Table 1.
Bacterial strains used in this study.
Table 2.
qPCR primers used in this study.
Table 3.
Selected genes from the microarray dataset and qRT-PCR confirmation*.
Figure 1.
Effect of synthetic sialic acid upon nan operon transcription.
F. nucleatum ssp. nucleatum was incubated in the presence of synthetic sialic acid at concentrations ranging from 0.02 mM to 2 mM before extracting RNA and measuring nan operon expression. Data are expressed relative to the cDNA abundance measured in the sample receiving no exogenous sialic acid. This sample was arbitrarily assigned a value of 1. All data were normalized using the DNA gyrase gene gyrA as a housekeeping control. Presented data are the average results from 3 independent experiments. Statistical significance was evaluated using an unpaired Student's t-test. *p<0.05, **p<0.01, ***p<0.001.
Figure 2.
F. nucleatum ssp. nucleatum actively sialylates its outer surface.
A) F. nucleatum ssp. nucleatum was grown in SDM before fluorescently labeling surface sialic acids. B) F. nucleatum ssp. nucleatum was grown in the presence of SDM and pretreated with neuraminidase before fluorescently labeling surface sialic acids. All pictures in the left panels are phase contrast images, whereas the right panel images are the corresponding images captured using epifluorescence microscopy. Total magnification is 1000x.
Table 4.
Representative neu genes of sequenced oral fusobacteria.
Figure 3.
Surface sialylation is conserved among oral fusobacteria.
Fusobacterial clinical isolates were grown in SDM to mid-log phase before fluorescently labeling surface sialic acids. Each set of images is shown with the phase contrast image on the left and the corresponding epifluorescence image on the right. The pictured species are: A) F. nucleatum ssp. animalis, B) F. nucleatum ssp. animalis pretreated with neuraminidase, C) F. nucleatum ssp. vincentii, D) F. nucleatum ssp. vincentii pretreated with neuraminidase, E) F. nucleatum ssp. polymorphum, F) F. nucleatum ssp. polymorphum pretreated with neuraminidase, G) F. periodonticum, and H) F. periodonticum pretreated with neuraminidase. Images were captured at 1000x total magnification.
Figure 4.
Comparison of surface sialylation abilities among oral fusobacteria.
Representatives of all of the F. nucleatum subspecies as well as F. periodonticum were grown to mid-log phase in SDM containing vegetable peptone before fluorescently labeling surface sialic acid. Average fluorescence intensity values were determined using ImageJ image analysis software. Data are expressed relative to the average fluorescence intensity values for erythrocytes labeled in parallel. Erythrocyte fluorescence values were arbitrarily assigned a value of 1. The species identities for the strains are as follows: F. nucleatum ssp. animalis (JM17 – FQG51A), F. nucleatum ssp. polymorphum (JMSY2 – JMTC3), F. nucleatum ssp. nucleatum (23726 – 25586), F. nucleatum ssp. vincentii (JMP2A), F. nucleatum unnamed subspecies (JMGN1), and F. periodonticum (JMSK2 and SYJL4).
Table 5.
Proportion of cells with detectable surface sialylation.
Figure 5.
Analysis of fusobacterial surface sialylation in subgingival plaque.
(A – D) Subgingival plaque samples were collected from both healthy and diseased sites and analyzed using a combination of fluorescence in situ hybridization (FISH) and fluorescent labeling of surface sialic acid. The health status of the patient sample had no discernable impact upon fusobacterial sialylation results and one representative sample is shown: A) phase contrast image of dispersed subgingival plaque, B) FISH results of subgingival plaque labeled with Alexa488-conjugated Fusobacterium genus probe FUS644, C) fluorescent image of sialylated bacteria in subgingival plaque, and D) merged image of FISH and sialylation images (arrows indicate doubly labeled cells). E) In rows 1–4, individual subgingival plaque samples were suspended in phosphate buffered saline (PBS), dispersed, and directly spotted onto a microscope slide in duplicate. Samples in the top row received 4-trifluoromethylumbelliferyl-α-D-N-acetylneuraminic acid (CF3MU-Neu5Ac), while samples in the bottom row received the same volume of ddH2O. In row 5, PBS containing CF3MU-Neu5Ac was spotted as a negative control. Samples were left at 25 °C for 3 min. before directly visualizing in the presence of UVA irradiation (356 nm). Visible CF3MU-Neu5Ac fluorescence is indicative of sialidase activity [39].