"Desvendando o papel de tiol

peroxidases no contexto da
interação patógeno-hospedeiro"

Prof. Dr. Diogo de Abreu Meireles

diogo@uenf.br
WhatsApp: (11) 96215-1015
 TEMA

• O2 final electron acceptor

O2 • Improved energetic efficiency
• Enzyme cofactor

Adaptated from: RS Edgar et al. Nature 000, 1-6 (2012)

Espécies Reativas de Oxigênio - EROs Termo coletivo para espécies derivadas
Reactive Oxygen Species - ROS do O2 que são mais reativas que o
Peroxynitrite próprio O2.
-
ONOO
Fenton chemistry
NO● Fe+2  Fe+3

e- e -
+ 2H +
e- + H+ • e- + H+
O2 O 2
•ˉ
H2O2 OH H2O
Molecular Superoxide Hydrogen Hydroxyl radical Water
oxygen anion radical peroxide

MPO
+ energy Cl-
(myeloperoxidase)

1
O2 HOCl
Singlet Hypochlorous
oxygen acid
Oxidative Stress
• Definition:
An imbalance between oxidants and antioxidants in favour of the oxidants, leading to a disruption
of redox signaling and control and/or molecular damage (Sies, 2018).

• Nonspecific oxidations (oxidative damages)

DNA
Structural/functional
Proteins

Lipids
modifications
 Sources of oxidative stress in eukaryotes
Endogenous oxidative stress

https://en.wikipedia.org/wiki/Mitochondrial_ROS
 Sources of oxidative stress in bacteria
Endogenous oxidative stress
- O2-. and H2O2 are primarily
produced by the accidental
autoxidation of flavoenzymes.
.

https://doi.org/ggkv2w
 Sources of oxidative stress in bacteria
Exogenous oxidative stress

Production of ROS by the duox

pathway in Drosophila gut

https://doi.org/ggjksf
 Sources of oxidative stress in bacteria
Exogenous oxidative stress

Aerobic metabolic of lactic acid

bacteria

https://doi.org/jh26
 Sources of oxidative stress in bacteria
Exogenous oxidative stress Fagosome
H+-channel
- Oxidative killing in fagosomes.
~0.25 µm ~ 1 µm V-ATPase

H2O2
1
O2

Adapted from: Antioxid. Redox Signal. 2014, 20 (6), 1000–1037. NADPH

steady state levels pH 7.4 – 7.8; oxidase
Proc. Natl. Acad. Sci. USA (1997) 94:10973–10978
[MPO] ~1 mM; complex
Thin section electron micrograph of a single
PMN with three bacteria visible within two [H2O2] ~ 2 µM; [Cl-] ~100 mM;
phagocytic vacuoles.

ꜙ
[O2 ] ~ 25 µM;
ꜙ
O2 generation rate ~ 5.2 mM/s;
 Cellular antioxidants systems
Fine tuning adjustment on [ROS]
• scavenging x production

Enzymatic systems:

• SOD (superoxide dismutase) 2 O2●- + 2 H+  H2O2 + O2

• Catalase (heme peroxidases) 2 H2O2   2 H2O + O2

• Thioredoxin (Trx)
• thiol-disulfide oxidoreductases
Glutaredoxin (Grx)
• Peroxiredoxin (Prx)
• Glutathione peroxidase (Gpx) thiol peroxidases
• Organic hydroperoxide resistance protein (Ohr)
10
 Thiol peroxidases
Reduced by thiols
Thiol dependent

Cys-based peroxiredoxins
Active site - cysteine Peroxide reduction

Catalytic
- Hydrogen peroxide (H2O2) cycle
- Peroxynitrite (ONOO-)

- Organic hydroperoxides (ROOH)

13S-hydroperoxy-9Z,11E-octadecadienoic acid
(13(S)-HPODE)
 E.coli MG1655 antioxidant arsenal

3 Superoxide dismutases
5 Thiol peroxidases
3 Heme peroxidases

CcpR (cytochrome c peroxidase)

