Mass Spectrometric Analysis of Whole Secretome and Amylase-precipitated Secretome Proteins from Streptococcus gordonii

Abstract

Oral biofilm (dental plaque) is formed by the initial adhesion of "pioneer species" to salivary proteins that form the dental pellicle on the tooth surface. One such pioneer species, Streptococcus gordonii, is known to bind salivary amylase through specific amylase-binding proteins such as amylase-binding protein A (AbpA). Recent studies have demonstrated that once bound, salivary amylase appears to modulate gene expression in S. gordonii. However, it is not known if this amylase-induced gene expression leads to secretion of proteins that play a role in plaque biofilm formation. In this study we examined the differences in secreted proteomes between S. gordonii KS1 (wild type) and AbpA-deficient (ΔAbpA) strains. We also examined the differentially precipitated secretome proteins following incubation with salivary amylase. The culture supernatants from KS1 and ΔAbpA were analyzed by nano-LC/MS/MS to characterize the whole secreted proteomes of the KS1 and ΔAbpA. A total of 107 proteins were identified in the KS1 and ΔAbpA secretomes of which 72 proteins were predicted to have an N-terminal signal peptide for secretion. Five proteins were differentially expressed between the KS1 and ΔAbpA secretomes; AbpA and sortase B were expressed exclusively by KS1, whereas Gdh, AdcA and GroEL were expressed only by ΔAbpA. Incubation of culture supernatants from KS1 and ΔAbpA with amylase (50 μg/ml) at room temperature for 2 h resulted in the differential precipitation of secretome proteins. Hypothetical protein (SGO_0483), cation-transporting ATPase YfgQ (Aha1), isocitrate dehydrogenase (Icd), sortase A (SrtA), beta-N-acetylhexosaminidase (SGO_0405), peptide chain release factor 1(PrfA) and cardiolipin synthase (SGO_2037) were precipitated by amylase from the KS1 culture supernatant. Among the identified secreted proteins and amylase-precipitated proteins, transcriptional regulator LytR (SGO_0535) and cation-transporting ATPase YfgQ (Aha1) are potential signaling proteins.

Keywords: AbpA; AbpB; Amylase-binding proteins; GtfG; Proteomic analysis; Secretome.

Figure 1

Functional distribution of proteins in whole secretome of Streptococcus gordonii.

Figure 2. Silver stained 10% SDS-PAGE gel showing amylase-precipitated proteins in various strains of S. gordonii strains

Lanes: 1-CH1 (WT), 2-KS1 (kanamycin resistant WT), 3-ΔAbpA, 4-ΔAbpB and 5-amylase (control).

Figure 3. Sample preparation for MS/MS analysis of amylase-precipitated proteins

Coomassie-stained gels were cut in the pattern depicted above to get 5 bands/slices per strain. Amylase and AbpA bands were avoided as they are well known from past experiments. The gel bands/slices were placed in microcentrifuge tubes containing 70 μl of double distilled water and sent for nano-LC/MS/MS analysis.