microbial redox systems - university of...

Microbial Redox Systems

Tomas N Gustafsson MD, PhD

Researcher, Resident Physician

Unit of Clinical Bacteriology

Department of Clinical Microbiology

Umeå University and

Infectious Disease Clinic

Sunderby Hospital, Luleå

Tomas Gustafsson ([email protected]) 2017-05-17 2

Why do microbes need redox systems?

• Respiration and synthesis of essential cellular building blocks

- Synthesis of deoxyribonucleotides

• Defence against against a hostile invironment

- Protection against attacks by the host immune system

• Neutralization of by-products from metabolism

• Communication

3

Many organisms have two major disulfide reductase systems

Flow of electrons

Tomas Gustafsson ([email protected]) 2017-05-17

4

DNA

Ribonucleotide Deoxyribonucleotide

• Only mechanism for de novo synthesis of dNTPs

• Essential in most organisms

• Exist in several different forms (classes)

Ribonucleotide reductase (RNR)


Different classes of RNR

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Class Ia Ib Ic II III

Oxygen dependence

Aerobic Aerobic Aerobic Aerobo indifferent

Anaerobic

Genes (subunits)

NrdA (R1, α) NrdB (R2, β)

NrdE (R1E, α) NrdF (R2F, β)

NrdA (R1, α) NrdB (R2, β)

NrdJ NrdD NrdG (activase)

Radical chemistry

NrdB: Tyrosyl radical and Fe-O-Fe

NrdF: Tyrosyl radical and Mn-O-Mn

Lacks tyrosyl radical. Unpaired e-

in Fe-O-Mn centre

NrdJ: radical generated from AdoCbl

NrdD: Glycyl radical.

Electron donor

Trx or Grx Trx or NrdH or Grx

Unknown Trx Formate

Occurence Eukariots (incl humans) Bacteria

Bacteria Virus

Bacteria Bacteria Archea Virus

Bacteria Archea Virus


Some organisms encode several RNRs

6

• E. coli encodes Ia, Ib and III

– Ia: The day-to-day aerobic workhorse

– Ib: Important under conditions of oxidative stress and iron-starvation (1).

– III: Essential for anaerobic growth (2).

• S. aureus encodes Ib, III

- Ib: Only aerobic RNR (most likely essential)

- III: Essential under anaerobic conditions (3) and null-mutants severly attenuated (4).

(1) Martin, J. E., and Imlay, J. A. 2011. Molecular Microbiology 80, 319–334 (2) Garriga, X et al. 1996. Biochem. Biophys. Res. Commun. 229, 189–192 (3) Masalha, M et al J. Bacteriol. 183, 7260–7272 (4) Kirdis, et al . Microb. Pathog. 43, 179–188


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Some organisms don not use Glutathione (GSH)


8

Glutathione (GSH) is absent in many microbes

• Dominant LMW reductant in higher eukariots such as mammals. • Typically present at millimolar concentrations (0.5-10 mM). • Absent in many bacteria.

-Staphylococcus aureus - Mycobacterium tuberculosis - Bacillus anthracis

• Absent in some parasites -Entamoeba histiolytica - Giardia lamblia -Trichomonas vaginalis

• Absent in Archea

Fig: Copley, SD and JK Dhillon. 2002. Genome Biol. 3(5):research0025.1-0025.16

The complex world of microbial LMW-thiols

9 Van Laer K Antioxidants & Redox Signaling 2013, 18(13): 1642-1653.

Trypanothione (Try(SH)2)

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TryR

NADPH+H+

NADP+

• Present in Kinetoplastida

- Trypanosoma brucie/cruzi (African sleeping disease / Chagas disease)

- Leishmania (Leishmaniasis)

• Synthesized from 2 molecules GSH and 1 molecule spermidine.

