The role of ergothioneine in mycobacteria
Date
2015-12
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
Description
Thesis (PhD)--Stellenbosch University, 2015
ENGLISH ABSTRACT : Glutathione (GSH) is a well-known anti-oxidant that prevents cellular damage caused by oxidative stress. It is synthesized by most eukaryotes but not by actinomycetes amongst which mycobacteria. However, mycobacteria synthesize two other thiols namely Mycothiol (MSH) and Ergothioneine (ERG). The role of MSH has been extensively investigated in mycobacteria and it has been shown that it protects mycobacteria from oxidative stress. However, MSH-deficient M. tuberculosis mutants have no growth defect ex vivo neither in vivo which indicates that MSH deficiency is compensated for. On the other hand, many studies have demonstrated the anti-oxidative role of ERG in eukaryotes but little is known of the role of ERG in mycobacteria. Recently, the enzymes involved in ERG biosynthesis have been identified. This enabled us to investigate the role of ERG in mycobacteria specifically M. tuberculosis. To achieve that we first generated an ERG deficient single mutant and an ERG/MSH deficient double mutant in M. smegmatis, a non-pathogenic fast growing mycobacteria used as a surrogate for M. tuberculosis. This was achieved by deleting the gene coding for the first enzyme involved in ERG biosynthesis (EgtD) in a wild type strain of M. smegmatis and a related MSH-deficient strain of M. smegmatis. Then we tested the susceptibility of the resulting mutants to oxidative stress, nitrosative stress and anti-tuberculosis drugs. We could show that ERG protects M. smegmatis from oxidative stress and that it compensates for MSH-deficiency in M. smegmatis. Then we investigated the role of ERG in M. tuberculosis. To achieve that, we generated mutants deficient in ERG biosynthetic enzymes, namely EgtA, EgtB, EgtC, EgtD and EgtE, a MSH-deficient single mutant and a MSH/ERG-deficient double mutant. Ergothioneine quantification in these mutants revealed that EgtE is not essential for ERG biosynthesis and may not be the only enzyme that catalyses the last step of ERG biosynthesis. On the other hand, though EgtB is essential for ERG biosynthesis, it may not be an ideal drug target since the loss of EgtB caused the accumulation of gamma glutamyl cysteine which potentially protected the mycobacteria from oxidative and nitrosative stress. The enzyme EgtC as well may not be an ideal drug target since the mutant deficient in this grew slightly better than the wild type during the exponential phase. The enzymes EgtA and EgtD could be the ideal drug targets since deleting the gene coding for these enzymes did not favour the survival of the resulting mutants under stress conditions. In addition, we were able to show that every CDC1551 ERG-deficient mutant generated in this study grew poorly during stationary phase. This indicates the potential protective role of ERG in dormant mycobacteria during latent tuberculosis.In addition; we have shown that ERG is secreted by mycobacteria (M.smegmatis, M.bovis and M.tuberculosis). This finding is an indication of the imperative role of ERG during infection. Taking into account the ability of ERG to modulate immune responses, it is possible that M.tuberculosis secretes it during infection to suits its survival and proliferation within the host.
AFRIKAANSE OPSOMMING : Glutatioon (GSH) is ‘n welbekende antioksidant en beskerm teen radikaal selskade as gevolg van oksidatiewe stres. Dit word in die meeste eukariote vervaardig maar nie deur aktinomycetes nie en waaronder mycobakterieë ook resorteer. Mycobakterieë vervaardig egter twee ander tiole naamlik mycothiol (MSH) en ergothioneine (ERG). Die rol wat MSH in mycobakterieë speel is reeds goed bestudeer waar dit bewys is dat MSH mycobakterieë beskerm teen oksidatiewe stres. MSH tekort M.tuberculosis (M. tuberculosis) mutante toon geen groeiafwykings ex vivo of in vivo nie wat daarop dui dat die MSH tekort op ‘n ander manier voor gekompenseer word. In vele studies is dit reeds aangetoon dat ERG ‘n anti-oksidatiewe rol speel in eukariote maar baie min is bekend omtrent ERG se rol in mycobakterieë. Onlangs is die ensiemsisteme betrokke by die biosintese van ERG geïdentifiseer wat ons in staat gestel het om die rol van ERG in mycobakterieë en spesifiek M. tuberculosis te ondersoek. Om dit te kon bereik is eerstens ‘n ERG enkelmutant in M.smegmatis geskep, gevolg deur ‘n ERG/MSH dubbelmutant. Mutante is eers in M.smegmatis geskep en daarna in M. tuberculosis om tegnieke te standardiseer en ook omdat M.smegmatis ‘n nie-patogeniese model is vir M.tuberculosis studies. Mutante is geskep deur die uitskakeling van die geen wat vir die eerste stap van ERG biosintese verantwoordelik is (EgtD) in M.smegmatis en ook in ‘n MSH tekort mutant