Discussion

N. meningitis normally colonizes the human nasopharynx and only causes bacteraemia and meningitis when the bacterium crosses the epithelium to reach the bloodstream, and the blood-brain barrier. Our aim was to, for the first time, use transposon mutagenesis to study gene fitness during traversal of the organism across the epithelium. To do this, we developed an in vitro epithelial model system that as closely as possible replicated the in vivo situation. This was achieved by growing polarized respiratory epithelial cells to form an intact barrier with functional tight junctions on a semipermeable insert, which allows sampling of cells capable of basolateral escape. Our preliminary work using 16HBE14o- cells under LCC or AIC conditions showed similar results to those previously reported (Forbes and Ehrhardt, 2005), namely that cells grown in AIC took longer to reach the same TEER levels as cells grown in LCC. This was up to 4 days longer in our study (data not shown). Ehrhardt et al. (2002) also reported that the presence of apical fluid is pertinent to formation of a functional 16HBE14o- cell layer with higher TEER and well-defined

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tight, adherens, and gap junctions. Thus, all further work was done with 16HBE14o- cells grown in liquid-liquid condition.

As previously mentioned, cell growth is also affected by the substrate they are grown on. The inserts are commonly coated with Vitrogen or bovine collagen prior to seeding, but various groups have reported that the cell barrier is adequately formed even in the absence of any filter coating (Ehrhardt et al., 2003, Forbes and Ehrhardt, 2005). When bovine collagen was used to coat the Transwell® _{inserts in our study,} no significant difference was seen in terms of the level of epithelial resistance obtained in the presence or absence of the coating. Nevertheless, all future work was carried out using collagen coated inserts as the epithelial resistance was marginally higher in this situation and more importantly, it is thought to prevent the possibility that cells may grow down into the pores located on the surface of the insert (Forbes et al. (2003)).

Next, we focused on establishing an intact barrier of differentiated, polarised 16HBE14o- cells. We defined an intact cell layer as one having an elevated TEER, low permeability to small molecules and the expected localization of apical tight junction proteins, occluding and ZO-1 markers. We monitored the growth of the 16HBE14o- cells through time, for up to 18 days, measuring epithelial resistance and the permeability of the formed layer to FITC labelled 70k Da dextran. No difference in paracellular diffusion was observed after day 6 concurrent with an increase in TEER to 475±16 Ω. This demonstrated that an intact barrier had been formed as early as day 6 even though a steady increase in TEER was seen up to the 12th _day post culture. By immunostaining for tight junction proteins, ZO-1 and occludin, we

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further confirmed that the difference in TEER and permeability was due to cell differentiation and polarization. Increase in TEER is indicative of higher differentiation and polarization of cells. Nevertheless, even an incompletely polarized cell layer can form a functional barrier with intact tight junctions and adherens junctions (Bucior et al., 2014).

Tight junctions are the most apical intercellular junction in epithelial cells as well as endothelial cells, and are central for the development and function of the barrier (Balda and Matter, 1998). ZO-1, claudin, and occludin are tight junction markers usually used to determine whether cells were differentiated and polarised (Bazzoni and Dejana, 2004, McClean and Callaghan, 2009). According to our results, the day 6 cell layers, with a TEER of ~500Ω were adequate for the traversal assay as they formed an intact barrier made of differentiated polarized 16HBE14o- cells as shown by the apical localization of ZO-1 and occludin and the characteristic ‘chicken wire’ pattern of staining connecting neighbouring cells. More importantly, only minimal cell stacking was observed at day 6 compared to the other tested time points. Therefore, all future experiments were conducted using cells grown on 24 mm Transwell® inserts for 6 days to a TEER >500 Ω.

Differentiated, polarized 16HBE14o- cells were then challenged with N. meningitidis L91543, the strain which was used to build the Tn5 based library, at a MOI of 200. At this MOI, meningococci migrated across the epithelial layer without breaching the barrier as shown by the TEER and 70kDa dextran permeability data (when compared to the control (non-infected wells). This is consistent with findings by Merz et al. (1996) and Sutherland et al. (2010), both of whom reported that the

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meningococci cross the epithelial layer by transcellular passage without disrupting the cell to cell contact. High levels of TEER were also maintained for other bacterial and viral transcytosis events that did not disrupt the cell barrier such as for N. gonorrhoeae (Wang et al., 1998) and adenovirus (Tang et al., 2007). In contrast, when bacteria or viruses traverse the epithelial cells by the paracellular route, this leads to tight junction protein disruption and/or cell disruption, and a drop in TEER (Chen et al., 2006, Fiorentino et al., 2014). Our findings also attest that this model is sufficiently sturdy to withstand infections with high MOI, i.e. MOI of 200, which is critical when working with mutant libraries to minimize selection bias.