• Reduced by Trypanothione reductase (TryR)


A closer look at the redox system in Trypanosoma brucie

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T(SH)2

TS2 NADPH

NADP+

TXN(SH)2

TXNS2

TryR

TXNPx(SH)2

TXNPxS2

ROOH

ROH + H2O

RNR(SH)2

RNRS2 dNDP

NDP

GrxS2 Grx(SH)2

Detoxification of xenobiotics and heavy metals

TryR: Trypanothione reductase T(SH)2: Trypanothione (reduced) TS2: Trypanothione (oxidized) TXN: Tryparedoxin TXNPx: Tryparedoxin peroxidase RNR: Ribonucleotide reductase NDP: Riboucleotide dNDP: Deoxyribonucleotide


Can this peculiar system be targeted?

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T(SH)2

TS2 NADPH

NADP+

TXN(SH)2

TXNS2

TryR

TXNPx(SH)2 ROOH

RNR(SH)2

RNRS2 dNDP

NDP

ROH + H2O TXNPxS2 2

1

• The most popular target is TryR (1) • Tryparedoxin has also been targeted (2) • Other targets include synthesis of spermidine via inhibition Ornithine Decarboxylase and direct inhibition of Trypanothione synthesis


Mycothiol (MSH)

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• Major thiol in Actinomycetes

- Mycobacteria (M. tuberculosis)

- Corynebacteria (C. diptheria)

- Streptomyces

• Oxidized mycothiol (MSSM) is reduced by Mycothiol Reductase (MtR)

• Important for protection against oxidative stress and resistance against antibiotics.


Bacillithiol (BSH)

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• Present in some AT-rich, G+ bacteria

- B. anthracis

- B. subtilis

- S. aureus

• Unclear physiological role

- Null-mutants with limited phenotype

- Resistance against fosfomycin (epoxide antibiotic)

- Resistance against NaOCl-stress


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Not all TR’s are created equal – bacterial vs mammalian

Bacterial type Mammalian type

Molecular weight 70-75 kDa 110-115 kDa

Subunits Dimer Dimer

(Terminal) active site

CAT/VC GCUG

Seleno enzyme No Yes

Movements during catalysis

66o rotation during catalysis

Floppy C-terminal tail

Subtrate specificity

Narrow Usually Trx and NrdH when present

Broad Trx, small molecules etc.

Easy to modify active site?

No Yes


An example of a bacterial TR – Helicobacter pylori TR

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Gustafsson TN, Sandalova T, Lu J, Holmgren A, Schneider G. Acta Crystallogr D Biol Crystallogr. 2007 Jul;63(Pt 7):833-43


A specific example: Bacillus anthracis

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• Causative agent of anthrax

• Capable of very rapid growth during an infection

• Gram-positive rod (AT-rich group)

• Belongs to the Bacillus cereus group

• Related to Bacillus subtilis

• Spore-forming


A closer look at the ”redox-situation” in Bacillus anthracis

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•Does not have glutathione or glutaredoxins •Does have Bacillithiol which is of unknown significance •Appears to have multiple thioredoxins/NrdH-redoxins and thioredoxin reductases


Bioinformatics – apparent redundancies in B. anthracis

19 Tomas Gustafsson ([email protected]) 2017-05-17

B. anthracis has one TR , two trxs and one NrdH-redoxin

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DTNB-assay Insulin-assay

0 50 100 1500.0

0.2

0.4

0.6

0.8

Trx1

Trx2

NrdH

Time (seconds)

A4

12

nm

(m

OD

/min

)

0 200 400 600 800

-0.3

-0.2

-0.1

0.0

Trx1

NrdH

Time (seconds)

A3

40

nm

(m

OD

/min

)


Trx1 is the most efficient substrate for B. anthracis TR

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Protein kcat (s-1) Km (µM) kcat/Km (M-1s-1) Relative catalytic efficiency

Trx1 13.5 8.41 1.61 x106 1

Trx2 10.9 19.2 5.68 x 105 0.35

NrdH 7.4 85.5 8.66 x 104 0.054

• DTNB-assay

• Different redoxins treated as substrates for BaTR


No r

edoxi

nTrx

1Trx

2Trx

3

Nrd

H

Nrd

H2

Ars

C-li

ke

0

10

20

30

40

MnF

FeF

Sp

ecif

ic a

cti

vit

y

Only Trx1 and NrdH can reduce B. anthracis ribonucleotide reductase

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• Activity measured following the conversion of H3CDP to dCDP.

