Transcript Poster Thomas Sutherland DMMI - Workspace

A Model System to Investigate Translocation of Neisseria meningitidis
Thomas C. Sutherland & Prof. C. Tang
Centre for Molecular Microbiology and Infection, Flowers Building, Imperial College London
Meningococcal
septicaemia
Translocation
across bloodbrain barrier
800
600
400
Meningitis
800
600
400
200
200
0
72
0
72
• Current studies limited to expensive, technical tissue explants and culture of cells on impermeable substrates.
• The human bronchial epithelial cell line ‘Calu-3’ can be grown into a differentiated epithelium, and has been
much used in aerosol medicine studies3, however, it has never been used to study N. meningitidis.
Aim
• Establish a physiologically relevant cell culture model of the upper respiratory epithelium and use it to
investigate translocation of N. meningitidis.
Method
1000
Apical Chamber
Cell culture insert
Porous membrane (1μm pore size)
Calu-3 cells
Basal Chamber
96
120
98
5.00E+07
4.50E+07
4.00E+07
3.50E+07
3.00E+07
2.50E+07
2.00E+07
1.50E+07
1.00E+07
5.00E+06
0.00E+00
Avg Bacteria per cell
(Normalised to input)
Avg Bacteria
per cell
10
5
AP
0
Chang
Calu-3
Cell Type
TJ
CCM
D
Monolayer of Calu-3 cells on the cell culture membrane
(CCM). Apical (AP)/basal (BA) differentiation, the
formation of tight junctions (TJ), desmosomes (D) and
microvilli (MV) are clearly all visible.
2
4
6
0
8
2
4
6
8
Time (hrs)
siaD::ery/wt
2.0
2.0
1.5
1.5
1.0
1.0
0.5
C
The Junctional
complex (JC)
consisting of tight
junctions, the
adhesion belt and
desmosomes.
D
• Calu-3 cells have not previously been infected with N. meningitidis. Test
infections demonstrate that they undergo a similar pattern of infection to the
more studied ‘Chang’ cell line (A).
DP
TJ
E
Occludin
C
0.0
Desmosome: showing
desmoplakins
(DP),
desmogleins (DG) and
intermediate
filament
(IF) accumulation.
24-25h
• Competitive infections, comparing wt N. meningitidis with a capsule mutant
(ΔsiaD) (A) and a pilus mutant (ΔpilE) (B). Both mutants show a defect for
traversal of the monolayer compared to wt, but the capsule mutant’s defect is
greater (average CI after 24hrs of 0.01 compared to for the 0.37 pilus mutant).
Conclusions
• A protocol to grow Calu-3 cells on cell culture inserts to form a polarised, differentiated, epithelial
monolayers has been optimised.
• The epithelial monolayers are impermeable to non-invasive bacteria and generate trans-epithelial electrical
resistance (TEER) showing that they are confluent and not ‘leaky’. Invasive N. meningitidis is able to
penetrate the monolayers.
• The pilus mutant shows a competitive disadvantage for translocation compared to wt, a result consistent
with a previous study performed in polarised intestinal epithelial cells4.
• Future work will involve use of the model to understand the host and pathogen roles in the translocation
process.
DG
Detail of Tight Junctions:
TEM shows tight association of
cell membranes, while IF staining
shows presence of tight junction
proteins Occludin and ZO-1.
24-25h
• We show for the first time that the capsule mutant has massive translocation defect compared to wt.
Previous work has shown that the capsule is essential for intracellular survival5. These data suggest N.
meningitidis translocates across the epithelium via an intracellular, rather than paracellular, route.
IF
IF
ZO-1
7-8h
• N. meningitidis can infect Calu-3 cells in a manner consistent with other cell lines used in the field.
Strain
• Type IV Pilus dependant adhesion, previously documented in other cell
lines2 is also observed in the Calu-3 cells (B & C).
1.E+00
7-8h
MV
TJ
MC58
1.E+01
AB
15
ΔpilE::kan
1.E+02
0.5
TJ
BA
0
No Cells
Cells
D
JC
5
1.E+03
Figure 5 – Translocation mutants
A
B
pilE::kan/wt
Figure 3 – Morphology of the cultured cell layers
B
A
Intracellular
10
1.E+04
• The N. meningitidis strain MC58, on the other hand is able to penetrate the
monolayer (B).
• Coating cell culture inserts with extracellular matrix proteins affects the
development of TEER. A 1:1 mixture of laminin and fibronectin is most effective
(A). Optimisation culture procedure allows reliable generation of TEER (B).
Figure 1 – N. meningitidis infection of Calu-3 cells
A 25
B 25
Adherent
Adherent
15
1.E+05
• The monolayer is impermeable to non-invasive bacteria (E. coli DH5α) (A).
0.0
20
1.E+07
1.E+06
Time (Hrs)
• Trans-epithelial electrical resistance (TEER) develops as tight junctions form
and is a good indicator of monolayer polarisation. Unpolarised cells generate
TEER readings of 0-100Ω.
• Cells are grown on inserts in cell culture plates. This creates basal and apical chambers separated by the
differentiated epithelium. Translocation can be analysed by infecting the apical chamber and monitoring the
passage of bacteria to the basal chamber.
20
1.E+08
No cells
Cells
Time (hrs)
Time after seeding (hrs)
Well of cell culture plate
Intracellular
1.E+09
0
122
CI
Colonisation of
the nasopharynx
1000
Laminin
Fibronectin
Fibronectin+Laminin
cfu/ml in basal chamber
TEER(Ω)
• Understanding translocation is vital to understanding the disease process.
Translocation
across
Nasopharyngeal
Epithelium
1200
CI
• Adherence and colonisation is type IV pilus dependant2 but pathway for translocation across the
nasopharyngeal epithelium is unknown.
cfu/ml in basal chamber
• Gram-negative diplococci, harmlessly colonises the nasopharynx of up to 40% of the population1.
Figure 4 – Permeability of the monolayer to bacteria
A
B
DH5α Infection
MC58 Infection
Figure 2 – Optimising the cell culture conditions
No Coating
A 1400
B 1200
TEER (Ω)
Pathogenesis of Neisseria meningitidis
Adhesion Belt:
(AB) Intracellular
and intercellular
attachment
proteins visible.
• TEM (cross sections, 120 hrs post seeding) and Confocal microscopy
(monolayers viewed from above, 120 hrs post seeding) demonstrate that the
cultured cells reliably form confluent, polarised monolayers with all the
morphological features of a polarised epithelium.
References
1.
K. A. Cartwright et al., Epidemiol. Infect. 99: 591 (1987).
2.
X. Nassif, C. Pujol, P. Morand, E. Eugene, Mol. Microbiol. 32: 1124 (1999).
3.
K. Foster, M. Avery, M. Yazdanian, K. Audus, Int. J. Pharm. 208: 1 (2000).
4.
C. Pujol, E. Eugene, L. de Saint Martin & X. Nassif, Infect. Immun. 65: 4836 (1997).
5.
M. Spinosa, C. Progida, A. Tala, L. Cogli, P. Alifano, C. Bucci, Infect. Immun. 75:3594 (2007).
Acknowledgements
Special thanks must go to Mike Hollinshead from the Henry Wellcome imaging suite on the
St Mary’s Campus for all his help with the Transmission Electron Microscopy.