Respiratory pathway choice in Neisseria meningitidis
Andrew Schofield, Jamie Wood and James Moir
Overview
Introduction: Neisseria and respiration
Organisation of respiratory chain components in N. meningitidis
Regulation: (i) Genetic control, (ii) Metabolic control
Biology of a microbial respiratory system
Early stages of mathematical model building: perspectives and approaches
Neisseria meningitidis
natural habitat –
the human
nasopharynx
(5-20 % of population
colonised)
Occasionally, invasive disease occurs …
Multiplication in blood
 septicaemia
Multiplication in
cerebrospinal fluid
 meningitis
meninges
Neisseria meningitidis
0.01
C. violaceum
N. weaveri
N. m eningitidisand N. gonorrhoeae459
N. flavescens
are more closely related to one
410
another than other, non-pathogenic
N. subflava
Neisseria. Phylogenetic
998
N. meningitidis
tree based on 16S rRNA
N. gonorrhoeae
810
sequenc e.
N. lactamica
610
N. pharyngis
1000
N. mucosa
494
N. flava
956
N. sicca
N. elongata
N. bacilliformans
985
762
Only these two species
are pathogenic,
the others are strictly
commensal. (Why?)
Colonisation of human nasopharynx necessary precursor to virulence
Survival in natural habitat and blood/CSF relies on virulence determinants
such as adhesion / immune evasion etc. but also on respiratory metabolism
Respiration
Oxidation of cellular reductants to generate biochemical energy
reduction
potential
+
½O2
+830 mV
H 2O
electrochemical (H+)
gradient across
biological membrane
2e-
-340 mV
-
NADH
NAD+
drives
synthesis of
ATP
In the mitochondrion
Respiratory electron transport
NADH
Q
cyt. bc1
cyt. c
aa3
O2
succinate
H+
H+
H+
H+
Q
½O2
NADH
NAD+
H2O
ADP
ATP
In bacterial plasma membrane
Respiratory electron transport
cbb3
NADH
succinate
hydrogen
formate
Q
cyt. bc1
nitrate
reductase
ba3
O2
methanol
methylamine
sulfur
dehydrogenase
cyt. c
aa3
O2
O2
nitrite
reductase
nitric oxide
reductase
nitrous oxide
reductase
*selected respiratory pathways of
Paracoccus denitrificans, a close
relative of the mitochondrion
In bacterial plasma membrane
Respiratory electron transport
predicted for N. meningitidis
cbb3
NADH
Q
cyt. bc1
O2
cyt. c
succinate
Cytochrome cbb3 oxidase, identified in:
Microaerophilic pathogens: Helicobacter, Campylobacter
Necessary for maintaining oxygen sensitive nitrogenase in root
nodule bacteroids of rhizobia
Expressed under microaerobic conditions in versatile bacteria such
as Paracoccus, Pseudomonas
Bradyrhizobium japonicum cbb3 has high affinity (Km = 7 nM) for O2
Oxygen and Neisseria environment
air-flow
O2
colonising the nasopharyngeal mucosa
Common bacterial cohabitants with Neisseria species:
Streptococcus, Staphylococcus
Veillonella, Prevotella, Fusobacterium (“strict” anaerobes)
Respiratory pathways in N. meningitidis
Predictions from genomes of Neisseria meningitidis
O2
NO3-
cbb3
NO2-
H2O
AniA
NO
microaerophilic oxidase
NorB
N2O
N2
partial denitrification
…function of denitrification genes verified genetically:
Denitrification of nitrite supports growth under
microaerobic conditions and requires AniA and NorB
Respiratory substrates in the environment of N. meningitidis
NO3- / NO2-
saliva
macrophages
NO
air-flow
O2
colonising the nasopharyngeal mucosa
Meningococcus exposed to variety of potential
respiratory electron acceptors
Must deal with NO generated in the host by NO
synthase
Questions
How is the respiratory pathway of N. meningitidis organised?
How does the bacterium regulate respiratory flux?
Is mathematical modelling necessary to appreciate a system
that consists of just three respiratory reactions?
Does analysis of respiration in N. meningitidis tell us anything
about its nature as a pathogen?