• Both Manganese (MnF) and Iron (FeF) forms of NrdF tested.

• Electrons supplied by 1mM DTT.

• Redoxins kept at 10µM.


Trx1 is the most efficient electron donor for B. anthracis ribonucleotide reductase

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Protein kcat (s-1) appKm (µM) kcat/Km (M-1s-1) Relative catalytic efficiency

Trx1-DTT 0.174 0.49 3.54 x 102 1

Trx1-TR 0.149 0.54 2.75 x 102 1

NrdH-DTT 0.150 2.68 5.60 x 101 0.16

NrdH-TR 0.119 3.27 3.64 x 101 0.13

• Electrons supplied by DTT or TR + NADPH.

• Relative catalytic efficiency calculated DTT vs DTT and TR vs TR


Trx1 is the predominant disulfide reductase in extracts of B. anthracis

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• Antibodies raised in rabbits.

• Extracts of B. anthracis Sterne 7700 (pOX1-, pOX2-) were incubated with antibodies.

• Activity was measured using the DTNB-assay.


Trx1 is present in a much higher concentration than both Trx2 and NrdH

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Lysate LysateStandards

22µg 22µg

44µg 44µg

66µg 66µg

22µg

44µg

66µg

2.5ng 5ng 7.5ng 10ng 15ng 20ng

0.4ng 0.8ng 1.6ng 2.4ng 4ng 6ng

0.3ng 0.5ng 1ng 1.5ng 2ng 3ng

Trx1

Trx2

NrdH

Unspecific

Recombinant

Native

Protein Level (ng/µg

total protein)

Relative

abundance

Trx1 0.71 1

Trx2 0.049 1/15

NrdH 0.012 1/60

• Western-blot determination of levels using recombinant standards.


B. anthracis summary

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• B. anthracis does not use glutathione

• B. anthracis has 1 TR, 2 Trx and 1 NrdH-redoxin

• Trx1 is the dominant disulfide reductase

• Trx1 is the physiologically relevant electron donor for RNR.

• TR and Trx1 is likely essential

- Essential in B.subtilis and (S. aureus)

Gustafsson TN, Sahlin M, Lu J, Sjöberg BM, Holmgren A. J Biol Chem. 2012 Nov 16;287(47):39686-97


Can we target thioredoxin systems in GSH-negative bacteria?

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• Ebselen is an inhibitor of bacterial TRs with activity against GSH-negative bacteria (1,2).

• Auranofin is an inhibitor of bacterial TRs with activity against various pathogenic bacteria (3,4).

(1) Lu J, Vlamis-Gardikas A, Kandasamy K, Zhao R, Gustafsson TN, Engstrand L, Hoffner

S,Engman L, Holmgren A. FASEB J. 2013 Apr;27(4):1394-403 (2) Gustafsson TN, Osman H, Werngren J, Hoffner S, Engman L, Holmgren A. Biochim Biophys Acta. 2016 Jun;1860(6):1265-71 (3) Harbut MB, Vilchèze C, Luo X, Hensler ME, Guo H, Yang B, Chatterjee AK, Nizet V, Jacobs WR Jr, Schultz PG, Wang F. Proc Natl Acad Sci U S A. 2015 Apr 7;112(14):4453-8 (4) Owings JP, McNair NN, Mui YF, Gustafsson TN, Holmgren A, Contel M, Goldberg JB, Mead JR. FEMS Microbiol Lett. 2016 Jul;363(14)

