The calcium-induced conformation and glycosylation of scavenger-rich cysteine repeat (SRCR) domains of glycoprotein 340 influence the high affinity interaction with antigen I/II homologs

Abstract

Oral streptococci adhere to tooth-immobilized glycoprotein 340 (GP340) via the surface protein antigen I/II (AgI/II) and its homologs as the first step in pathogenesis. Studying this interaction using recombinant proteins, we observed that calcium increases the conformational stability of the scavenger-rich cysteine repeat (SRCRs) domains of GP340. Our results also show that AgI/II adheres specifically with nanomolar affinity to the calcium-induced SRCR conformation in an immobilized state and not in solution. This interaction is significantly dependent on the O-linked carbohydrates present on the SRCRs. This study also establishes that a single SRCR domain of GP340 contains the two surfaces to which the apical and C-terminal regions of AgI/II noncompetitively adhere. Compared with the single SRCR domain, the three tandem SRCR domains displayed a collective/cooperative increase in their bacterial adherence and aggregation. The previously described SRCRP2 peptide that was shown to aggregate several oral streptococci displayed limited aggregation and also nonspecific adherence compared to SRCR domains. Finally, we show distinct species-specific adherence/aggregation between Streptococcus mutans AgI/II and Streptococcus gordonii SspB in their interaction with the SRCRs. This study concludes that identification of the metal ion and carbohydrate adherence motifs on both SRCRs and AgI/II homologs could lead to the development of anti-adhesive inhibitors that could deter the adherence of pathogenic oral streptococci and thereby prevent the onset of infections.

Keywords: Bacterial Adherence, Antigen I/II, Streptococcus mutans, Streptococcus gordonii, Oral Streptococci; Bacterial Adhesion; Bacterial Pathogenesis; Calcium; Cell Surface Protein; Cell Surface Receptor; Glycosylation; Innate Immunity; Surface Plasmon Resonance (SPR).

FIGURE 1.

Schematic representation of primary sequence of S. mutans UA159 AgI/II and S. gordonii DL1 SspB, including the extents of the recombinant fragments shown here.

FIGURE 2.

Schematic representation of primary sequence of GP340's 13 tandem SRCR domains, the CUB, and ZP are shown here. The figure also includes the extents of the recombinantly expressed iSRCR₁ and iSRCR₁₂₃ (33).

FIGURE 3.

Competition experiments with FL^AgI/II, A₃VP₁^AgI/II, and C₁₂₃^AgI/II were conducted with immobilized iSRCR₁ (A) and immobilized iSRCR₁₂₃ (B) to determine the multiple adherence sites of the SRCRs. Although the direct adherence of the fragment is shown in bold (such as A₃VP₁^AgI/II), the observed adherence inhibition in the presence of competing fragments (FL^AgI/II and C₁₂₃^AgI/II) are plotted in subsequent bar graphs. Similarly, the competition of FL^SspB, A₃VP₁^SspB, and C₁₂₃^SspB with immobilized iSRCR₁, iSRCR₁₂₃, and SAG are shown in C–E. All experiments were carried out in triplicate, and the error bars represent standard deviations.

FIGURE 4.

Illustrated here are the calcium-mediated adherences of (2 μm) AgI/II of S. mutans with iSRCR₁ (A) and iSRCR₁₂₃ (B) on a CM5 sensor chip. The adherence of AgI/II fragments was observed only in the presence of calcium. Devoid of calcium, AgI/II did not adhere to SRCRs. A similar result was observed with SspB of S. gordonii (C and D).

FIGURE 5.

A, these curves illustrate the direct adherence of SRCRP2 peptide to FL^AgI/II A₃VP₁^AgI/II, and C₁₂₃^AgI/II of S. mutans (bold lines) as well as FL^SspB, A₃VP₁^SspB, and C₁₂₃^SspB of S. gordonii (dotted lines). Fluorescein-tagged SRCRP2 (FAM) was serially diluted (0.03–1 mg/ml), and its interaction with immobilized AgI/II and SspB and their subfragments was measured at OD₅₁₉. The results illustrated that the SRCRP2 peptide adheres well with AgI/II and SspB. B, adherence/inhibition of FL^AgI/II and FL^SspB and subfragments (2 μm) in the presence of SRCRP2 peptide at various concentrations (0.005 to 0.200 mm) with immobilized iSRCR₁₂₃. In control experiments, SRCRP2 peptide alone does not display any measurable interaction with iSRCR₁₂₃. More importantly SRCRP2 does not inhibit the adherence of AgI/II and SspB (and their subfragments) to the SRCRs. Surprisingly, SRCRP2 increased the adhesiveness of FL^SspB confounding us, as it could be interpreted as nonspecific adherence/aggregation.

FIGURE 6.

Self-interactions of SRCR domains at various concentrations (0.250–2 μm) with immobilized iSRCR₁ (A and C) and iSRCR₁₂₃ (B and D) on CM5 sensor chip were analyzed using BIA-Evaluation software. The analytes iSRCR₁ and iSRCR₁₂₃ demonstrated nanomolar affinity interaction with SRCRs, clearly indicating the self-adhesion property among SRCRs.

FIGURE 7.

Aggregation of S. mutans UA159 (A) and S. gordonii DL1 (B) cells in the presence of iSRCRs, SAG, and SRCRP2 peptide was analyzed. The results are plotted as percentage of aggregation measured at OD₇₀₀ at 5-min intervals for 1 h. Bacterial cells in buffer alone were used as control. Differences in aggregation detected between groups were analyzed using one-way analysis of variance, where *, p < 0.05 was considered significant, and error bars represent the standard deviation.

FIGURE 8.

Interaction between S. mutans UA159 (A) and S. gordonii DL1 (B) cells with iSRCR₁ and iSRCR₁₂₃ was analyzed using confocal microscopic images. The images display S. mutans and S. gordonii cells stained with DAPI (blue) and anti-His tag Alexa Fluor 488 antibody (green). The observed green fluorescence depicts the binding of iSRCR₁ and iSRCR₁₂₃ to S. mutans and S. gordonii, whereas the control S. mutans and S. gordonii were counterstained by DAPI alone. Also, it is evident from the images that iSRCR₁₂₃ adhered more profoundly than iSRCR₁.

FIGURE 9.

Schematic model displaying the conformational change on the SRCR domains of GP340 in the presence of calcium, and particularly implicating the role of glycosylation in this high affinity adherence to AgI/II homologs. The model also includes the possible self-association of the SRCR domains that could potentially sandwich AgI/II homologs of oral streptococci.