SodA (superoxide dismutase) KatE (catalase E)

SodB Fe
SodC Cu/Zn

KatG (bifunctional)

Tpx (thiol peroxidase)

Prx AhpC (alkyl hydroperoxide reductase)
Bcp (bacterioferritin co-migratory protein)
Ohr/OsmC OsmC (osmotically inducible protein C)
GPx
Gpx (glutathione peroxidase)
cytoplasm

periplasm
extracellular space
Antioxidant systems lower ROS levels to avoid cellular damage
Escherichia coli exponential growth in glucose

H2O2 20 - 50 nM

Intracellular
10-14 μM
steady state
per sec
concentration H2O2

5-10 106–107 M−1 s−1

μM per
sec O2-.

O2-. ~ 0.1 - 0.2 nM

Adapted from Nature Reviews Microbiology, (2013) 11:443-454.

Responses to specific oxidants are orchestrated by specific sensors

H2O2 ROOH
Oxidant
scavenging
and repair
responses
O2•ˉ H2O2

Adapted from: J Bacteriol Mycol. 2014;1(1): 2.

Pseudomonas aeruginosa
 Ubiquitous microorganism with versatile metabolism;
 Opportunistic pathogen.
PA14 strain
 Clinical isolate (burn)
 Multi-host virulence pathogen – excellent model;
 P. aeruginosa PA14 antioxidant arsenal
- Much more complex than E.coli!

11 thiol peroxidases
5 Heme peroxidases
2 Superoxide dismutases
Tpx (thiol peroxidase)

AhpC (alkyl hydroperoxide reductase) Cpo(chloroperoxidase)

AhpA Prxs CcpR(cytochrome c peroxidase)
AhpB KatE(catalase E)
Bcp (bacterioferritin co-migratory protein);
LsfA Gpx
Gpx (glutathione peroxidase)
Ohr/OsmC
OsmC (osmotically inducible protein C)
Ohr (organic hydroperoxide resistance protein)

cytoplasm
periplasm
Extracellular space
Why so many enzymes?
Ohr = “Organic Hydroperoxide Resistance Protein”

 It was first described in a genetic screening that aimed to find genes involved with
organic hydroperoxide detoxification in Xanthomonas campestris pv. phaseoli.

tBOOH H2O2

Mongkolsuk et al., (1998) J. Bacteriol. 180: 2636.

 Ohr/OhrR system

P. aeruginosa cell

reduced OhrR
ohr
ohrR ohrR ohr

oxidation
ROOH

ohrR ohr

cytoplasm
periplasm
Extracellular space
Pa Δohr mutant cells are sensitive to fatty acid hydroperoxides

Linoleic Acid hydroperoxide (LAOOH) Oleic Acid hydroperoxide (OAOOH)

mM 0 0.04 0.08 0.14 0.16 0.18 0.2 mM
0 0.2 0.4 0.5 0.6 0.7 0.8 1 2 3 0.06 0.1 0.12

Wt

Δohr

ΔahpC

Δtpx

Δgpx

Meireles et al., Proc. Natl. Acad. Sci. 2017, 114 (2), E132–E141.
 The biological meaning of Ohr
occurrence
Table 1- Second order rate constants between XfOhr and organic hydroperoxides

AhpE /Ohr kox

oxidant Main occurrencea Oxidation mechanism
(M-1.s-1)

OAOOH (18:1) 2.0 x 108 Biological membranes non enzymatic oxidation?

