2.5. Putative Virulence Proteins Expressed by an Oral C. concisus Strain Cultured under AnaeroH2− and AnaeroH2+ Conditions
Proteins expressed by C. concisus cultured under AnaeroH2− and AnaeroH2+ conditions were analysed using mass spectrometry. C. concisus strain P6CDO-S1 was used in this experiment. P6CDO-S1 is an oral strain previously isolated from saliva of a patient with CD. Our previous studies showed that this oral C. concisus strain was genetically close to a C. concisus strain isolated from the intestinal biopsies of a patient with CD [6]. Therefore, we decided to investigate whether this oral C. concisus strain expresses putative virulence proteins and whether these proteins are expressed differentially when the strain is grown under AnaeroH2− and AnaeroH2+ conditions.
Briefly, C. concisus P6CDO-S1 strain was grown on HBA plates for 48 hours under AnaeroH2− and AnaeroH2+ conditions, respectively. C. concisus bacteria were collected and washed with PBS and then 19 μg whole cell proteins were separated on 12% SDS-PAGE as described previously [16]. The gel lane of each sample was cut into 10 slices. In-gel protein trypsin digestion was performed. The extracted peptides were separated by liquid chromatography and analysed by MS/MS as previously described [16, 17].
Mascot Daemon program (Matrix Science, London, UK) was used for bacterial protein identification against the NCBI database. The spectral counts of the same proteins expressed by P6CDO-S1 under AnaeroH2− and AnaeroH2+ conditions were compared using the Scaffold-3 software (Proteome software, OR, USA) [18]. The experiment was carried out in duplicate and repeated twice.
Mass spectrometry was conducted at the Bioanalytical Mass Spectrometry Facility, University of New South Wales, Australia.