Fusobacterium nucleatum secretes amyloid-like FadA to enhance pathogenicity (2021)
Figure 4

Figure 4. Functional analysis of amyloid-like FadA in vitro.

A. Facilitation of Fn biofilm formation by amyloid-like FadA. An aliquot of 10 μg each of the detergent-resistant pellets prepared from of Fn 12230 (wild type), US1 (∆fadA) and lam, or PBS, was added to the 96-well plates and incubated at 370C for 5 hours. Fresh culture of Fn 12230 was diluted to OD600 of 0.2 and an aliquot of 200 μl was added to the wells, followed by incubation at 37 0C for 48 or 72 hours under anaerobic conditions. Following washes, the biofilms were incubated with 100 μl 0.1% crystal violet for 15 min. After washing, 100 μl 95% ethanol was added to each well and the optical density at OD550 was measured in a microplate reader. The results shown are the average of four experiments each performed in duplicate. The error bars indicate SD. A representative image following 72 hours of incubation is shown in the top panel. ** p<0.01. ***p<0.001 (t-test).

B. Inhibition of biofilm formation mediated by detergent-resistant Fn pellets by Congo Red. An aliquot of 10 μg detergent-resistant pellets prepared from Fn 12230 (pellet) was added in 96-well plates in the presence of 0, 10 or 50 µg/ml Congo Red (CR), followed by addition of Fn 12230 and incubated as described above. The results shown are the average of four experiments each performed in duplicate. The error bars indicate SD. A representative image following 72 hours of incubation is shown in the top panel. ** p<0.01, ***p<0.001 (t-test).

C. Effects of pH on Fn survival. Wild type Fn 12230 (Fn) or its fadA deletion mutant US1 (∆fadA) grown to log or stationary phase were washed and incubated in PBS at pH7 or pH4 for 1 or 2 hours. The live bacterial counts at time 0 were designated as 100%, and those after 1 or 2 hrs of incubation were expressed relative to time 0. The results shown are the average of 5 (for log) or 4 (for stationary) independent experiments, each performed in triplicate. The error bars indicate SD. *p<0.05, ** p<0.01, ***p<0.001 (One-way ANOVA).

D. Effects of Congo Red on Fn survival in acidic environment. Wild type Fn 12230 (Fn) or its fadA deletion mutant US1 (∆fadA) grown to stationary phase were washed and incubated in PBS at pH4 in the presence of absence of 50 μg/ml Congo Red (CR) for 1 or 2 hours. The live bacterial counts at time 0 were designated as 100%, and those after 1 or 2 hrs of incubation were expressed relative to time 0. The results shown are the average of 5 independent experiments, each performed in duplicate. The error bars indicate SD. *p<0.05, ** p<0.01, ***p<0.001 (One-way ANOVA).

E. Attachment of Fn 12230 grown to log or stationary phase to CRC cells DLD1. Fn 12230 (Fn) was either unwashed or washed twice with PBS or 1 mg/ml Congo Red (CR), or mixed with 20 μl mAb 7H7, before adding to the monolayers at multiplicity of infection (MOI) of 50:1. US1 (∆fadA) was included as a negative control. The attachment level by untreated log phase Fn was designated as 100%, which all other values were expressed relative to. Data shown are mean values ± SEM. The experiment was performed in duplicate and repeated four times. *p < 0.05, **p < 0.01, ***p < 0.001 (t-test).

F. Inhibition of Fn attachment by FadAc in the presence or absence of anti-amyloid antibody OC. An aliquot of 50 μg purified recombinant FadAc was pre-incubated with DLD1 for 45 min either alone, or mixed with 20 μl OC or rabbit IgG control, before Fn was added and the attachment assay performed as above. The attachment value by Fn alone was designated as 100%, with all other values expressed relative to it. FadA mutant L14A was included as a negative control. Data shown are mean values ± SEM. The experiment was performed in duplicate and repeated three times. **p< 0.01 (One-way ANOVA).