Research ArticleBACTERIAL ADHESION

An intramembrane sensory circuit monitors sortase A–mediated processing of streptococcal adhesins

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Science Signaling  07 May 2019:
Vol. 12, Issue 580, eaas9941
DOI: 10.1126/scisignal.aas9941
  • Fig. 1 The roles of substratum and LPXTG adhesin C-peps in biofilm biomass.

    (A) Quantification of biofilm formation by the indicated S. gordonii mutants in wells that were uncoated (filled bars) or saliva-coated (unfilled bars). Wild-type (WT) cells were compared to mutants lacking LPXTG adhesins SspA and SspB (ΔSspAB) or SGO_0707 (ΔSGO_0707) and to each deletion mutant expressing the SLC form of the deleted adhesin, which contains the engineered polypeptide: signal peptide, LPXTG motif, and C-pep (adhesin domain missing). Each substrata dataset was analyzed separately using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. (B) Quantification of biofilm formation by mutant S. gordonii in which the SGO_0707 C-pep production was eliminated by deleting the SrtA cleavage site, LPNTG, from the endogenous sgo_0707 locus (SGO_0707∆LPNTG). Data were analyzed using an unpaired, two-tailed Student’s t test. All data represent the means ± SD of at least three independent experiments, with at least three technical replicates for each strain per experiment. Significant differences are denoted by asterisk(s). ****P ≤ 0.0001.

  • Fig. 2 Colocalization of LPXTG motif C-peps with the cell membrane.

    (A) Representative SIM showing a field of S. gordonii cells fluorescently to show the cell membrane (FM 4-46FX; red), chromosome (Hoechst 33342; blue), and the Venus-tagged SGO_0707 C-pep (green). Scale bar, 5 μm. (B to E) Higher-magnification views of the boxed area in (A) showing overlays of the indicated stains. Scale bar, 2 μm. n = 3 independent experiments. (F) ClustalW alignment of SspA, SspB, SGO_0707, and PavB C-peps showing the conserved hydrophobic transmembrane α helix. Color coding indicates amino acid hydrophobicity intensity based on the Kyte-Doolittle hydrophobicity scale (red, hydrophobic; blue, hydrophilic).

  • Fig. 3 The SGO_1180 and SGO_1181 TCS is required for enhanced biofilm formation in the absence of SspAB C-peps.

    (A) Quantification of biofilm formation by S. gordonii lacking SGO_1180 and SGO_1181 (Δ1180-81) on uncoated (filled bars) or saliva-coated (unfilled bars) wells relative to WT. Biofilm formation was quantified in Δ1180-81 mutants in combination with deletion of SspAB (ΔSspAB Δ1180-81), with replacement of SspAB with SspASLC (SspASLC Δ1180-81), with deletion of SGO_0707 (ΔSGO_0707 Δ1180-81), or with replacement of SGO_0707 with SGO_0707SLC (SGO_0707SLC Δ1180-81). Data represent the means ± SD of at least three independent experiments with at least three technical replicates per experiment. (B) Quantification of biofilm formation in SGO_1181 D53N point mutants in the ∆SGO_0707 background relative to WT. Data represent the means ± SD of at least three independent experiments with at least three technical replicates per experiment. (C) Quantification of the competitiveness of S. gordonii mutants lacking SspAB adhesins, SGO_1180, or SrtA in ex vivo multispecies dental plaque biofilms. S. gordonii were added to supragingival multispecies plaque biofilms and cultured overnight. The amount of each strain that colonized the plaque ex vivo community was quantified using quantitative PCR (qPCR). Data represent the means ± SD of n ≥ 2 experiments with at least duplicate biofilms. Horizontal hatched line indicates WT S. gordonii. Each dataset was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Significant differences are denoted by asterisk(s). *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001.

  • Fig. 4 LPXTG-adhesin C-peps interact with SGO_1180.

    (A) Schematic of the interaction between SGO_0707 C-pep (red helical ribbon) and transmembrane domains (TM1 and TM2) of SGO_1180. The N- or C-terminal (N or C, respectively) domain of Venus YFP was inserted in-frame between the penultimate and stop codon of sgo_0707 and on the cytoplasmic side of TM1 or TM2 of sgo_1180. All four combinations of the constructs that could reconstitute active YFP were tested in cells, but only the two that produced fluorescence are shown in the diagram. (B) Quantification of fluorescence emission from the single- and dual-labeled bifluorescent strains shown in (A). Fluorescence was only detected when the N-terminal domain of YFP fused to the SGO_0707 C-pep (SGO_0707-V-N) was combined with the C-terminal domain of YFP fused to TM1 of SGO_1180 (1180-V-C-TM1) or when the C-terminal domain of YFP fused to the SGO_0707 C-pep (SGO_0707-V-C) was combined with the N-terminal domain of YFP fused to TM2 of SGO_1180 (1180-V-C-TM2). RFU, relative fluorescence units, fluorescence units divided by the culture optical density at λ = 600 nm. Data represents the means ± SD of three independent experiments with three technical replicates per experiment. Data were analyzed using an unpaired Student’s t test. Significant differences between strains are denoted by asterisk(s). ****P ≤ 0.0001. (C to F) Magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy of SspA C-pep and SGO_1180 (1:1) in artificial lipid membranes. The one-dimensional (1D) cross-polarization (CP) spectrum to detect the transmembrane helical domains (C), the overlay of 2D–N to Cα (NCA) spectra of free and SspA C-pep–bound SGO_1180 (D), the rINEPT of free and SspA C-pep–bound SGO_1180 to capture the dynamic residues of SGO_1180, and the 2D-rINEPT spectrum of SGO_1180 alone to detect the dynamic residues. Red, SGO_1180 alone; black, SGO_1180 + SspA C-pep (1:1).