Can analysis of respiration in Neisseria tell us anything more
general about the nature of respiratory chain structure and
function in other bacteria?
Part 1
Organisation of Neisseria respiratory chains:
architecture and function of respiratory chain components
Focus on:
electron transport pathways and topological arrangements
Respiratory pathways in N. meningitidis
Predictions from genomes of Neisseria meningitidis
cbb3
NADH
Q
cyt. bc1
succinate
H2O
cyt. c
NO
O2
AniA
NorB
N2O
*confirmation of bc1 complex dependent pathway to oxygen
and nitrite using inhibitor sensitivity with myxothiazol
NO2NO
Respiratory pathways in N. meningitidis
Predictions from genomes of Neisseria meningitidis
outer
membrane
4H+
periplasm
4H+
2H+
inner
NorB
membrane
?
oxidase
NADH
dehydrogenase
2NO
+2H+
NAD+
NADH
N2O
-0.2
NADH/NAD+
2H+
4H+
Thermodynamic considerations:
-0.4
0
0.2
0.4
NO2-/NO
0.6
2H+
bc1
Q
Q
AniA
NO2+2H+
NO
0.8
½O2/H2O
potential (V)
1.0
1.2
NO/N2O
2H+ ½O
2
+2H+
H2O
NADH/O2
NADH/NO2NADH/NO
H+:e5
3
3
?
Potential pathways of electrons to O2 and nitrite
OM
(* like cytochrome-NIR
fusion in Bdellovibrio)
$
*
Periplasm
$
like extra domain
in NG cbb3 oxidase
IM
?
Cytochrome cx
NMB0717
monohaem,
Cytochrome c4
NMB1805
di-haem,
predicted
periplasmic
Cytochrome c5
NMB1677
di-haem, predicted
predicted
periplasmic
membrane-associated
similar to
NGO0292
similar to
NGO0101 (near
(different C
termini)
identical)
identical to
NGO1328
To cut a long story short…
Organisation of respiratory chain in N. meningitidis
outer
membrane
NO2-
periplasm
NO
inner
membrane
Q
NADH
Q
NAD+
2NO
N2O
H2O
½ O2
electron flow between membranes in N.
meningitidis
outer membrane
~50 Å
AniA
~50 Å
e~60 Å
e- cyt. c
5
e-
inner membrane
N. meningitidis versus all the other Neisseria sp.
Same respiratory components except…
…different domain structure of cbb3 oxidase
N. meningitidis
N. gonorrhoeae, N. lactamica, etc.
CcoP
CcoP
CcoO
CcoO
H2O
H2O
½ O2
CcoN
½ O2
CcoN
>99% sequence conservation between conserved domains, but loss of domain between
other Neisseria and evolution of N. meningitidis –strong selection pressure –for what?
Nitrite reductase activity –dependency on c5
Strain
Nitrite reductase activity
N. meningitidis wild type
+
+
+
N. meningitidis c5-
N. gonorrhoeae wild type
N. gonorrhoeae c5-
Why are multiple pathways necessary for oxygen / nitrite reduction?
Experimental approaches are vital, but can modelling provide additional insight?
Once we have predicted qualitative behaviour we should be able to test
this with whole cell UV/visible spectroscopy …
Redox difference spectra of N. meningitidis intact cells
0.16
absorbance
0.12
c-type cytochrome
0.08
b-type cytochrome
0.04
0
350
-0.04
400
450
500
550
600
650
700
Wavelength (nm)
-0.08
Reduced cells oxidised by oxygen
Reduced cells oxidised by nitrite
Reduced cells oxidised by nitric oxide
Part 2
Regulation:
Genetic and metabolic
Respiratory pathways in N. meningitidis
O2
cbb3
NO2-
H2O
AniA
NO
microaerophilic oxidase
NorB
N2O
partial denitrification
this doesn’t look too complicated...
