampton M.B. Dickerhof N. Glutathione utilization protects Streptococcus pneumoniae against lactoperoxidase-derived hypothiocyanous acid.Free Radic. Biol. Med. 2022; 179: 24-33Crossref PubMed Scopus (2) Google Scholar). These findings suggested the presence of additional HOSCN protective mechanisms in S. pneumoniae.HOSCN is known to be well tolerated by mammalian cells, owing to the presence of thioredoxin reductase (TrxR), a flavoprotein disulfide reductase (FDR) that can directly reduce HOSCN using NADPH (25Chandler J.D. Nichols D.P. Nick J.A. Hondal R.J. Day B.J. Selective metabolism of hypothiocyanous acid by mammalian thioredoxin reductase promotes lung innate immunity and antioxidant defense.J. Biol. Chem. 2013; 288: 18421-18428Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Interestingly, an enzyme that consumes NAD(P)H in the presence of HOSCN has also been reported for streptococcal species present in human oral cavities (26Carlsson J. Iwami Y. Yamada T. Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxide.Infect. Immun. 1983; 40: 70-80Crossref PubMed Scopus (158) Google Scholar). The HOSCN oxidoreductase activity was correlated with the ability of oral streptococci to recover from exposure to HOSCN in vitro (26Carlsson J. Iwami Y. Yamada T. Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxide.Infect. Immun. 1983; 40: 70-80Crossref PubMed Scopus (158) Google Scholar). Because SCN− levels reach low millimolar levels (17Schultz C.P. Ahmed M.K. Dawes C. Mantsch H.H. Thiocyanate levels in human saliva: quantitation by Fourier transform infrared spectroscopy.Anal. Biochem. 1996; 240: 7-12Crossref PubMed Scopus (81) Google Scholar, 18van Haeringen N. Ensink F. Glasius E. The peroxidase-thiocyanate-hydrogenperoxide system in tear fluid and saliva of different species.Exp. Eye Res. 1979; 28: 343-347Crossref PubMed Scopus (39) Google Scholar) and LPO is an abundant peroxidase in saliva (10Seidel A. Parker H. Turner R. Dickerhof N. Khalilova I.S. Wilbanks S.M. et al.Uric acid and thiocyanate as competing substrates of lactoperoxidase.J. Biol. Chem. 2014; 289: 21937-21949Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 11Thomas E.L. Jefferson M.M. Joyner R.E. Cook G.S. King C.C. Leukocyte myeloperoxidase and salivary lactoperoxidase: identification and quantitation in human mixed saliva.J. Dent. Res. 1994; 73: 544-555Crossref PubMed Scopus (85) Google Scholar, 12Vilja P. Lumikari M. Tenovuo J. Sievers G. Tuohimaa P. Sensitive immunometric assays for secretory peroxidase and myeloperoxidase in human saliva.J. Immunol. Methods. 1991; 141: 277-284Crossref PubMed Scopus (29) Google Scholar), the ability of oral streptococci to tolerate HOSCN is vital for their survival in this niche. The aim of the present study was to identify enzymes in S. pneumoniae with HOSCN reductase activity and to determine if they have any role in protecting this serious human pathogen from HOSCN.ResultsEvidence for reduction of HOSCN by an FDR in S. pneumoniaeTo investigate whether S. pneumoniae has an enzyme that uses NADPH to reduce HOSCN, we added NADPH to bacterial lysates and observed its consumption by the loss of absorbance at 340 nm upon the addition of HOSCN (Fig. 1A). NADH was also oxidized when HOSCN was added to lysates (Fig. 1B), and NAD(P)H consumption was not observed when either lysate or HOSCN was omitted from the reaction mixture (Fig. 1, A and B). NADH was consumed 20-fold faster than NADPH and while its consumption was significantly higher in the presence of HOSCN, it was also rapidly oxidized in the absence of HOSCN (Fig. 1B). NADH consumption in the absence of oxidant is possibly because of the oxygen-reducing NADH oxidase present in S. pneumoniae (27Auzat I. Chapuy-Regaud S. Le Bras G. Dos Santos D. Ogunniyi A.D. Le Thomas I. et al.The NADH oxidase of Streptococcus pneumoniae: its involvement in competence and virulence.Mol. Microbiol. 1999; 34: 1018-1028Crossref PubMed Scopus (104) Google Scholar).To confirm that HOSCN reduction was occurring in this system, we monitored the rate of HOSCN decline in S. pneumoniae lysates (Fig. 1, C and D). There was no significant difference between the rates of HOSCN and NADPH consumption measured concurrently on the same lysates (0.11 ± 0.03 and 0.09 ± 0.01 μM/min/mg, mean ± SD; n = 4; paired two-tailed t test). As a control, no appreciable loss of HOSCN was observed over the same period in the absence of the bacterial lysate or NADPH (Fig. 1, C and D). Lysates consumed HOSCN slightly more rapidly in the presence of NADH instead of NADPH (0.15 ± 0.02 versus 0.09 ± 0.01, mean ± SD, p < 0.01, unpaired two-tailed t test).Varying the concentrations of HOSCN and NADPH at fixed concentrations