Effect of alkaline growth pH on the expression of cell envelope proteins in Fusobacterium nucleatum

Abstract

Fusobacterium nucleatum is a Gram-negative anaerobic organism that plays a central role in the development of periodontal diseases. The progression of periodontitis is associated with a rise in pH of the gingival sulcus which promotes the growth and expression of virulence factors by periodontopathic bacteria. We have previously reported that the expression of specific cytoplasmic proteins is altered by a shift in growth pH. In the present study we have compared cell envelope protein expression of F. nucleatum during chemostat growth at pH 7.2 and 7.8. From a total of 176 proteins resolved from the cell envelope, 15 were found to have altered expression in response to an increase in growth pH and were identified by MS. Upregulated proteins included an outer membrane porin which has been identified as playing a role in virulence, a periplasmic chaperone which assists in the folding of outer membrane proteins, and a transporter thought to be involved with iron uptake. Proteins downregulated at pH 7.8 were consistent with our previous findings that the bacterium reduces its catabolism of energy-yielding substrates in favour of energy-storage pathways. Among the downregulated proteins, two transporters which are involved in the uptake of C4 dicarboxylates and phosphate were identified. A putative protease and an enzyme associated with the metabolism of glutamate were also identified. A high proportion of the cell envelope proteins suggested by these data to play a role in the organism's response to alkaline growth pH may have arisen by lateral gene transfer. This would support the hypothesis that genes that provide an ability to adapt to the changing conditions of the oral environment may be readily shared between oral bacteria.

A proteomic investigation of Fusobacterium nucleatum alkaline-induced biofilms

“…The increased intracellular concentration of TRAP transporters and increased concentration of ABC transporter binding proteins could be considered to be energy conserving as TRAP transporters rely on proton-motive force instead of ATP hydrolysis (ABC transporters) to drive the uptake of solutes from the environment. In contrast to our results, the production of TRAP transporter binding proteins was suppressed 10-fold in planktonic cells cultured at pH 7.8 [27]. The authors explained that the decreased abundance of TRAP binding proteins in planktonic cells may be due to a reduced proton gradient [27].…”

“…Earlier studies in our laboratory showed that the regulation of proteins associated with energy production, transport and protein folding occurred in planktonic cells cultured at pH 7.8 [26,27]. While the present study reports a similar change in protein expression patterns at pH 8.2, we have identified 32 proteins that are altered in response to growth at pH 8.2.…”

“…Previous studies resolved whole cell- or cytoplasmic-protein subsets within a 4–8 pI range [26,37-39]. We have also reported the expression of cell envelope proteins in F. nucleatum (pI 4–10) grown at pH 7.8 [27]. In comparison, the present study examined both cytoplasmic and cell membrane protein expression (pI range 4–10) following growth at pH 8.2.…”

“…The excised protein spots were digested with trypsin, destained and digested as described before [27]. …”

“…The characterisation of biofilms has revealed that cells within them exhibit different concentrations in proteins involved in metabolism, transport and regulation [22-25]. Protein regulation in F. nucleatum in response to acidic (pH 6.4) and mild alkaline (pH 7.4 and 7.8) has been reported [26,27]. …”