**Statement of Work**

**Dr. Gilad Bachrach**

Dr. Gilad Bachrach is an expert in the field of genetics of
*Fusobacterium nucleatum*, a bacterium associated with human oral
gingivitis, and in coaggregation of this organism with other oral
bacteria. This individual's specific experience and knowledge on
fusobacterial genetics and coaggregation will enhance the collaboration
of research between our laboratories. Genomic DNA microarrays are now
available for *Fusobacterium nucleatum* ATCC100953. Dr. Bachrach will
use the microarray format currently employed in Dr. Kolenbrander's
laboratory to identify target genes involved in coaggregation and
communication signals between human oral bacteria.

**Background**

It is now evident that coaggregation (cell-cell recognition between
genetically distinct microorganisms) is an important factor in the
development of oral biofilms. Coaggregation can enhance the
multi-species dental plaque formation and maintenance in a number of
ways. First, coaggregation can promote retention of bacteria that are
not equipped for binding directly to oral surfaces. Without attachment
to bacteria already attached to the dental plaque, such late oral
colonizers will be washed away by the saliva and swallowed. Second,
coaggregation can enhance metabolic synergism for the break down and
consumption of complex nutrients. Third, coaggregation can also create
spatial proximity that might enable cross-species exchange of genetic
material. Such genetic exchange is expected to improve species
adaptation to fluctuation in biofilm environmental conditions.

Accumulating *in vitro* experimentation data demonstrate that
coaggregation is a common trait among oral bacteria. In most cases, a
lectin-like protein adhesin exposed on the surface of one bacterium
binds a specific carbohydrate receptor located on the surface of the
coaggregation partner. Some coaggregating pairs of oral bacteria unable
to grow by themselves on saliva as sole nutrient showed abundant
mutualistic biofilm growth when they are inoculated together in an *in
vitro* flowcell system.

Recent work performed on biofilms developed *in vivo* on retrievable
enamel chips worn by volunteers supplied evidence for coaggregation in
early stages of dental biofilm formation. Unlike other common
disease-associated pathogens such as *Pseudomonas aeruginosa,
Streptococcus pyogenes* or *Staphylococcus aureus* that form
mono-species biofilms, the initial dental biofilm is characterized by
micro colonies comprising multiple species suggesting that synergism and
mutualism are beneficial (if not required) for dental plaque formation.

Work performed using fluorescently labeled antibodies demonstrated the
involvement of coaggregation components (adhesins, receptors) on
adjacent species in the *in vivo* grown early biofilms.
Micromanipulation combined with species identification using
fluorescently labeled probes (antibody or DNA) has been recently
proposed and utilized in the laboratory of Dr. Kolenbrander as a
powerful approach to study coaggregation among oral bacteria in biofilms
grown *in vivo*.

Members of the genus *Fusobacterium* coaggregate with every oral
bacterial isolate; this is a feature that distinguishes fusobacteria
from all other oral bacteria that coaggregate with only a few species.
Fusobacteria are not initial colonizers of the enamel surface. They
appear in dental plaque after about 8 hours following oral hygiene
procedures. At later times, fusobacteria are the predominant
gram-negative bacteria in healthy sites and in gingivitis and
periodontal diseased sites. However, the role of fusobacteria and their
coaggregation properties have not been explored from a temporal
viewpoint in the development of dental plaque. We propose that
fusobacteria are a key group of bacteria in the transition from healthy
sites to diseased sites and that examination of initial communities
containing fusobacteria will shed light on their role in this
transition.

Genomic DNA microarrays are now available for *Fusobacterium nucleatum*
ATCC100953. The microarray format is the same as the format used in Dr.
Kolenbrander's laboratory for his study of *Porphyromonas gingivalis*,
and the microarray reader is located in Dr. Kolenbrander's laboratory.
Fusobacterial gene regulation can be studied by coaggregating
fusobacteria with their partners and conducting microarray analyses.
This approach can be particularly revealing when examining initial
communities containing fusobacteria and their in situ partners.

**Proposed Research Project**

***Our long-range goal*** is to understand the role of fusobacterial
coaggregations in the development, maintenance and architecture of the
multi-species dental biofilm.

We plan to investigate the timing of the first appearance of
fusobacteria in dental plaque formed on retrievable enamel chips and to
examine the cell-cell interactions of fusobacteria with their community
neighbors isolated at this first appearance. To complement the studies
of the initial communities of fusobacteria in dental plaque, we will
include microarray analyses of coaggregating fusobacteria and their
partners.

Our specific aims are:

1.  Use existing anti-fusobacterial antibodies (in Dr. Kolenbrander's
    laboratory) conjugated to fluorescent dyes to determine the timing
    of first appearance of fusobacteria on retrievable enamel chips.

2.  Isolate an early fusobacterium-containing biofilm community from the
    enamel surface using a micromanipulator.

3.  Identify the microbial composition of the isolated community using
    16S rRNA gene amplification followed by cloning and sequencing. If
    needed RT-PCR of 16S rRNA will be tested for improving sensitivity.

4.  Determine the coaggregation properties of the fusobacterial isolate
    with the members of its community.

5.  Use fusobacterial genomic DNA microarrays to analyze fusobacterial
    gene regulation when fusobacteria are coaggregated with their
    partners.

6.  Prepare specific fluorescently labeled nucleotides for fluorescence
    in situ hybridization (FISH) and determine the spatial arrangement
    of fusobacteria and their community neighbors on enamel chips.

7.  By using pairwise coaggregation properties, reconstitute in vivo
    communities in the in vitro flowcell model to determine the role of
    fusobacterial coaggregation with each member in the community.

Understanding the mechanisms involved in the transition of the
indigenous dental biofilm to one involved in inducing dental diseases is
expected in the long run to enable the development of novel approaches
for the prevention and treatment of dental diseases and of systemic
ailments affected by them.