of either NADPH (200 μM) or HOSCN (100 μM), respectively, produced Michaelis–Menten saturation curves consistent with an enzymatic reduction of HOSCN by NADPH (Fig. 1, E and F). The apparent KM values for HOSCN and NADPH added to lysates were 5 (2.5–9.0, 95% confidence interval) and 107 (76–151) μM and the Vmax 0.12 (0.1–0.14) and 0.13 (0.12–0.15) μM/min/mg, respectively. When no NADPH was added, HOSCN was still consumed at approximately 15% of the maximal measured rate, which is likely facilitated by endogenous NAD(P)H present in the lysates.HOSCN oxidizes GSH to GSSG, which is reduced by glutathione reductase (GR) at the expense of NADPH (21Shearer H.L. Paton J.C. Hampton M.B. Dickerhof N. Glutathione utilization protects Streptococcus pneumoniae against lactoperoxidase-derived hypothiocyanous acid.Free Radic. Biol. Med. 2022; 179: 24-33Crossref PubMed Scopus (2) Google Scholar, 22Potter A.J. Trappetti C. Paton J.C. Streptococcus pneumoniae uses glutathione to defend against oxidative stress and metal ion toxicity.J. Bacteriol. 2012; 194: 6248-6254Crossref PubMed Scopus (73) Google Scholar). To test whether the observed NADPH consumption in response to HOSCN in S. pneumoniae lysates was mediated by GSH, we used ΔgshT and Δgor mutants of S. pneumoniae, which lack GSH or GR, respectively (22Potter A.J. Trappetti C. Paton J.C. Streptococcus pneumoniae uses glutathione to defend against oxidative stress and metal ion toxicity.J. Bacteriol. 2012; 194: 6248-6254Crossref PubMed Scopus (73) Google Scholar). NADPH utilization still occurred in lysates from the mutant strains, indeed to a greater extent than in WT, indicating that it was independent of GSH oxidation and recycling by the GR system (Fig. 2A). To rule out that the observed HOSCN reductase activity involved another low molecular weight thiol such as coenzyme A, we passed lysates through a centrifugal filter with a molecular weight cutoff (MWCO) of 3 kDa and demonstrated that NADPH consumption in response to HOSCN still occurred in the retentate (Fig. 2B). Next, we tested the involvement of TrxR in reducing HOSCN in bacterial lysates as mammalian TrxR is known to have HOSCN reductase activity (25Chandler J.D. Nichols D.P. Nick J.A. Hondal R.J. Day B.J. Selective metabolism of hypothiocyanous acid by mammalian thioredoxin reductase promotes lung innate immunity and antioxidant defense.J. Biol. Chem. 2013; 288: 18421-18428Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The TrxR inhibitor auranofin did not affect HOSCN reductase activity in S. pneumoniae lysates when added at 20 μM (Fig. 2B), despite having previously been shown to inhibit recombinant bacterial TrxR at a 25-fold lower concentration (28Harbut M.B. Vilchèze C. Luo X. Hensler M.E. Guo H. Yang B. et al.Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 4453-4458Crossref PubMed Scopus (185) Google Scholar). Similarly, the activity was not inhibited by 500 μM of 1-chloro-2,4-dinitrobenzene (Fig. 2B), a thiol-alkylating reagent that reacts preferentially with TrxR rather than with GSH and fully inhibits human TrxR at 100 μM (29Arner E.S. Bjornstedt M. Holmgren A. 1-Chloro-2,4-dinitrobenzene is an irreversible inhibitor of human thioredoxin reductase. Loss of thioredoxin disulfide reductase activity is accompanied by a large increase in NADPH oxidase activity.J. Biol. Chem. 1995; 270: 3479-3482Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar).Figure 2HOSCN reductase activity in Streptococcus pneumoniae is not carried out by glutathione reductase or thioredoxin reductase. A, reductase activity in lysates from S. pneumoniae WT, GSH transporter substrate-binding protein, and glutathione reductase mutants (ΔgshT and Δgor) was measured as described for Figure 1, A and B. B, lysates from WT S. pneumoniae were passed through a MWCO 3 kDa centrifugal filter before measuring the reductase activity of the filter retentate. Auranofin (20 μM) and DNCB (500 μM) were preincubated with WT lysates for 5 min in the presence of NADPH before measuring the reductase activity. Reductase activity in each case was expressed relative to that obtained for untreated lysate. Each symbol represents a separate experiment using lysates obtained from independent cultures, and the bar is the mean ± SD. A significant difference compared with WT in (A) and to the 100% control in (B) was determined by one-way ANOVA with Dunnett’s multiple comparison test and is indicated by ∗p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We hypothesized that the observed HOSCN reductase activity in S. pneumoniae was due to direct reduction by another member of the FDR/pyridine nucleotide-disulfide reductase family. FDRs, such as GR or TrxR, shuttle electrons from NADPH to a tightly bound flavin adenine dinucleotide and then to a redox-active disulfide (Fig. 3A). One of the two resulting reduced thiols then reacts with an oxidized substrate, for example, GSSG or oxidized thioredoxin, to form a mixed disulfide. Subsequent reaction with the adjacent active site thiol results in regeneration of the active site disulfide and release of the reduced substrate (Fig. 3A). HOSCN reacts with thiols to produce sulfenyl thiocyanate derivatives (30Aune T.M. Thomas E.L. Oxidation of protein sulfhydryls by products of peroxidase-catalyzed oxidation of thiocyanate ion.Biochemistry. 