N

Se

O


Not all bacterial TRs are created equal

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Ba 1 ---MSEEKIYDVIIIGAGPAGMTAAVYTSRANLSTLMLERGIPGGQMANTEDVENYPGYE

Bc 1 MNSVSEEKIYDVVIIGAGPAGMTAAVYTSRANLSTLMLERGIPGGQMANTEDVENYPGYE

Bs 1 ---MSEEKVYDVIIIGAGPAGMTAAVYTSRANLSTLMIERGIPGGQMANTEDVENYPGFE

Sa 1 ----MTEIDFDIAIIGAGPAGMTAAVYASRANLKTVMIERGIPGGQMANTEEVENFPGFE

Ec 1 ---MGTTKHSKLLILGSGPAGYTAAVYAARANLQPVLITGMEKGGQLTTTTEVENWPGDP

Ba 58 -SILGPDLSNKMFEHAKKFGAEYAYGDVKEVIDGKEYKTIIAGKKEYKARAIIVASGAEY

Bc 61 -SILGPDLSNKMFEHAKKFGAEYAYGDVKEVIDGKEYKTIIAGKKEYKARAIIVASGAEY

Bs 58 -SILGPELSNKMFEHAKKFGAEYAYGDIKEVVDGKEYKVVKAGSKEYKARAVIIAAGAEY

Sa 57 -MITGPDLSTKMFEHAKKFGAVYQYGDIKSVEDKGEYKVINFGNKELTAKAVIIATGAEY

Ec 58 NDLTGPLLMERMHEHATKFETEIIFDHINKVDLQNRPFRLNGDNGEYTCDALIIATGASA

Ba 117 KKIGVPGETELGGRGVSYCAVCDGAFFKGKELVVIGGGDSAVEEGVFLTRFASKVTIVHR

Bc 120 KKIGVPGETELGGRGVSYCAVCDGAFFKGKELIVIGGGDSAVEEGVFLTRFASKVTIVHR

Bs 117 KKIGVPGEKELGGRGVSYCAVCDGAFFKGKELVVVGGGDSAVEEGVYLTRFASKVTIVHR

Sa 116 KKIGVPGEQELGGRGVSYCAVCDGAFFKNKRLFVIGGGDSAVEEGTFLTKFADKVTIVHR

Ec 118 RYLGLPSEEAFKGRGVSACATCDGFFYRNQKVAVIGGGNTAVEEALYLSNIASEVHLIHR

Ba 177 RDTLRAQKILQDRAFQNEKVDFIWNHTIKEINEANGKVG---SVTLVDVN-SGEEKEVKT

Bc 180 RDTLRAQKILQDRAFQNEKVDFIWNHTIKEINEASGKVG---SVTLVDVN-SGEEKEVKT

Bs 177 RDKLRAQSILQARAFDNEKVDFLWNKTVKEIHEENGKVG---NVTLVDTV-TGEESEFKT

Sa 176 RDELRAQRILQDRAFKNDKIDFIWSHTLKSINEKDGKVG---SVTLTSTK-DGSEETHEA

Ec 178 RDGFRAEKILIKRLMDKVENGNIILHTNRTLEEVTGDQMGVTGVRLRDTQNSDNIESLDV

Ba = B. anthracis

Bc = B. cereus

Bs = B. subtilis

Sa = S. aureus

Ec = E. coli

Gustafsson TN, Osman H, Werngren J, Hoffner S, Engman L, Holmgren A. Biochim Biophys Acta. 2016 Jun;1860(6):1265-71


Ebselen and derivatives thereof are potent inhibitors of BaTR


Ebselen and derivatives are potent, bactericidal antibacterials


More findings…

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• Ebselen and derivatives are potent antibacterials active against several medically important pathogens

- S. aureus

- M. tuberculosis

- B. cereus

• General, bactericidal mode of action

• High barrier towards resistance development

Gustafsson TN, Osman H, Werngren J, Hoffner S, Engman L, Holmgren A. Biochim Biophys Acta. 2016 Jun;1860(6):1265-71


Conclusions

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• There is redox outside of the mammalian world

• Redox systems can be quite different in the microbial world - Trypanothione - Mycothiol - Bacillithiol - Dependence on just the thioredoxin system for core

processes

• Microbial redox systems can be viable drug

targets - Bacterial thioredoxin reductases in GSH-negative bacteria - Trypanothione system in Trypanosoma and Leichmania


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