Oxylipin intermediary/Biological
LAOOH (18:2) 3.7 x 107 LOXs, non enzymatic oxidation
membranes

5(S)-HpETE (20:4) 2.6 x 107 Inflammation reponse intermediary

ALOX5, ALOX15, non enzymatic
oxidation
15-HpETE (20:4) 6.0 x 107 Inflammation ressponse intermediary

Ch-OOH not detected mammalian membranes Non enzymatic oxidation

tBOOH 7.0 x 106 artificial molecule -
H2O2 2.30 x 102 incomplete product of reduction of O2 -
product of reaction of nitric oxide and
ONOO- (2 ± 0.3) x 107
superoxide radicals
-

Meireles et al., Proc. Natl. Acad. Sci. 2017, 114 (2), E132–E141.
Meireles et al., Proc. Natl. Acad. Sci. 2017, 114 (2), E132–E141.
 Proposed model for PaOhr biological significance during host-pathogen
interaction
Infectious milieu Host phospholipds
Phospholipases, PLs, host and bacteria derived – ex. via ExoU (2, 3)

Arachidonic acid release

into extracelular medium
Secreted bacterial 15-LOX (1, 4)

Hydroperoxide Initiation of host anti-

formation, ex.: 15- inflamatory signalling
HpETE (1, 4)
Ohr (5)/Gpx
(cytosolic proteins)

Detoxification and use of fatty

acid products as C source to reach Expression induced by
High Cell Density, HCD (6, 7) OhrR and OspR,
Adapted from (1) respectively (8).
1- Deschamps, JD. et al. “Biochemical and Cellular Characterization and Inhibitor Discovery of Pseudomonas aeruginosa 15-Lipoxygenase.” Biochemistry 55.23 (2016): 3329–3340.
2- Vromman, F, and Agathe Subtil. “Exploitation of Host Lipids by Bacteria.” Current Opinion in Microbiology 17.1 (2014): 38–45.
3- da Cunha LG Jr, Ferreira MF, et al (2015) ExoU-induced redox imbalance and oxidative stress in airway epithelial cells during Pseudomonas aeruginosa pneumosepsis. Med Microbiol Immunol. 204(6):673-80
4- Banthiya, Swathi et al. “Structural and Functional Basis of Phospholipid Oxygenase Activity of Bacterial Lipoxygenase from Pseudomonas aeruginosa.” Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids 1861.11 (2016): 1681–1692.
5- Alegria, TG, Meireles, DA et al (2016) Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite. Submitted to PNAS
6- Sun, Z, et al. “Blocking Phosphatidylcholine Utilization in Pseudomonas aeruginosa, via Mutagenesis of Fatty Acid, Glycerol and Choline Degradation Pathways, Confirms the Importance of This Nutrient Source in Vivo.” PLoS ONE 9.7 (2014).
7- Turner, Keith H. et al. “Requirements for Pseudomonas Aeruginosa Acute Burn and Chronic Surgical Wound Infection.” PLoS Genetics 10.7 (2014): n. pag. Web.
8- Atichartpongkul, S et al. “Regulation of Organic Hydroperoxide Stress Response by Two OhrR Homologs in Pseudomonas aeruginosa.” Plos One 11.8 (2016): e0161982.
 Lack of Ohr render cells sensitive to peroxynitrite (ONOO-)

WT SIN-1 + 5 mM 3-ATZ
150

relative sensibility (% c.f.u.)

t

**
en

125
m
at

H
tre

100
SO

R
O

H
HP

hr
1

hr
O
No

eO
N-
DM

Δo
Δo
LA

t-B
SI

M
75
α-Ohr 50

25
loading
control
0
wild type ohr complemented

Meireles et al., Proc. Natl. Acad. Sci. 2017, 114 (2), E132–E141.
 Group 5

Ohr/OsmC family
Group 4

Group 6

1.1 Group 3
OsmC
(968 seqs)

1.2
1.3 Group 2
Group 1 1.4
Ohr
(1498 seqs) Meireles et al, ongoing
 Residue conservation analysis– Ohr (3295 sequences) HIS
TYR 134, A
CYSr
CYS, 127, A
Chain A 125, A
61A

