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


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


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


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


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


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-


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


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 ]


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


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?


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


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:


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


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


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


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


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?


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


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


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


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!

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