Biology Reference
In-Depth Information
followed by peroxidase-conjugated secondary antibody (1:3,000), each for 1 hr at
37°C. Antigen-antibody binding was determined by a colorimetric reaction using
the 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate, and absorbance at 655
nm.
Porphyromonas gingivalis
antibody binding to the fusobacterial biofilm alone
was subtracted as background. (iii) Confocal microscopy assay. (A) Single species.
Porphyromonas gingivalis
was stained with 4',6-diamidino-2-phenylindole (50 μg ml
-
1
) and 2 × 10
6
cells in rPBS incubated anaerobically at 37°C for 16 hr with rocking in
individual chambers of the CultureWell coverglass system (Grace Bio Labs). Cham-
bers were washed three times in rPBS. (B) Dual-species. Heterotypic
P. gingivalisS.
gordonii
communities were generated as described previously [15].
Streptococcus
gordonii
cells were labeled with hexidium iodide (15 μg ml
-1
), then cultured anaero-
bically at 37°C for 16 hr with rocking in CultureWell chambers.
Porphyromonas
gingivalis
was stained with 5-(and-6)-carboxyfluorescein, succinimidyl ester (10 μg
ml
-1
), and 2 × 10
6
cells in rPBS were reacted with the surface attached
S. gordonii
for 24 hr anaerobically at 37°C with rocking. (C) Three species. Surface attached
hexidium iodide-stained
S. gordonii
were generated as above.
Fluorescein
stained
F.
nucleatum
(2 × 10
6
cells in rPBS) reacted with
S. gordonii
for 24 hr anaerobically
at 37°C with rocking. The coverglass was then washed with rPBS to remove non-
attached bacteria.
Porphyromonas gingivalis
was stained with 4',6-diamidino-2-phe-
nylindole (50 μg ml
-1
) and 2 × 10
6
cells in rPBS were added and further incubated for
24 hr anaerobically at 37°C with rocking. Communities were observed on a Bio-Rad
Radiance 2,100 confocal laser scanning microscope (Blue Diode/Ar/HeNe) system
with a Nicon ECLIPSE TE300 inverted light microscope and 40 × objective using re-
flected laser light of combined 405, 488, and 543 nm wavelengths where appropriate.
A series of fluorescent optical x-y sections were collected to create digitally recon-
structed images (z-projection of x-y sections) of the communities with Image J V1.34s
(National Institutes of Health) or Laser Sharp software (Bio-Rad). Z stacks of the x-y
sections of CLSM were converted to composite images with “Iso Surface” functions
of the “Surpass” option on Imaris 5.0.1 (Bitplane AG; Zurich, Switzerland) software.
Iso Surface images of
P. gingivalis
were created at threshold of 20 and smoothed with
Gaussian Filter function at 0.5 width, and
P. gingivalis
biovolume was calculated.
Biofi lm assays were repeated independently three times with each strain in trip-
licate. Crystal violet results were compared by t-tests. Biovolume calculations were
compared with a t-test using the SPSS statistics software.
CONCLUSION
Complex multi-species biofilms such as pathogenic dental plaque accumulate through
a series of developmental steps involving attachment, recruitment, maturation, and
detachment. Choreographed patterns of gene and protein expression characterize
each of these steps. In this study we developed a model of the early stages of plaque
development whereby three compatible species accreted into simple communities.
Porphyromonas gingivalis
increased in biomass due to attachment and recruitment, and
this allowed us to catalog differential protein expression in
P. gingivalis
consequent
to contact dependent interbacterial signaling and communication through short range