Consurf

ARG
19, B
ASP GLU TYR
Chain B PRO
49, B 51, B 9,B
37, B
PRO50,B

WebLogo
9 19 27 37 49 51 61
analysis

96 122 125 127 134

 Residue functional/structural analysis – Ohr subfamily
L1 L2 L3 L4 L5 L6

S1 S2 S3 H1 H2 S4 S5 H3
1 2

4
3

Fig. 3 - A. Consensus sequence logo of Ohr sub-family. The numbers below selected conserved residues refer to those in the protein sequence of 1ZB8 from Xylella fastidiosa. The logo was constructed from alignment of 4129 non-redundant sequences extracted from 21880
Ohr/OsmC initial pool. The alignment was produced by MUSCLE (Edgar, 2004) and cured by eye. Gaps present in more than 90 % of sequences were removed to construct the input file for Skylign tool (Wheeler et al., 2014). B. and C. Distances and spacial localization of each
interacting residue pair. Molecular graphics and analyses were performed with the UCSF Chimera package.
1N2F – co-crystalized w/
DTT
Y126 mutation impairs protein stability and affects peroxidase activity

Tm SD
wt 58,69 0,06366
Y126A 53,44 0,2133
Y126F 57,08 0,1292

Meireles et al, ongoing

 Δohr strain of Pa is not attenuated for virulence

BALB/c mice were infected intratracheally with 2×106 bacteria per mouse with wild-type strain PA14, ΔohrR, Δohr and
Δohr/ΔohrR (n = 5 per group). Control mice were inoculated with PBS (n = 5) and the survival of the mice was followed for the
course of the experiment, around 60 hours.

Anita Martins Fontes, et al 2019 to be published

 P. aeruginosa PA14 antioxidant arsenal
- Much more complex than E.coli!

11 thiol peroxidases
5 Heme peroxidases
2 Superoxide dismutases
Tpx (thiol peroxidase)

AhpC (alkyl hydroperoxide reductase) Cpo(chloroperoxidase)

AhpA Prxs CcpR(cytochrome c peroxidase)
AhpB KatE(catalase E)
Bcp (bacterioferritin co-migratory protein);
LsfA Gpx
Gpx (glutathione peroxidase)
Ohr/OsmC
OsmC (osmotically inducible protein C)
Ohr (organic hydroperoxide resistance protein)

cytoplasm
periplasm
Extracellular space
Why so many enzymes?
AhpC1 is essential for P. aeruginosa full virulence in vivo

https://doi.org/gmdx3h
LsfA is essential for P. aeruginosa full virulence in vivo

https://doi.org/f6pdwr
 P. aeruginosa PA14 antioxidant arsenal
- Much more complex than E.coli!

11 thiol peroxidases
5 Heme peroxidases
2 Superoxide dismutases
Tpx (thiol peroxidase)

AhpC (alkyl hydroperoxide reductase) Cpo(chloroperoxidase)

AhpA Prxs CcpR(cytochrome c peroxidase)
AhpB KatE(catalase E)
Bcp (bacterioferritin co-migratory protein);
LsfA Gpx
Gpx (glutathione peroxidase)
Ohr/OsmC
OsmC (osmotically inducible protein C)
Ohr (organic hydroperoxide resistance protein)

cytoplasm
periplasm
Extracellular space
Why so many enzymes?
Creation of nonpolar library of mutants in thiol peroxidases
Antibiotic resistance profile

Single mutant Galleria mellonella as

alternative infection
model

Double, triple
mutant Phenotypic analysis;
generation
Motility– swarming e twitching

Screening for altered

phenotypes
Growth in the presence of antioxidants

in vitro and in
Biofilm assays vivo virulence
assays
Meireles, on going
G. mellonella é amplamente aceita como um organismo modelo para estudo da
patogênese microbiana
Vantagens do uso de G. mellonella como modelo alternativo de infecção para estudar
patógenos bacterianos
Isolados mutantes de PA14 que são menos virulentos em G. mellonella também tendem a
ser menos virulentos em camundongos.
Ohr proteins are also present among eukaryotes
MfOhr is a mitochondrial protein
"Nothing in Biology Makes Sense
Except in the Light of Evolution" 
Dobzhansky, Theodosius (March 1973) The American Biology
Teacher 35 (3): 125–129, JSTOR 4444260