A. C. difficile R20291 and F.
nucleatum subspecies nucleatum were incubated
together anaerobically in co-aggregation buffer. Aggregation was calculated
using the equation: 100%- ((OD600nm at 0 hr
–OD600nn at 1 hr) x 100%). For inhibition assays,
F. nucleatum subspecies nucleatum was
pre-incubated for 5 min with 50 mM L-arginine, D-galactose, or D-glucose
anaerobically, followed by addition of C. difficile (n=3-4
replicates, repeated 4 independent times). Significance was determined by
Multi-way ANOVA. B. Aggregation was examined with 11 C.
difficile strains, wild-type F. nucleatum
subspecies nucleatum, as well as F. nucleatum
subspecies nucleatum Δfap2 and
F. nucleatum subspecies nucleatum
ΔradD. C. For visualization, cuvettes
were examined for aggregation in C. difficile alone, C.
difficile with wild-type F. nucleatum subspecies
nucleatum, Δfap2 and
ΔradD. D. Microscopy of CFDA-SE tagged
C. difficile R20291 (green) and F.
nucleatum subspecies nucleatum (wild-type (WT),
Δfap2 or ΔradD) (red) (scale
bar =50 μm) (n=3). Co-aggregation was also examined with C.
difficile strains and (E) F.
nucleatum subspecies polymorphum and
(F) F. nucleatum subspecies
animalis (n=4 replicates, repeated 3 independent times).
One Way ANOVA, * p <0.05.