van M.smegmatis. Hierna is die mutante geëvalueer vir vatbaarheid vir oksidatiewe stres, stikstofradikaal stres en anti-tuberkulose middels. Ons kon aantoon dat ERG in M.smegmatis ‘n beskermingsfunksie het teen oksidatiewe stres en dat ERG kompenseer vir MSH tekort in M.smegmatis. Om die rol van ERG in M.tuberculosis te bestudeer is mutante geskep wat ‘n tekort het aan ensieme in die ERG biosintese pad nl. EgtA, EgtB, EgtC, EgtD, EgtE, ‘n MSH tekort en ‘n MSH/ERG dubbelmutant. Kwantifisering van ERG in hierdie mutante het getoon dat EgtE nie belangrik is vir ERG biosintese nie en is moontlik dat dit deur n ander ensiem vervang word vir die finale stap in ERG biosintese in M. tuberculosis. Alhoewel EtgB essensieël is vir ERG biosintese sal dit nie ‘n goeie middelteiken wees nie aangesien die verlies aan EgtB die akkumulasie van gamma-glutamiel sisteïen tot gevolg het wat op sy beurt weer mycobakterieë teen oksidatiewe stres beskerm. Die ensiem EgtC is moontlik ook nie ‘n goeie middelteiken nie omdat M. tuberculosis mutante van EgtC effens beter gegroei het in die eksponensiële fase in vergelyking met die intakte kontrolestam. Daarteenoor sal EgtA en EgtD beter middelteikens wees omdat M. tuberculosis mutante wat tekorte het aan hierdie ensieme baie sensitief was vir en moeilik oorleef het onder oksidatiewe toestande. Ons kon ook aantoon dat elke CDC1551 ERG mutant, wat in hierdie studie gegenereer is, baie swak groei getoon het in die stasionêre groeifase. Dit is ‘n sterk aanduiding van die potensiële beskermings rol wat ERG kan speel in dormante M. tuberculosis gedurende sluimerende tuberkulose.
ENGLISH ABSTRACT : Glutathione (GSH) is a well-known anti-oxidant that prevents cellular damage caused by oxidative stress. It is synthesized by most eukaryotes but not by actinomycetes amongst which mycobacteria. However, mycobacteria synthesize two other thiols namely Mycothiol (MSH) and Ergothioneine (ERG). The role of MSH has been extensively investigated in mycobacteria and it has been shown that it protects mycobacteria from oxidative stress. However, MSH-deficient M. tuberculosis mutants have no growth defect ex vivo neither in vivo which indicates that MSH deficiency is compensated for. On the other hand, many studies have demonstrated the anti-oxidative role of ERG in eukaryotes but little is known of the role of ERG in mycobacteria. Recently, the enzymes involved in ERG biosynthesis have been identified. This enabled us to investigate the role of ERG in mycobacteria specifically M. tuberculosis. To achieve that we first generated an ERG deficient single mutant and an ERG/MSH deficient double mutant in M. smegmatis, a non-pathogenic fast growing mycobacteria used as a surrogate for M. tuberculosis. This was achieved by deleting the gene coding for the first enzyme involved in ERG biosynthesis (EgtD) in a wild type strain of M. smegmatis and a related MSH-deficient strain of M. smegmatis. Then we tested the susceptibility of the resulting mutants to oxidative stress, nitrosative stress and anti-tuberculosis drugs. We could show that ERG protects M. smegmatis from oxidative stress and that it compensates for MSH-deficiency in M. smegmatis. Then we investigated the role of ERG in M. tuberculosis. To achieve that, we generated mutants deficient in ERG biosynthetic enzymes, namely EgtA, EgtB, EgtC, EgtD and EgtE, a MSH-deficient single mutant and a MSH/ERG-deficient double mutant. Ergothioneine quantification in these mutants revealed that EgtE is not essential for ERG biosynthesis and may not be the only enzyme that catalyses the last step of ERG biosynthesis. On the other hand, though EgtB is essential for ERG biosynthesis, it may not be an ideal drug target since the loss of EgtB caused the accumulation of gamma glutamyl cysteine which potentially protected the mycobacteria from oxidative and nitrosative stress. The enzyme EgtC as well may not be an ideal drug target since the mutant deficient in this grew slightly better than the wild type during the exponential phase. The enzymes EgtA and EgtD could be the ideal drug targets since deleting the gene coding for these enzymes did not favour the survival of the resulting mutants under stress conditions. In addition, we were able to show that every CDC1551 ERG-deficient mutant generated in this study grew poorly during stationary phase. This indicates the potential protective role of ERG in dormant mycobacteria during latent tuberculosis.In addition; we have shown that ERG is secreted by mycobacteria (M.smegmatis, M.bovis and M.tuberculosis). This finding is an indication of the imperative role of ERG during infection. Taking into account the ability of ERG to modulate immune responses, it is possible that M.tuberculosis secretes it during infection to suits its survival and proliferation within the host.
