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cmi12163-sup-0001-figs1.pdf204K

Fig. S1. Effect of inhibitors on N. meningitidis adhesion and FaDu cell viability.

A. FaDu cells treated with the cholesterol-sequestering agent nystatin or inhibitors of actin polymerization (cytochalasin D), Rac1, ROCK, Cdc42, PKC or PI3K (wortmannin and LY294002) were infected with FAM20 at an moi of 5. After 1 h, unbound bacteria were removed by washing and infection continued for a further 2 h. Bacterial adhesion was determined by plating of viable counts after saponin lysis of host cells. The relative adhesion to inhibitor-treated cells compared with adhesion to untreated control cells is shown.

B. FaDu cell viability after treatment with chemical inhibitors was analysed by using an MTT assay. Formaldehyde-fixed samples (PFA) were used as the blank and 0.5% Triton X-100 was used as the positive control. Cell viability was expressed as the relative absorbance of inhibitor-treated cells compared with untreated controls. Bars represent means + standard errors of ≥ two independent experiments.

cmi12163-sup-0002-figs2.pdf357K

Fig. S2. RhoA and Cdc42 signalling is required for adhesion-mediated LL-37 resistance.

A. LL-37 killing of N. meningitidis strain FAM20 (moi = 5) adhered to untreated FaDu cells or cells transfected with a non-targeting siRNA (siNT) or siRNAs targeting RhoA or Cdc42. Survival of adhered bacteria was determined by plating of viable counts after saponin lysis of host cells. The relative survival compared with untreated control cells is shown.

B and C. mRNA expression of RhoA (B) and Cdc42 (C) in transfected FaDu cells was analysed by qPCR using GAPDH as an internal standard. For each target, two separate siRNAs and a siRNA pool were compared.

Bars represent the means + standard errors of a single experiment performed with triplicate samples.

cmi12163-sup-0003-figs3.pdf438K

Fig. S3. Effect of host cells on LL-37 resistance of Streptococcus pyogenes, Neisseria lactamica and capsule- or adhesion-deficient N. meningitidis.

A–C. FaDu cells were infected with S. pyogenes (A), N. lactamica (B) or N. meningitidis strain FAM20 Cap (C) and non-adhered or adhered bacteria treated with 0 (control) or 10 μM LL-37 for 2 h. Results are presented as mean relative survival of LL-37-treated bacteria compared with controls treated with 0 μM LL-37 + standard errors of three independent experiments.

D. Adhesion of FAM20 wild-type (wt) and isogenic mutants lacking PilC1 or PilC2 to FaDu cells. Bacteria were added at an moi of 5 and incubated for 2 h. The relative adhesion compared with FAM20 wt + standard errors of ≥ two independent experiments is shown.

E. LL-37 killing of N. meningitidis FAM20 wild-type (wt), FAM20 PilC1 and FAM20 PilC2 mutants in the presence of FaDu cells. Bacteria (moi = 0.05, reflecting the number of adhered bacteria in previous assays) were pre-incubated with the host cells for 1 h before addition of LL-37. A washing step was excluded due to the low adhesion of the PilC1 mutant. Alternatively, bacteria (moi = 0.05) and LL-37 were added to host cells simultaneously. After 2 h of incubation with LL-37 (0 or 10 μM), survival was determined by plating of viable counts.

F. LL-37 killing of FAM20 wt in the presence of untreated FaDu cells or cells treated with inhibitors of ROCK or Cdc42. LL-37 was added at the time of infection (simultaneous) or after a 1 h pre-incubation as described above. Bars represent the mean relative survival of LL-37-treated bacteria to controls + standard error of ≥ three independent experiments (wt and PilC1), or two independent experiments (PilC2).

cmi12163-sup-0004-figs4.pdf6753K

Fig. S4. Visualization of the infection set-up.

A. Live microscopy of FaDu cells infected with FAM20. Bacteria were pre-stained with DyeLight 594 NHS ester (red). Adhered: FaDu cells were infected at an moi of 5. After 1 h, unbound bacteria were removed by washing and infection continued for a further 2 h. Non-adhered: bacteria were added to FaDu cells at a concentration corresponding to the number of adhered bacteria (moi = 0.05) and incubated for 2 h. Bright-field and fluorescence images were taken at the end of the infection period. Scale bars indicate 20 μm.

B. Live microscopy of FAM20 at low (3 × 104), middle (3 × 105) or high (3 × 106) cfu ml−1 incubated with 0 (ctrl) or 10 μM LL-37 for 2 h. Low cfu ml−1 represent concentrations used for infecting cells with bacteria in non-adhered settings. Scale bars indicate 10 μm.

cmi12163-sup-0005-figs5.pdf390K

Fig. S5. Adhesion to host cells protects from LL-37 at higher multiplicities of infection (moi) and varying LL-37 concentrations.

A and B. Adhered: bacteria were allowed to attach for 1 h and unbound bacteria removed by washing before LL-37 treatment. Non-adhered: bacteria were added to FaDu cells and treated with LL-37 at the time of infection. Bacterial survival was determined by plating.

A. LL-37 killing of bacteria at 10× higher moi. Adhered (moi = 50) and non-adhered (moi = 0.5) FAM20 were incubated with 10 μM LL-37 in the presence of host cells.

B. Adhered (moi = 5) and non-adhered (moi = 0.05) FAM20 were incubated with LL-37 (0–15 μM) in the presence of host cells in serum-free DMEM or DMEM with 10% FBS.

C. FaDu cells were infected with FAM20 at 1× moi (0.05) or a 10× higher moi (0.5) and treated with LL-37 at the time of infection for 2 h. Results are presented as mean relative survival of LL-37-treated bacteria compared with controls + standard errors of two or more independent experiments.

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