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 Figure 1 – N. meningitidis infection of Calu-3 cells 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). Type IV Pilus dependant adhesion, previously documented in other cell lines 2 is also observed in the Calu-3 cells (B & C). AB C Figure 2 – Optimising the cell culture conditions AB 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. 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 3 – Morphology of the cultured cell layers 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. 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. CCM BA AP MV TJ D D AB The Junctional complex (JC) consisting of tight junctions, the adhesion belt and desmosomes. JC A B TJ ZO-1Occludin Detail of Tight Junctions: TEM shows tight association of cell membranes, while IF staining shows presence of tight junction proteins Occludin and ZO-1. C Desmosome: showing desmoplakins (DP), desmogleins (DG) and intermediate filament (IF) accumulation. DP DG IF D Adhesion Belt: (AB) Intracellular and intercellular attachment proteins visible. E Figure 4 – Permeability of the monolayer to bacteria The monolayer is impermeable to non-invasive bacteria (E. coli DH5α) (A). The N. meningitidis strain MC58, on the other hand is able to penetrate the monolayer (B). BA DH5α InfectionMC58 Infection Figure 5 – Translocation mutants BA 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). Pathogenesis of Neisseria meningitidis Colonisation of the nasopharynx Translocation across Nasopharyngeal Epithelium Meningococcal septicaemia Translocation across blood- brain barrier Meningitis Gram-negative diplococci, harmlessly colonises the nasopharynx of up to 40% of the population 1. Adherence and colonisation is type IV pilus dependant 2 but pathway for translocation across the nasopharyngeal epithelium is unknown. Understanding translocation is vital to understanding the disease process. 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 studies 3, 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 Apical Chamber Basal Chamber Calu-3 cellsPorous membrane (1μm pore size) Well of cell culture plate Cell culture insert 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. Conclusions N. meningitidis can infect Calu-3 cells in a manner consistent with other cell lines used in the field. 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 cells 4. 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 survival 5. These data suggest N. meningitidis translocates across the epithelium via an intracellular, rather than paracellular, route. Future work will involve use of the model to understand the host and pathogen roles in the translocation process. 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. (Ω)(Ω) (Ω)(Ω)