AFRIKAANSE OPSOMMING : Glutatioon (GSH) is ‘n welbekende antioksidant en beskerm teen radikaal selskade as gevolg van oksidatiewe stres. Dit word in die meeste eukariote vervaardig maar nie deur aktinomycetes nie en waaronder mycobakterieë ook resorteer. Mycobakterieë vervaardig egter twee ander tiole naamlik mycothiol (MSH) en ergothioneine (ERG). Die rol wat MSH in mycobakterieë speel is reeds goed bestudeer waar dit bewys is dat MSH mycobakterieë beskerm teen oksidatiewe stres. MSH tekort M.tuberculosis (M. tuberculosis) mutante toon geen groeiafwykings ex vivo of in vivo nie wat daarop dui dat die MSH tekort op ‘n ander manier voor gekompenseer word. In vele studies is dit reeds aangetoon dat ERG ‘n anti-oksidatiewe rol speel in eukariote maar baie min is bekend omtrent ERG se rol in mycobakterieë. Onlangs is die ensiemsisteme betrokke by die biosintese van ERG geïdentifiseer wat ons in staat gestel het om die rol van ERG in mycobakterieë en spesifiek M. tuberculosis te ondersoek. Om dit te kon bereik is eerstens ‘n ERG enkelmutant in M.smegmatis geskep, gevolg deur ‘n ERG/MSH dubbelmutant. Mutante is eers in M.smegmatis geskep en daarna in M. tuberculosis om tegnieke te standardiseer en ook omdat M.smegmatis ‘n nie-patogeniese model is vir M.tuberculosis studies. Mutante is geskep deur die uitskakeling van die geen wat vir die eerste stap van ERG biosintese verantwoordelik is (EgtD) in M.smegmatis en ook in ‘n MSH tekort mutant van M.smegmatis. Hierna is die mutante geëvalueer vir vatbaarheid vir oksidatiewe stres, stikstofradikaal stres en anti-tuberkulose middels. Ons kon aantoon dat ERG in M.smegmatis ‘n beskermingsfunksie het teen oksidatiewe stres en dat ERG kompenseer vir MSH tekort in M.smegmatis. Om die rol van ERG in M.tuberculosis te bestudeer is mutante geskep wat ‘n tekort het aan ensieme in die ERG biosintese pad nl. EgtA, EgtB, EgtC, EgtD, EgtE, ‘n MSH tekort en ‘n MSH/ERG dubbelmutant. Kwantifisering van ERG in hierdie mutante het getoon dat EgtE nie belangrik is vir ERG biosintese nie en is moontlik dat dit deur n ander ensiem vervang word vir die finale stap in ERG biosintese in M. tuberculosis. Alhoewel EtgB essensieël is vir ERG biosintese sal dit nie ‘n goeie middelteiken wees nie aangesien die verlies aan EgtB die akkumulasie van gamma-glutamiel sisteïen tot gevolg het wat op sy beurt weer mycobakterieë teen oksidatiewe stres beskerm. Die ensiem EgtC is moontlik ook nie ‘n goeie middelteiken nie omdat M. tuberculosis mutante van EgtC effens beter gegroei het in die eksponensiële fase in vergelyking met die intakte kontrolestam. Daarteenoor sal EgtA en EgtD beter middelteikens wees omdat M. tuberculosis mutante wat tekorte het aan hierdie ensieme baie sensitief was vir en moeilik oorleef het onder oksidatiewe toestande. Ons kon ook aantoon dat elke CDC1551 ERG mutant, wat in hierdie studie gegenereer is, baie swak groei getoon het in die stasionêre groeifase. Dit is ‘n sterk aanduiding van die potensiële beskermings rol wat ERG kan speel in dormante M. tuberculosis gedurende sluimerende tuberkulose.
Keywords
Ergothioneine (ERG), Mycobacteria, Tuberculosis, Oxidative stress, UCTD