Pneumococcal fratricide: possible
impact on horizontal gene transfer
and virulence
Natural genetic transformation
Free DNA
R.V. Miller, Scientific American 1998
Chlorobi
?
?
Deinococcus/
Thermus
65 bacterial species are known to be competent for natural transformation
Prevalence of natural
transformation in the
genus Streptococcus
pyogenic group
Competence in Streptococcus
pneumoniae is strictly regulated
CSP
STP
pheromone
pheromone
Sensor
kinase
ComD
Cell
membrane
14 required for transformation
Late-gene promoter
> 100 late genes
Alternative
sigma factor
ComX
~ 20 early genes
7 required for transformation
Early-gene promoter
Response
regulator
ComE
CSP
STP
pheromone
pheromone
Sensor
kinase
ComD
Cell
membrane
14 required for transformation
Late-gene promoter
> 100 late genes
Alternative
sigma factor
ComX
~ 20 early genes
7 required for transformation
Early-gene promoter
Response
regulator
ComE
The term quorum-sensing describes the ability of a microorganism
to perceive and respond to microbial population density
DNA uptake depends on cell-cell communication
through a quorum-sensing mechanism.
Why?
Probably to ensure that homologous extracellular DNA is present during
competence.
DNA
How can competence regulation through a
quorum-sensing mechanism influence the level
of extracellular homologous DNA during
natural transformation?
Passive release
Since the quorum-sensing mechanism ensures that competence
development only takes place in a relatively dense population,
DNA from members of the population that has died from
natural causes will always be present.
Alternative mechanism for release of DNA to the external
milieu:
Active release coupled to competence development
Induction of the competent state triggers a DNA release
mechanism.
a) altruistic suicide
b) predation
Beta-galactosidase release assay for measuring cell lysis
S. pneumoniae strain EK4166 : Unable to self-induce (ComA-)
β-gal negative (ebg-)
Harbours a constitutively expressed cytoplasmic E.coli β-gal fusion
protein which do not affect DNA uptake and transformation
Addition of CSP-1
Incubation, 37°C
30 min.
EK4166
Removal of cells by sentrifugation
Filtration of supernatant (0.2 um)
Determination of
beta-galactosidase
activity
Analysis of beta-galactosidase activity and NovR DNA contents
in the same supernatant.
Beta-galactosidase
release
NovR DNA
release
Steinmoen et al.,
PNAS, 2002
Kinetics of beta-galactosidase release
Beta-galactosidase activity
(Absorption at 420 nm)
0,25
0,2
CSP-1 added at time 0
0,15
0,1
0,05
No CSP-1 added
0
0
20
40
Minutes
Steinmoen et al.,
PNAS, 2002
60
80
Kinetics of beta-galactosidase release
Beta-galactosidase activity
(Absorption at 420 nm)
0,25
0,2
CSP-1 added at time 0
0,15
0,1
0,05
No CSP-1 added
0
0
20
40
Minutes
60
80
What is the mechanism behind the competence-induced
cell lysis?
Does the observed cell lysis involve cell wall degrading
enzymes?
Known murein hydrolases in Streptococcus pneumoniae
Choline-binding domains
LytA
LytB
LytC
R. Lopez, Res. Microbiol.,2000.
Amidase
Glucosaminidase
Lysozyme
Yother et al., J. Bacteriol,1998.
The murein hydrolases LytA, LytB and LytC, plus the putative
murein hydrolases CbpD are choline-binding proteins.
Addition of choline to the growth medium has a
negative effect on competence induced cell lysis
LytA and LytC are involved in competence induced
cell lysis
Involved in
lysis
LytA
+
LytB
–
LytC
+
Choline binding protein D (CbpD) is a key
component of the lysis mechanism.
CHAP = cysteine, histidine
dependent amidohydrolases/
peptidases
Choline binding
repeats
Beta-galactosidase reporter assay
50
45
40
35
Millerunits
30
25
no csp
20
csp
15
10
5
0
-5
0
5
10
15
20
25
30
35
minutes after added csp
Kinetics of competence induced transcription of the cbpD gene
Release of DNA into media from csp induced cultures
Beta-galactosidase
release in wild type and CbpD- cells.
90
Release of DNA (Millerunits)
80
70
Wild type
60
50
cbpD/csp
40
no cbpD/csp
30
20
CbpD- mutant
10
0
0
1
2
3
OD550
4
5
6
DNA release measured by Real-Time PCR
35
30
Relative DNA release
25
20
15
10
5
0
Competence
wt/csp
induced
Uninduced
wt/-
Competence
mut/csp
induced
Uninduced
mut/_
Wild type
CbpD mutant
Competence induced
release of DNA
Bistability?
Altruistic suicide?
