Fusobacterium nucleatum is a saccharolytic Gram-negative anaerobic organism
belonging to the so-called ‘orange complex’ which is believed to play an important role in the
microbial succession associated with the pathogenesis of periodontal disease. Its genome
contains niche-specific genes shared with the other inhabitants of dental plaque, which may
help to explain its ability to survive and grow in the changing environmental conditions
experienced in the gingival sulcus during the progression from health to disease. The pH of
the gingival sulcus increases during the development of periodontitis and is thought to occur
by the metabolism of nutrients supplied by gingival crevicular fluid. Studies have shown that
F. nucleatum is partly responsible for the rise in pH and have concluded that in comparison to
other plaque inhabitants, F. nucleatum has the greatest ability to neutralise acidic
environments. In common with a number of other oral bacteria, F. nucleatum has also been
shown to produce intracellular polyglucose (IP) from simple sugars such as glucose, galactose
and fructose. Its response and adaptation to stressful environmental conditions such as pH is
unknown. The overall aim of this study was, therefore, to determine how F. nucleatum copes
with environmental stresses induced by pH changes.
F. nucleatum was grown by continuous culture in a chemically defined medium at a
growth rate corresponding to those measured in vivo. The effect on protein expression, and IP
synthesis was examined during steady-state growth at high (>7.2<7.8) or low pH (pH 6.4). The
present study also investigated the response of F. nucleatum to growth at pH 8.2. It was found
that the organism grew as a biofilm and this corresponded with an increase in cellular
hydrophobicity and decreased IP levels.
Optimal growth pH’s differed between the different sub-species used in this study. In
response to pH stress, F. nucleatum changed its amino acid and glucose utilisation and increased
IP synthesis at the expense of cell numbers. Pulsing the chemostat with glutamic acid or serine
produced an increase in IP synthesis and the pattern of end-products observed was dependent
upon the amino acid being fermented. The effect on IP synthesis in response to increased levels
of exogenous fermentable amino acids was also compared during concomitant fructose or
glucose fermentation. Growth media containing fermentable amino acids and supplemented with
fructose produced higher cell numbers and non-detectable levels of IP compared to media
containing glucose.
The differential expression of cytoplasmic- and cell envelope-proteins induced by
changes in pH were identified by two-dimensional gel electrophoresis. The results represent the
first proteomic investigation of F. nucleatum. Twenty-two cytoplasmic proteins were found to
have altered expression in response to external pH. At low (sub-optimal) pH, proteins associated
with the generation of ATP and ammonia were up-regulated, the latter contributing to the
alkalinisation of the gingival sulcus. Conversely, neutral to alkaline pH conditions led to the upregulation
of enzymes involved in energy storage. The study also identified several proteins
associated with iron limitation and fatty acid synthesis which might not otherwise have been
identified as part of the pH-dependent response.
In response to growth at pH 7.8, 14 cell envelope proteins were identified as having
significantly altered expression. Down-regulated proteins included those associated with uptake
of C4 di-carboxylates and phosphorus, a potential membrane protease and an enzyme associated
with amino acid fermentation. The up-regulation of a transcriptional regulator linked to the
repression of sugar metabolism was also reported along with proteins linked to the transport of iron. The periplasmic chaperone, peptidyl prolyl cis trans isomerase, which is responsible for the
folding of outer membrane proteins, was also found to be up-regulated.
In conclusion, the proteomic investigation of protein expression by F. nucleatum
identified gene products which form part of the organism’s coordinated stress response to
changes in environmental pH. In addition to these, the physiological based studies also presented
help to explain the organism’s persistence during the transition from health to disease in vivo.Thesis (Ph.D.) - University of Adelaide, Dental School, 200
