Comment les bactéries dét i t l fdéterminent leur forme...Budding and appendaged bacteria its...
Transcript of Comment les bactéries dét i t l fdéterminent leur forme...Budding and appendaged bacteria its...
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
1
College de France _ 27 janvier 2016
Comment les bactéries Comment les bactéries dét i t l fdét i t l fdéterminent leur formedéterminent leur forme
une cible pour de nouveauxune cible pour de nouveauxantibiotiques ?antibiotiques ?
Rut CARBALLIDO LOPEZRut CARBALLIDO LOPEZ
Prokaryotic Cell Development LabProkaryotic Cell Development Lab
2 µm
Kevin D. Young Microbiol. Mol. Biol. Rev. 2006;70:660-703
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
2
Coccus
Variety of prokaryotic cell shapes
Rod
Spirillum
“… Space must be cut off, shaped, defined, for us to inhabit.
From cradle to coffin, it's enclosure that
Spirochete
Budding and appendaged bacteria
it s enclosure that defines us”
— Robert Morgan (1992)
Cell Wall Peptidoglycan
Glycanchains
ide
NAM
OH
O
NAG
Peptide cross-links
Pen
tape
pti
L-alanine
D-alanine
D-alanine
D-glutamate
L-lysine/mDAP
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
3
L2
Flipase(PBPs)
Transglycosylasemembrane
OUT(cell wall)
Transpeptidase
L2
PG-precursor syntheticenzymes (Mur & DAP) IN
(cytoplasm)
The enzymatic steps involved in cell wall synthesis are the targets of cell wall antibiotics.
Pencillins, β-lactams
gy2
011
moenomycin
glycopeptides
De
ngl
er
et a
l. B
MC
Mic
rob
iolo
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
4
The Cell Wall is a major determinant of shape
Isolated cell wall sacculi retain bacterial cell shape
B. subtilis sacculus (AFM)(Hayhurst, et al. PNAS 2008)
The Cell Wall is a major determinant of shape
+ LYSOZYME
Removal of the cell wall produces spherical cells (protoplasts)
© Arnaud Chastanet
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
5
Classical genetics of cell shape in rod-shaped bacteria
rod mutations
• rodA Peptidoglycan (wall) synthesis rodA, pbpA
• rodC Teichoic Acid (wall) synthesis tagF
• rodB Function unknown mreBCD
Bacteria with complex shapes tend to contain MreB-like proteins
Nr. organisms example/group Shape
8 / 2* B. subtilis (3 genes), E. coli,
3 Vibrio cholera, Caulobacter crescentus
1 Streptomyces coelicolor (3 genes)
5 Helicobacter pylori, Borrelia burgdorferi
1 Chlamydia (2 species)
mre
Bpr
esen
t
Rod
Curved rod
Filamentous
Helical
Pleiomorphic
5 / 3* Strep. pneumoniae, Staph. aureus, Methanococcus jannaschii*
2 Mycoplasma (2 species)
* Archaebacterium
mre
Bab
sent
Round
Pleiomorphic
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
6
Bacillus subtilis has three MreB isoforms: MreB, Mbl and MreBH
mreDmreCmreBmreB57%
67%mreCmreBmreB mreD
in E. coli in B. subtilis
Gram -- Gram ++
67%
mblmbl mreBH51%76%
mreB-wild-type
mreBH-mbl-
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
7
unprocessed
Subcellular localization of MreBs by IFM (Immunofluorescence Microscopy)
Indirect immunofluorescence
The ‘Point Spread Function’ (PSF)
z
bead 175 nm (diameter)
acquired image(1.4NA/100 x Zeiss Plan/Apo/Widefield)
PSF3D blurry image of such a single
point light source
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
8
Mathematical operation used in image analysis to removeout-of-focus light above and below the focal plane (PSF)
Deconvolution
drawnto scale
acquired imageunder the
microscope
deconvolvedimage
unprocessed deconvolved
‘MreB proteins form filamentous helical structures
that encircle the cell’[ Jones, Carballido-López & Errington (2001) Cell ]
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
9
Subcellular localization of GFP-MreBs fusions imaged by epifluorescence in live cells
GFPGFP
GFP
GFP
Subcellular localization of GFP-MreBs fusions imaged by conventional epifluorescence
Bacillus subtilis
GFP-MreBHGFP-MblGFP-MreB
Escherichia coli Caulobacter crescentus
GFP-MreBGFP-MreB
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
10
Eukaryotic ACTINMreB (T maritima)
MreB proteins are structural homologues of eukaryotic actin
Sub domain 4(IIB)
Sub domain 2(IB)
Eukaryotic ACTINMreB (T. maritima)
(IA)Sub domain 1(IIA)
Sub domain 3
[van den Ent F, Amos LA, Lowe J. Nature 2001, 413:39-44]
Actin-like MreB proteins and the peptidoglycan celll wall are major determinants of shape
in non-spherical bacteria
MreBs( ti t k l t )
Peptidoglycan ll ll(actin cytoskeleton) cell wall
What is their relationship?
