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.

Autoaggregation inStreptococcus intermediusis driven by the Pel polysaccharide

“…Glycoside hydrolases are a common feature of exopolysaccharide biosynthetic systems (60) and in P. aeruginosa the hydrolase activity of PelA has been associated with both production of cell free Pel, as well as matrix disruption and dispersal (19, 61). Our bioinformatics analyses suggest that SIR_1591 will be found on the extracellular surface of the bacterium, in keeping with the identification of a homologue of this protein in the secretome of Streptococcus gordonii (62) and the location of the mature Pel polymer. At present it is unclear whether this attachment is via a lipid anchor or a transmembrane helix.…”

“…Additional bioinformatics analyses reveal that a homologue of the first of the additional three genes, SIR_1594, is also found in the secretome of Streptococcus gordonii (62) and has been identified as a putative surface protein in Streptococcus salivarious F60-1 (66). Our analyses suggest that this gene encodes a secreted protein whose N-terminal region (residues 62-472) has significant with homology (22% identity) to Bacillus subtilis and Bacillus cereus CotH, a serine kinase involved in the regulation of spore coat formation.…”