Can one cell cause lysis of another cell ?
- Expresses beta-galactosidase
constitutively
- Competent when
CSP is added
- Competence
Target strain deficient (ComE-)
Attacker strain
= Beta-galactosidase
Co-cultivated
A
+
B
+
No CSP
C
+
+ CSP
Non-competent
target cells
+ CSP
Competent
attacker cells
No beta-gal.
release
No beta-gal.
release
Steinmoen et al., J. Bacteriol., 2003
Possible explanation of experimental data:
a) Both suicide and predation takes place in a competent
population of Streptococcus pneumoniae cells.
b) Only predation (also called fratricide) takes place
How was suicide (altruism) excluded?
It was discovered that competent cells protect themselves against
their own toxins by producing an immunity protein
The ComM immunity protein
ComM is an integral membrane protein with six transmembrane helices.
ComM is embedded in the
membrane and has no cytoplasmic
or extracellular domains
If competence induced cell lysis is due to predation (fratricide) only,
how come that CSP-induced lysis also takes place in a monoculture
of competent cells ?
Addition of CSP-1
Incubation, 37°C
30 min.
EK4166
Removal of cells by sentrifugation
Filtration of supernatant (0.2 um)
Determination of
beta-galactosidase
activity
Effect of competence induction on growth of comM mutant cells
Wt
ΔcomM
Havarstein et al.,
Mol. Microbiol., 2006
Proteins known to be involved in fratricide in liquid culture:
Competent cell
CbpD: Putative murein hydrolase
LytA:
LytC:
Autolysin
Autolysin
ComM: Immunity against autolysins
or CbpD
Lysis
?
Lysis
Non-competent
cell
Non-competent cell
What is the specific roles and relative contributions
of LytA, LytC and CbpD with respect to fratricide?
Non-competent
S. pneumoniae
Competent
S. pneumoniae
Transformed
S. pneumoniae
Lysins
Bacterial
β-galactosidase
chromosome
comE–
hirL::lacZ
ebg–
comA–
Termed Wt mixed cultures in subsequent experiments
Co-cultivation assay
Addition of CSP
2.5 ml
Attacker cells
2.5 ml
Incubation, 30°C
30 min
Mix of attacker
and target cells
Target cells
producing
cytoplasmic
β-galactosidase
Removal of cells by sentirfugation
Filtration of supernatant (0.2 um)
Determination of
beta-galactosidase
activity
Curiously, LytA and LytC in both attacker and target cells
contributes to fratricide.
Why?? What is the role of LytA and LytC?
Non-competent
cell
Activation of
competence
by CSP
CbpD
Competent
cell
CbpD (Murein hydrolase?)
LytA (Autolysin)
Non-competent
cell
LytC
Lysis of
non-competent
cell
(Autolysin)
ComM
(Immunity protein)
Is CbpD secreted into the growth medium, or will it stay attached
to the cell-wall teichoic acid of the producer cells?
CbpD
Competent
cell
Competent
cell
Non-competent
cell
Lysis of
non-competent
cell
Non-competent
cell
Lysis of
non-competent
cell
CbpD (Murein hydrolase?)
LytA (Autolysin)
LytC
(Autolysin)
ComM
(Immunity protein)
Does fratricide require cell-cell contact?
Non-competent
target
cell
Competent
predator
cell
ComM
immunity
protein
CbpD
LytA
LytC
Is CbpD triggering autolysis by activating
LytA and LytC?
CbpD
LytA
LytC
Two different sources of donor DNA for competent pneumococci
A: Natural cell death
B: Predation. Competent streptococci kill
non-competent streptococci and steal
their DNA.
Prevalence of CbpD in the genus Streptococcus
CSP induced genes and virulence
14 proteins involved
DNA
binding,
in
DNA
uptake and
Processing,
uptake
recombination
And recombination
~80 proteins,
~ 40 of totally
unknown function
LytA
ComAB
CbpD
LytC
7 proteins
needed for
competence
11 proteins of
unknown function
Fratricide
ComM
CSP-induced virulence and possible virulence genes: lytA, comD, comB, cbpD, endA, coiA, cglB, cinA
References: Berry et al., 1989; Bartilson et al., 2001; Lau et al., 2001; Hava and Camilli, 2002
Competence induced cell lysis – what is it for?
I
II
Through predatory behaviour (fratricide) competent
pneumococci get access to transforming DNA and
nutrients, and at the same time get rid of competitors.
It is possible that competence induced cell lysis may
contribute to virulence in Streptococcus pneumoniae
through the release of pneumolysin and cell wall-derived
inflammatory components.
Acknowledgements
Vegard Eldholm
Kristine Haugen
Ola Johnsborg
Hilde S. Ohnstad