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
11
Fluorescent labelling of new cell wall insertion
GFP-MreB
Sidewall elongation Midcell division
( Daniel & Errington. Cell 2003, 113:767-776 )
MreB (actin)-dependentHelical insertion
Sidewall elongation Midcell division
2001-2011, a decade of ‘helical’ research:
Model for the organization of cell wall synthesis by actin-like MreB proteins
AFM
MEMBRANE
CELL WALL
Adapted from Carballido-López et al. (2006) Dev. Cell 4:19-28
MEMBRANE
CYTOPLASM
MreB
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
12
A fresh look to the spatial and temporal organisation of the MreB cytoskeleton by TIRF
Total Internal ReflectionFluorescence Microscopy
EpifluorescenceMicroscopy
TIRF BF EPI
MreBs form diffraction-limited motile patches in exponentially growing cells
circumferentially bidirectionally processively
MreBs move:
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
13
3 MAJOR NEW INSIGHTS:
1. MreBs form dynamic patches and not extended helices during exponential growthduring exponential growth
2. MreB-associated elongation complexes display processive, circumferential movement
3. Motion of MreB patches is Xnot driven by treadmillingbut by PG synthesis itself
X
Inhibition of PG synthesis stops MreB patches motility
reversible inhibition of MreB movement
wash out
+ vancomycin (100 µg/ml)
Vancomycin
Ph h f i
CELL WALL
wash outPhosphosfomycin
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
14
Wh t th i th bi l i l f tiWhat, then, is the biological function
of the essential actin-like MreB?
In the absence of MreB, cell wall synthesis complexes run amok
Patches move much faster & exhibit less uniform directionality
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
15
In the absence of MreB, cell wall synthesis complexes run amok
without MreB
with MreB
PBPs & autolysins
MreB
YkuR & PG-precursor enzymesPG precursor enzymes
Model for the coordination of extra- and intra-cellular PG-synthesizing machineries by MreB
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
16
How does circumferential motion of
cell wall elongation machineries
correlate with cell wall growth?
B. subtilis cell length (but not cell width) is regulated in response to nutrient availability
5 S
(73 min)
Medium(doubling time)
2
3
4
Cel
l siz
e (
m)
cell length
cell width
(73 min)
TS (58 min)
CH(40 min)
0.5 1 1.5 2 2.50
1
Growth rate (1/hr)
C
Sharpe M E et al. (1998) J. Bacteriol.
CHG (30 min)
TSTSSS CHCH CHGCHG
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
17
E. coli cell length and cell width are regulated in response to nutrient availability
S(73 min)
Medium(doubling time)
5
6
B. subtilisCell length
(µm)(73 min)
TS (58 min)
CH(40 min)
1 1
1,2
B subtilis
1
2
3
4
5
0 1 2 3
B. subtilis length
E. coli length
(µm)
Cell width( )
Growth rate (1/h)
Sharpe M E et al. (1998) J. Bacteriol.
CHG (30 min)
0,7
0,8
0,9
1
1,1
0 1 2 3
B. subtilis width
E. coli width
(µm)
Growth rate (1/h)
In response to nutrient availability,
do rod-shaped cells regulate:
1. rotation speed of MreB patches ?
2. density of patches per unit surface area?
3. size of the patches/polymers?
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
18
E. coli B. subtilis
Native GFP fusions as only copy of the corresponding MreB protein in the genome
Richmedium(LB)
wt MreB-RFPSW wt GFP-MreB Mbl-GFP
Poormedium(S)
P-M
reB
Rich medium(LB) Poor medium(S)
FP
nativ
eGF
Pna
tiveM
bl-G
F
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
19
Ri h di (LB) P di (S)
Diffraction-limited patches are the physiological form of MreB in both conditions
Rich medium(LB) Poor medium(S)
ativ
eG
FP
-Mre
Bn
a
MreBs patch speed is proportional to growth rate in B. subtilis, but not in E. coli
LBLB
CHCH
S S
M9SE M9SE
B. subtilis
E. coli
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
20
MreBs patch density is constant at different growth rates in B. subtilis, but not in E. coli
E. coli B. subtilis
TAKE-HOME MESSAGE:
B. subtilis regulates patch rotation speed, not patch density, in response to growth rate
E. coli regulates patch density, not rotation speed, in response to growth rate
Rut CARBALLIDO LOPEZ College de France, 27 janvier 2016
21
Distinct regulatory mechanisms reflect different cell wall integrity constraints in Gram+ and Gram-
B. subtilis E. coli
Gram -- Gram ++
m
MreB MreBHMreBMbl
2-7
nm 25-3
0 n m
Peptidoglycan(PG)
The actin-like MreB cytoskeleton and the cell wall are major determinants of shape in rod-shaped bacteria
MreB proteins form motile patches in growing cells
SUMMARY
MreB proteins form motile patches in growing cells
MreB assemblies organize and restrict the motility of cell wall synthesizing machineries in the membrane
Movement of MreB-associated cell wall elongation machineries and cell wall expansion are correlated
The MreB cytoskeleton and cell wall synthesis are prominent targets for new antibiotics!