As shown in Figure 4.5A, the bacteria crossed the barrier as early as 2 h incubation and no significant difference was seen between the time points even after 12 h of incubation, making it difficult to determine the best incubation time for the infection. Even though the number of meningococci crossing the epithelial barrier was a ~4 log increase at 24 h, this time point was not used as this may be more reflective of intracellular survival compared to traversal. Furthermore we may be looking at a different population of mutants compared to the adhesion and invasion assays at 6 h (as optimized in Chapter 3). Hey et al. (2013) demonstrated the transcriptome of adherent meningococci obtained at 24h was more similar to those at 96h, compared to an earlier time point (4 h) during co-cultivation of N. meningitidis MC58 strain with confluent monolayers of 16HBE14o- cells. Thus, 8 h incubation, as used by Pujol et al. (1997) and Sutherland et al. (2010), as well as the 12 h incubation which provided higher cumulative bacterial counts, were both used to verify the model with the L91543 strains and the phoP mutant.

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Nearly half (44%) the wells contained no traversed bacteria, despite efforts to increase the sensitivity of detection, indicating that the absence of meningococci was real, and not due to sampling error. The possibility that this was due to the formation of a multi-layered cell barrier was ruled out by testing the correlation between the TEER and CFU counts, since Lu et al. (1996) demonstrated by electron microscopy that higher TEER values were indicative of multiple layered cell barriers of Caco-2 cells. However, no correlation was observed between TEER and CFU in our experiments. Moreover, Pujol et al. (1997) who worked with T84 mono layered barrier showed 70% and 15% of inserts had zero bacterial transmigration at 8 and 23 h of incubation respectively. Thus it does not seem likely that multi-layered barrier forming in the tested inserts is responsible for zero bacterial traversal and the phenomenon remains unexplained.

The optimized traversal model using 16HBE14o- cells was successfully verified with the L91543 strain and the phoP mutants. Using this model, translocation of the phoP mutant was reduced (<1.5%) when compared to the wild type strain, even at a high infectious dose. These results are consistent with published work (Johnson et al., 2001), in which <2.5% of the phoP cells crossed an epithelial cell barrier composed of Chang human conjunctiva epithelial cells compared to 42% of the wild type meningococci. The magnitude change in translocation between L91543 and the phoP mutant demonstrates that this system can successfully differentiate between strains differing in transversal capability, and is thereby suitable for the transposon library screen. Since no significant difference was seen in the bacterial counts between the two tested time points, we opted for the 12 h incubation as this was marginally better (~22% difference in the relative percentage of traversed phoP

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mutants compared to 8 h) and was also more suitable to the restricted working hours of the meningococcal laboratory.

Thus, based on the outcomes of the optimisation experiments, the following methodology will be used during the transposon mutagenesis experiments:

1. 16HBE14o- cells are seeded at a density 5x105 _{cells onto 24 mm diameter,} polyester, 3 µm pore sized Transwell® _{inserts coated with bovine collagen.} 2. After 6 days the polarized 16HBE14o- cells are confirmed to have a TEER

more than 500 Ω and can be used for the traversal assays.

3. The cells are challenged in the upper chamber with a 4 h culture grown in CBA at a MOI of 200

4. The wells are incubated for 12 h, after which they are transferred to new plates with fresh medium in the basal chamber.

5. The bacterial counts are set up from both upper and lower chambers after 2 h incubation.

6. The TEER is monitored at all the tested time points and the permeability to 70 kDA dextran is checked at the end of the experiment.

Even with all this optimization, the data to be obtained from the Tn5 derived mutant library has to be interpreted with care as this would be a snapshot of the traversal at only hours 12-14. This population might differ from the bacteria crossing the epithelial barrier at other time points.

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CHAPTER 5:

GENOME WIDE IDENTIFICATION OF GENES

INVOLVED IN ADHESION, INTERNALIZATION,

AND TRAVERSAL OF NEISSERIA MENINGITIDIS

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5 GENOME WIDE IDENTIFICATION OF GENES INVOLVED IN ADHESION,