1978; 17: 1005-1010Crossref PubMed Scopus (87) Google Scholar, 31Thomas E.L. Aune T.M. Lactoperoxidase, peroxide, thiocyanate antimicrobial system: correlation of sulfhydryl oxidation with antimicrobial action.Infect. Immun. 1978; 20: 456-463Crossref PubMed Google Scholar). We propose that in the case of a HOSCN reductase, a sulfenyl thiocyanate intermediate is formed, which is then attacked by the other active site thiol resulting in the release of SCN− (Fig. 3A). S. pneumoniae lysates lost their ability to consume NADPH in response to HOSCN when they were boiled or passed over Cibacron Blue beads, an affinity absorbent for proteins with a dinucleotide fold (32Thompson S.T. Cass K.H. Stellwagen E. Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins.Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 669-672Crossref PubMed Scopus (447) Google Scholar) (Fig. 3B). These results suggested that HOSCN reduction was due to a heat-sensitive enzyme with a dinucleotide-binding site. To further probe the involvement of an FDR-type reduction mechanism, any potential thiol-containing FDR substrates were alkylated with iodoacetamide (IAM), rendering them unavailable for oxidation by HOSCN. For this, S. pneumoniae lysates were incubated with IAM prior to monitoring reductase activity (Fig. 3B). Only a partial inhibition by IAM (25%) was observed when it was incubated with S. pneumoniae lysates for 10 min prior to the addition of NADPH and HOSCN, and this effect was not significant. In contrast, the alkylating reagent significantly inhibited the reductase activity when it was coincubated with the lysate in the presence of NADPH to generate the IAM-reactive di–thiol state in the active site (Fig. 3, A and B). These results are consistent with the direct reduction of HOSCN by an FDR (Fig. 3A).Figure 3Reduction of HOSCN by a flavoprotein disulfide reductase (FDR) present in Streptococcus pneumoniae. A, proposed mechanism for the reduction of HOSCN analogous to other known FDRs, in which NADPH reduces the tightly bound flavin adenine dinucleotide, which transfers electrons to a redox-active disulfide present in the conserved CXXXC motif. In the case of low molecular weight thiol disulfide reductases or TrxR, the thus reduced N-terminal thiol can undergo a dithiol-disulfide exchange reaction with a bound oxidized substrate RS-SR (e.g., GSSG or oxidized thioredoxin). We propose that in the case of a HOSCN reductase, the thiol reacts with HOSCN to form a sulfenyl thiocyanate intermediate, which is then attacked by the C-terminal thiol to generate the disulfide resulting in the release of SCN−. B, consumption of 200 μM NADPH by S. pneumoniae lysates in the absence of oxidant (lysate alone) and after the addition of 100 μM H2O2 or HOSCN. Lysates were boiled for 5 min at 95 °C or incubated with Cibacron (C) Blue agarose beads before measuring reductase activity of the boiled lysates and bead supernatant in response to 100 μM HOSCN. IAM (20 mM) was preincubated with the lysates for 10 min in the presence of NADPH before measuring the reductase activity. IAM# (20 mM) and lysates were preincubated for 10 min in the absence of NADPH before monitoring the reductase activity. NADPH consumption, that is, reductase activity, in each case was expressed relative to that obtained for the control system (untreated lysate, 100 μM HOSCN). Each symbol represents a separate experiment using lysates obtained from independent cultures and the bar the mean ± SD. A significant difference compared with the 100% control was determined by one-way ANOVA with Dunnett’s multiple comparison test and is indicated by ∗p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Given that S. pneumoniae cope with H2O2 generated during their metabolism, we tested whether S. pneumoniae lysates can also use NADPH to reduce H2O2. NADPH consumption in S. pneumoniae lysates was not observed in response to H2O2 (Fig. 3B). MPO can use H2O2 not only to produce HOSCN but also to oxidize chloride to the potent bactericidal hypochlorous acid (HOCl) (14van Dalen