Human common salivary protein 1 (CSP-1) promotes binding of Streptococcus mutans to experimental salivary pellicle and glucans formed on hydroxyapatite surface

Abstract

The saliva proteome includes host defense factors and specific bacterial-binding proteins that modulate microbial growth and colonization of the tooth surface in the oral cavity. A multidimensional mass spectrometry approach identified the major host-derived salivary proteins that interacted with Streptococcus mutans (strain UA159), the primary microorganism associated with the pathogenesis of dental caries. Two abundant host proteins were found to tightly bind to S. mutans cells, common salivary protein-1 (CSP-1) and deleted in malignant brain tumor 1 (DMBT1, also known as salivary agglutinin or gp340). In contrast to gp340, limited functional information is available on CSP-1. The sequence of CSP-1 shares 38.1% similarity with rat CSP-1. Recombinant CSP-1 (rCSP-1) protein did not cause aggregation of S. mutans cells and was devoid of any significant biocidal activity (2.5 to 10 μg/mL). However, S. mutans cells exposed to rCSP-1 (10 μg/mL) in saliva displayed enhanced adherence to experimental salivary pellicle and to glucans in the pellicle formed on hydroxyapatite surfaces. Thus, our data demonstrate that the host salivary protein CSP-1 binds to S. mutans cells and may influence the initial colonization of this pathogenic bacterium onto the tooth surface.

Figure 1. Human salivary proteins eluted from the surface of S. mutans

Venn diagram showing the human salivary proteins differentially bound to the surface of S. mutans, as grouped by the number of overlapping and non-overlapping proteins in the different affinity pools: 0.85% salt (weakly bound, pool A), 1 M salt (moderately bound, pool B) and 2M urea (tightly bound, pool C) solutions.

Figure 2. Comparison of overlapping human salivary proteins eluted from the surface of S. mutans

A comparison of the fifteen human salivary proteins bound to the surface of S. mutans present in all three affinity pools was performed based on their spectral counts: 0.85% salt (weakly bound, pool A), 1 M salt (moderately bound, pool B) and 2M urea (strongly bound, pool C) solutions. (A) Comparison of the nine most abundant proteins (spectral counts >160) identified in pools A, B and C. (B) Comparison of the six least abundant proteins (spectral counts <160) detected in pools A, B and C. CSP-1 = common salivary protein-1; Salivary agglutinin (gp340) = deleted in malignant brain tumor 1; Polymeric-Ig precursor = Polymeric-immunoglobulin precursor; P-I protein precursor = Prolactin-inducible protein precursor; Basic salivary PRP 1 precursor = Basic salivary proline rich protein 1 precurser; CA VI precursor = Carbonic anhydrase VI precursor; SPLNEC = short palate, lung & nasal epithelium carcinoma.

Figure 3. Multiple sequence alignment

The amino acid sequences were aligned for HRPE773 (also known as human CSP-1, GenBank AAQ89380.1) and rat CSP-1 (Accession no. NP598306.1) sourced from from NCBI_nr. Amino acid identities across both species are indicated by an asterisk. Individual peptides identified through MS analysis are mapped to the HRPE773 protein sequence (amino acids in bold italics) and provide 51% sequence coverage, validating the identification of the protein as CSP-1.

Figure 4. Structure, purification and validation of the recombinant human CSP-1 protein (rCSP-1)

Human CSP-1 was cloned and the recombinant protein expressed in S2 cells. (A) The rCSP-1 protein contains an N-terminal secretory signal peptide (SP; black box), FLAG epitope tags (grey box), six histidine residues (dotted black box), metal chelate tag (empty oval), S-peptide tag (striped oval), 3c protease cleavage site (dotted box) and soluble region (white box). Note that the CSP-1 portion of the recombinant protein is not to scale so that the N-terminal region can be more easily visualized. (B) SDS-PAGE (4–12%) analysis of rCSP-1 stained with Simply Blue. Lane 1, metal chelate affinity chromatography of rCSP-1; lane 2, molecular weight markers; lane 3, rCSP-1 re-purified using con A lectin affinity chromatography.

Figure 5. Recombinant human CSP-1 protein (rCSP-1) enhances bacterial adherence to experimental salivary pellicle (sHA) and to glucans synthesized in the pellicle (gsHA) formed on hydroxyapatite surface

Modulation of bacterial adherence to sHA (panel A) and gsHA (panel B) by S. mutans exposed to rCSP-1 (in PBS or in clarified human whole saliva, left and right, respectively). The data shown are mean values ± standard deviations (n=6). Values marked with an asterisk are significantly different from each other (P<0.05, ANOVA, comparison for all pairs using Tukey’s test).