The “switch” from aerobic to anaerobic is a complex one
1000
60
NO
800
50
O2
40
30
600
400
[NO] (nM)
[O2] (% air saturation)
70
20
200
10
0
0
0
0.5
50
1
1.5
2 2.5
3
3.5 4 4.5
100 150 200 250 300 350 Time
400 (h)
450
1. Oxygen becomes depleted
0
NO2-
AniA
NO
NorB
N2O
2. Nitrite reductase induced, NO accumulates
3. NO inhibits oxidase, O2 rises
O2
cbb3
H2O
4. NO induces NO reductase, NO falls
5. Oxygen falls, steady state respiration under oxygen limitation ensues
The “switch” from aerobic to anaerobic is a complex one
transition from aerobic to denitrifying growth:
high failure rate in N. meningitidis in batch growth
1.6
1.4
1.4
1.2
1.2
1.6
1.4
1.4
1.4
1.4
1.2
1.2
1.2
1
1
1
0.8
0.8
0.8
0.6
0.6
0.6
0.4
0.4
0.4
0.2
0.2
0.2
0
0
1.2
1
1
1
0.8
0.8
0.8
0.6
0.6
0.6
0.4
0.4
0.4
0.2
0.2
0
0
0.2
0
2
4
6
8
time(h)
10
0
2
4
8
time(h)
6
10
0
0
2
4
6
8
10
time(h)
0
2
4
6
8
time(h)
10
0
0
2
4
6
8
time(h)
10
0
2
4
6
8
10
time(h)
[nitrite]
cell density
N. meningitidis is under stress during the
transition and does not always achieve transition to
denitrification readily
This effect can be independent of culture history /
genotype (different behaviour of duplicates)
1.6
1.8
1.4
1.6
1.4
1.2
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
0
Unpredictability disappears in absence of active
nitrite reductase pathway
2
4
6
8
time(h)
10
0
2
4
In aniA- strains
6
8
time(h)
10
Metabolic control of respiration
NO2-
AniA
NO
-
NorB
-
Thermodynamic considerations:
-
-0.4
-
O2
N2O
Oxygen, nitrite and nitric oxide
compete for the same pool of
electrons
cbb3
-0.2
NADH/NAD+
0
0.2
0.4
NO2-/NO
0.6
0.8
½O2/H2O
potential (V)
1.0
1.2
NO/N2O
H2O
NO is a competitive inhibitor of the oxidase
(O2 and NO have similar molecular
structures) and thus denitrification
intermediate accumulation can inhibit
oxidase activity.
Genetic control of respiration
Synthesis of denitrification reductases is governed by availability of
respiratory substrates oxygen, nitrite and nitric oxide
O2 inhibits aniA expression
NO2- activates aniA expression
NO activates norB expression
NO2-
AniA
NO
NorB
+
+
-
O2
cbb3
H2O
N2O
Genetic control of respiration
Synthesis of denitrification reductases is governed by availability of
respiratory substrates via transcriptional regulators FNR, NarQP and NsrR
384 base promoter region
nitric oxide reductase
norB
-
NarP
NsrR
-
+
-
nitrite reductase
aniA
NsrR FNR
-
+
NO2 -
NO
+
O2
NO
NO
Effect of NO on mRNA measured using RT-PCR
12
14
norB
12
aniA
10
10
8
1.4
FNR +ve
control
1.2
1.0
8
6
6
10
8
6
0.6
4
2
2
aniA in nsrR-
12
0.8
4
4
14
2
0.4
0
10
20
30
40
[NO]
50
0
10
20
30
40
50
[NO]
0
10
20
30
40
50
[NO]
0
10
20
30
40
50
[NO]
Genetic control of respiration
Synthesis of denitrification reductases is governed by availability of
respiratory substrates via transcriptional regulators FNR, NarQP and NsrR
384 base promoter region
nitric oxide reductase
norB
-
NsrR
NO
NarP
+
+
NO2 -
-
+
nitrite reductase
aniA
NsrR FNR
NO
O2
NO
Positive and negative regulation of aniA by NO should ensure aniA synthesis
can be initiated before NO accumulation, can be optimal during
denitrification, and can be switched off if NO approaches toxic concentration.
Complex regulation of a system with only three reactions:
respiration in N. meningitidis
NsrR NsrR
NarP
+
NO2-
+
+
AniA
NO
NorB
N2O
- FNR- FNR
-
O2
cbb3
-
H2O
And...
