Membrane Topology and Structural Insights into the Peptide Pheromone Receptor ComD, A Quorum-Sensing Histidine Protein Kinase of Streptococcus mutans

Abstract

Quorum sensing activation by signal pheromone (CSP) in Streptococcus mutans depends on the membrane-associated receptor ComD, which senses the signal and triggers the signaling cascade for bacteriocin production and other cell density-dependent activities. However, the mechanism of the signal recognition via the ComD receptor in this species is nearly unexplored. Here, we show that the membrane domain of the ComD protein forms six transmembrane segments with three extracellular loops, loopA, loopB and loopC. By structural and functional analyses of these extracellular loops, we demonstrate that both loopC and loopB are required for CSP recognition, while loopA plays little role in CSP detection. A deletion or substitution mutation of four residues NVIP in loopC abolishes CSP recognition for quorum sensing activities. We conclude that both loopC and loopB are required for forming the receptor and residues NVIP of loopC are essential for CSP recognition and quorum sensing activation in S. mutans.

Figure 1. A schematic diagram describes the ComCDE quorum sensing system and its regulated genes in S. mutans.

The comC encodes a signal peptide precursor, which is cleaved and exported to release a 21-residue peptide through a peptide-specific ABC transporter encoded by cslAB. The 21-aa peptide is further modified by an extracellular protease SepM to remove the C-terminal 3 residues and generate an 18-residue functional peptide or competence-stimulating peptide (CSP). The comDE encode a two-component transduction system that specifically senses and responds to CSP. When it reaches a critical concentration, CSP interacts with the ComD receptor protein of the neighboring cells and activates its cognate response regulator, ComE, through autophospharylation. The phospharylated ComE in turn activates downstream genes, triggering the signaling cascade for bacteriocin production and other cell density-dependent activities.

Figure 2. A hypothetical topology model of the ComD receptor protein in S. mutans.

The membrane-spanning domain of ComD protein from S. mutans UA159 is predicted to form six transmembrane segments (TMSs) with three extracellular loops, loopA, loopB and loopC, and two intracellular loops. An arrow indicates a potential cleavable side in loopA. Small open circles indicate insertion locations by a dual phoA-lacZ fusion reporter in frame after selected codons corresponding to the amino acid residues L38, A70, T110, S150, P187 and A224. Open rectangles indicate the amino acid residues of loopA, loopB and loopC involved in the construction of in-frame deletion or substitution mutants. The conserved histidine residue (H252) in the C-terminal domain of the ComD protein inside the cytoplasm is also indicated.

Figure 3. Experimental determination of the ComD membrane topology.

(A) A schematic diagram indicates the reporter fusion points at L38 (pKTop-ComD_1-L38), A70 (pKTop-ComD_1–A70), T110 (pKTop-ComD_1–T110), S150 (pKTop-ComD_1–S150), P187 (pKTop-comD_1–P187) and A224 (pKTop-ComD_1–A225) of the ComD membrane-spanning region. Blue boxes indicate extracellular locations, while pink boxes indicate cytosolic locations as predicted by the hypothetical model. (B) The strains expressing the ComD_L38-Pho/Lac (GF-L38), ComD_T110-Pho/Lac (GF-T110) and ComD_P187-Pho/Lac (GF-P187) exhibit the higher levels of phosphatase activity (blue color), indicating the extracellular location of the reporter fusion points. The strains expressing the ComD_A70-Pho/Lac (GF-A70), ComD_S150-Pho/Lac (GF-S150) and ComD_A225-Pho/Lac (GF-A224) exhibit the higher levels of β-galactosidase activity (pink color), indicating the cytosolic location of the reporter fusion points. E. coli DH5α without pKTop (negative control) shows no color, while E. coli DH5α with pKTop or GF-pKTop (positive control) also shows pink color.

Figure 4. A schematic representation of the precise amino acid residues involved in the construction of extracellular loopA, loopB and loopC mutants.

Only amino acid residues that constitute the extracellular loopA, loopB and loopC are shown. The bold and underlined residues indicate an in-frame deletion of each mutant. The residues AA in loopC3 and loop4 mutants indicate alanine substitution mutations.

Figure 5. Western blot analysis of the membrane fractions of the S. mutans strains that constitutively express a His-tagged mutant loopA, loopB or loopC protein.