C.J. Whitehouse M.W. Winterbourn C.C. Kettle A.J. Thiocyanate and chloride as competing substrates for myeloperoxidase.Biochem. J. 1997; 327: 487-492Crossref PubMed Scopus (343) Google Scholar, 33Harrison J.E. Schultz J. Studies on the chlorinating activity of myeloperoxidase.J. Biol. Chem. 1976; 251: 1371-1374Abstract Full Text PDF PubMed Google Scholar). HOCl rapidly oxidizes NADPH directly and could therefore not be tested as a possible substrate for the bacterial HOSCN-reducing enzyme. Ammonia chloramine, a less reactive secondary oxidant derived from HOCl (34Coker M.S. Hu W.P. Senthilmohan S.T. Kettle A.J. Pathways for the decay of organic dichloramines and liberation of antimicrobial chloramine gases.Chem. Res. Toxicol. 2008; 21: 2334-2343Crossref PubMed Scopus (42) Google Scholar, 35Grisham M.B. Jefferson M.M. Melton D.F. Thomas E.L. Chlorination of endogenous amines by isolated neutrophils. Ammonia-dependent bactericidal, cytotoxic, and cytolytic activities of the chloramines.J. Biol. Chem. 1984; 259: 10404-10413Abstract Full Text PDF PubMed Google Scholar), did not appear to act as a substrate as the rate of NADPH oxidation was not enhanced in the presence of S. pneumoniae lysates (Supporting information, Fig. S1).Identification of a candidate HOSCN reductase in S. pneumoniae by LC–MS and confirmation by gene deletionTo enrich the HOSCN reductase for identification by LC–MS, we performed pull-down experiments on S. pneumoniae lysates using the Cibacron agarose beads. Attempts at eluting the activity off the beads using high salt, NADPH, and/or flavin adenine dinucleotide were unsuccessful. Instead, we directly loaded the beads onto an SDS-PAGE gel and following Coomassie staining, excised the protein bands for in-gel tryptic digestion and LC–MS/MS analysis (Fig. S2). Numerous proteins were identified when searching the peptide spectra against the S. pneumoniae proteome using database search engines (Supporting information, Protein hits.xlsx). Protein hits were manually filtered for enzymes with oxidoreductase activity, provided they had a high confidence score and the predicted molecular weight was consistent with their position on the SDS-PAGE gel (Table S1). Our IAM results suggested that a redox active disulfide was involved in the reduction of HOSCN. Of the shortlisted oxidoreductases, only an uncharacterized class I pyridine nucleotide reductase, GR and TrxR, belong to the family of FDRs, the latter two of which we had already ruled out using inhibitors and mutant bacteria. The class I pyridine nucleotide reductase that appeared in band 8 (37–50 kDa) is encoded by gene SPD_1415 in S. pneumoniae strain D39. In the S. pneumoniae TIGR4 and R6 strains, this gene is annotated as SP1588 and spr1442, respectively. To confirm that the enzyme encoded by S. pneumoniae strain D39 gene SPD_1415 is responsible for the HOSCN reductase measured in lysates, we targeted the gene for deletion. This was done by replacing the gene with a spectinomycin resistance gene using overlap extension PCR (Fig. S3A) (36Horton R.M. Ho S.N. Pullen J.K. Hunt H.D. Cai Z. Pease L.R. Gene splicing by overlap extension.Methods Enzymol. 1993; 217: 270-279Crossref PubMed Scopus (424) Google Scholar). Genetic replacement was confirmed by restriction digest (Fig. S3B) and sequencing (Fig. S4). Lysates of the constructed S. pneumoniae mutant strain no longer consumed NADPH in response to HOSCN confirming that a HOSCN reductase is encoded by this gene (Fig. 4A). We therefore named the gene har and the protein it encodes Har (hypothiocyanous acid reductase).Figure 4Deletion of the SPD_1415/har gene encoding a putative flavoprotein disulfide reductase (FDR) abolishes HOSCN reductase activity in Streptococcus pneumoniae and in combination with compromised GSH utilization abolishes growth in the presence of the LPO/SCN− system. A, reductase activity, that is, NADPH consumption, was measured in lysates of S. pneumoniae D39 WT, Δhar, ΔgshT-Δhar, and Δgor-Δhar in the presence (“+,” closed symbols) and absence (“−,” open symbols) of HOSCN as described for Figure 1. Each symbol represents a separate experiment using lysates obtained from independent cultures and the bar the mean ± SD. A significant difference between activities in the absence versus presence of HOSCN for each strain was determined by unpaired t test and is indicated by ∗ for p < 0.05. A significant difference of activities in the presence of HOSCN between" @default.
• W4290703693 created "2022-08-09" @default.
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• W4290703693 title "A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid" @default.
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