[Disproportionation of nitrite to NO
under acidic conditions]
[Reaction of NO with oxygen]
[Irreversible inhibition of oxidase by
peroxynitrite]
keeping it “simple” by ignoring genetic regulation
using this…
…to attempt to explain this
d [O2 ]
[O2 ]
 k1[O2 ][Ca ]  (1  sat
)
dt
K O2
d [ NO]
 l1[ NO][ Ba ]  m1[ NO2 ][ Aa ]   [ NO]
dt
d [ NO2 ]
 m1[ NO2 ][ Aa ]
dt
d [Qa ]
 g[Qi ]  f [Qa ][ L]  l3[Qa ][ Bi ]
dt
i
k3 K NO
[ E ][Ci ]
d[E]
 f [Qa ][ L] 
 m3[ E ][ Ai ]
i
dt
[ NO]  K NO
E  L  X, Qi  Qa  Q
d [ Aa ]
 m3 [ E ][ Ai ]  m1[ NO2 ][ Aa ]
dt
d [ Ba ]
 l3 [ E ][ Bi ]  l1[ NO][ Ba ]
dt
i
d [Ca ] k3 K NO
[ E ][Ci ]

 k1[O2 ][Ca ]
i
dt
[ NO]  K NO
Ai  Aa  A, Bi  Ba  B, Ci  Ca  C
NO2O2
NO
NO2NO
e-
-
O2
H2O
-
Transition from oxygen to denitrification appears
unstable under laboratory conditions.
What about in vivo?
Some patients recovering from meningococcal disease do
make antibodies against AniA
But, some clinical isolates show pseudogenization of aniA*
i.e. Nitrite reduction can be a useful strategy for N.
meningitidis in vivo, but it can also be selected against,
presumably because the short term adaptation has a high
failure rate
[*norB always conserved
and plays a role in
protecting bacterium from
host immune system in
tissue/organ culture models]
aniA is *very highly conserved* in N. gonorrhoeae and other commensal Neisseria
and aerobic/anaerobic switch consistently proceeds predictably in N. gonorrhoeae
-why the difference?
Transition from oxygen to denitrification appears
unstable under laboratory conditions
What factors affect this transition?
N. meningitidis
N. gonorrhoeae, N. lactamica, etc.
CcoP
cbb3
Structure of respiratory chain (components)?
-differences between N. meningitidis and the rest of Neisseria
Transcriptional regulatory network?
-differences between N. gonorrhoeae and the rest of Neisseria
Population structure / density?
CcoP
cbb3
Regulation and gonococcus versus meningococcus
Same components, same result, different mechanism
FNR binding to N. gonorrhoeae aniA promoter weak c.f. that of
N. meningitidis
Compensated for by cis effect on narP binding site
AND
trans effect on nitrite sensitivity of NarQ
N. gonorrhoeae
N. meningitidis
NO2-
NO
O2
NO2-
O2
NO2-
NO
O2
NsrR
FNR
NarQ
NarQ
NsrR
FNR
+
NarP
NarP
PaniA
PaniA
PaniA
Summary
Respiratory pathway choice in N. meningitidis is controlled in a complex manner
metabolically and genetically
An ODE model for respiratory pathway choice is providing good qualitative fits to
explain metabolic regulation but requires further parameterisation
This approach should be able to inform us regarding the key control features in
this system, and help us appreciate issues relating to different adaptations
between closely related Neisseria species
Neisseria uses multiple respiratory electron carriers to shuttle electrons from bc1
complex to terminal electron acceptor reductases
Adapting TASEP for application to branched respiratory chains may provide
insight regarding organisational principles with relevance to other bacteria
/synthetic biology
Acknowledgements
University of York
Jamie Wood
Andrew Schofield
Elizabeth Thomas
Yujiang Wang
Manu Deeudom
Melanie Thomson
Karin Heurlier
Jonathan Rock
Diana Quinn
University of Oslo
Mike Koomey
Ashild Vik
University of Sheffield
Robert Read
University of Leeds
Chris Needham