All the strains were grown in THYE medium with addition of CSP to prepare the membrane proteins, which were then resolved on 10% SDS-PAGE gels and transferred onto PVDF membranes for Western blotting using the anti-His antibody. (A) Lanes 1–4, XT-D-H (ComD⁺), XT-A1H (loopA1⁻), XT-A2H (loopA2⁻) and XT-Pldh-H (ComD⁻); (B) Lanes 1–4, XT-D-H (ComD⁺), XT-Pldh-H (ComD⁻), XT-B1H (loopB1⁻) and XT-B2H (loopB2⁻); and (C) Lanes 1–4, XT-C1H (loopC1⁻), XT-C2H (loopC2⁻), XT-C3H (loopC3⁻) and XT-C4H (loopC4⁻). Arrow indicates 51-kDa proteins detected by Western blotting.

Figure 6. Effects of a deletion or mutation of loopA, loopB or loopC on CSP-dependent quorum-sensing activation.

Two CSP-inducible promoters, PcipB and PnlmAB, were monitored for luciferase reporter activities. (A) Luciferase reporter activities (RLU/OD₅₉₀) of PcipB::luxAB reporter strains, XT-Lx20 (ComD⁺), XT-Lx21 (loopA1⁻), XT-Lx22 (loopA2⁻), XT-Lx23 (loopB1⁻), XT-Lx24 (loopB2⁻), and XT-Lx29 (ComD⁻), were assayed with addition of CSP (CSP+) and without CSP (CSP−). (B) Luciferase reporter activities (RLU/OD₅₉₀) of PcipB::luxAB reporter strains, XT-Lx20 (ComD⁺), XT-Lx25 (loopC1⁻), XT-Lx26 (loopC2⁻), XT-Lx27 (loopC3⁻), XT-Lx28 (loopC4⁻) and XT-Lx29 (ComD⁻), were assayed under the same conditions. (C) Luciferase reporter activities (RLU/OD₅₉₀) of PnlmAB::luxAB reporter strains, XT-Lx30 (ComD⁺), XT-Lx31 (loopA1⁻), XT-Lx32 (loopA2⁻), XT-Lx33 (loopB1⁻), XT-Lx34 (loopB2⁻) and XT-Lx39 (ComD⁻), were assayed under the same conditions. (D) Luciferase reporter activities (RLU/OD₅₉₀) of PnlmAB::luxAB reporter strains, XT-Lx30 (ComD⁺), XT-Lx35 (loopC1⁻), XT-Lx36 (loopC2⁻), XT-Lx37 (loopC3⁻), XT-Lx38 (loopC4⁻), and XT-Lx39 (ComD⁻), assayed under the same conditions.

Figure 7. Effects of natural point mutations in loopB or in both loopB and loopC on CSP perception and quorum-sensing activation.

(A) A sequence alignment of loopA, loopB and loopC among four S. mutans strains showing point mutations in loopB (T₁₁₀ >N₁₁₀ and G₁₁₆ >D₁₁₆) in strain GS-5 or in both loopB (T₁₁₀ >N₁₁₀ and G₁₁₆ >D₁₁₆) and loopC (T₁₇₈ >V₁₇₈) in strains KK23 and R221. (B) Luciferase report activities (RLU/OD₅₉₀) of S. mutans GS-5-derived strains XT-Lx40 (white, PcipB) and XT-Lx41 (black, PnlmAB), XT-D0_GS5 (ΔComD)-derived strains XT-Lx42 and XT-Lx43, R221-derived strains XT-Lx44 and XT-Lx45, and XT-D0_R211 (ΔComD)-derived strains XT-Lx46 and XT-Lx47. The luciferase report activities (RLU/OD₅₉₀) of PcipB::luxAB and PnlmAB::luxAB reporter strains were assayed in THYE medium with addition of CSP.

Figure 8. Effects of a deletion or mutation of loopA, loopB and loopC on bacteriocin production.

A deferred antagonism assay was used to assess production of CSP-induced bacteriocins by S. mutans strains. The cell suspensions of each strain were stabled onto THYE agar plates and overlaid with an indicator strain S. sanguinin SK108 by mixing the cells (10⁷ CFU/ml) in low-melting agarose. The overlaid plates were incubated anaerobically at 37 °C for 20 hours before examining bacteriocin production. (A) ComD⁺ (XT-C0), loopA1⁻, loopB1⁻, loopC1⁻, ComD⁻ (ΔComD); (B) loopC1⁻, loopC2⁻, loopC3⁻, loopC4⁻; (C) UA159 (wt), R211 (wt), GS5 (wt), ΔComD_R211 and ΔComD_GS-5.