  • Fig. 5 SGO_1180 conformational integrity and functional conservation across species.

    (A) ClustalW alignment of transmembrane helix 1 of SGO_1180, consensus S. aureus SaeS, and S. aureus Newman SaeS. The red arrow indicates the SaeS L18P mutation that constitutively activates signaling in the Newman SaeS. (B) Quantification of S. gordonii SGO_1180 L16P biofilm biomass in saliva-coated polystyrene wells. The L16P substitution was engineered into the endogenous sgo_1180 locus and corresponds to the L18P substitution in S. aureus Newman SaeS. Data represent the means ± SD of three independent experiments with at least three technical replicates per experiment. Data were analyzed using an unpaired Student’s t test. Significant differences between strains are denoted by asterisk(s). ****P ≤ 0.0001. (C) Sequence alignment of the first 120 amino acids of SGO_1180 with homologs from S. pneumoniae, S. agalactiae, and S. aureus using ClustalW. (D) Functional complementation of S. gordonii SGO_1180 (Sg) with orthologs from S. pneumoniae (Sp), S. agalactiae (GBS), and S. aureus (SaeS). Biofilm formation in saliva-coated wells was quantified for ΔSspABΔ1180 S. gordonii expressing each of the homologs from an Escherichia coli–streptococcal shuttle vector. Data represent the means ± SD of at least three independent experiments with at least three technical replicates per experiment. Data were analyzed using Kruskal-Wallis with Dunn’s multiple comparisons test. Significant differences are denoted by asterisk(s).

  • Fig. 6 Model of the SspAB C-pep–SGO_1180–SGO_1181 regulatory circuit for monitoring successful processing of LPXTG-containing proteins by SrtA.

    After cleavage of the LPXTG motif by SrtA, the mature adhesin is anchored to the cell wall and the C-pep is retained in the cell membrane. Within the cell membrane, the C-peps bind to the transmembrane domains of the HK SGO_1180, thereby negatively regulating the phosphorylation cascade and preventing compensatory gene expression. In the absence of one or more C-peps, kinase activity of SGO_1180 is increased and induces compensatory gene expression through SGO_1181 to maintain biofilm formation. This circuit indirectly monitors proper placement of LPXTG-containing proteins on the cell wall.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/580/eaas9941/DC1

    Fig. S1. Adhesin expression in S. gordonii cells isolated from saliva and plaque.

    Fig. S2. Growth curves of S. gordonii strains.

    Fig. S3. Biofilm biomass of PavB mutant strains.

    Fig. S4. Additional examples of colocalization of LPXTG motif C-peps with the cell membrane.

    Fig. S5. pavB, SGO_1181, and SGO_1180 are cotranscribed.

    Fig. S6. ScaA contributes to enhanced biofilm formation of ∆SspAB S. gordonii.

    Fig. S7. Conservation of SGO_1180 homologs across streptococci.

    Fig. S8. Conservation of SGO_1180 homologs in Firmicutes.

    Table S1. Characteristics of potential S. gordonii sensor HKs.

    Table S2. Transcriptome and RT-qPCR fold change in gene expression values for biofilms.

    Table S3. RT-qPCR fold-change gene expression values for free-growing bacteria.

    Table S4. Oligonucleotides used in this study.

    Table S5. Bacterial strains used in this study.

    Table S6. Plasmids used in this study.

    Movie S1. Colocalization of C-pep in the S. gordonii cell membrane.

    Movie S2. Colocalization of C-pep in the S. gordonii cell membrane at high magnification.

    Reference (67)

  • The PDF file includes:

    • Fig. S1. Adhesin expression in S. gordonii cells isolated from saliva and plaque.
    • Fig. S2. Growth curves of S. gordonii strains.
    • Fig. S3. Biofilm biomass of PavB mutant strains.
    • Fig. S4. Additional examples of colocalization of LPXTG motif C-peps with the cell membrane.
    • Fig. S5. pavB, SGO_1181, and SGO_1180 are cotranscribed.
    • Fig. S6. ScaA contributes to enhanced biofilm formation of ∆SspAB S. gordonii.
    • Fig. S7. Conservation of SGO_1180 homologs across streptococci.
    • Fig. S8. Conservation of SGO_1180 homologs in Firmicutes.
    • Table S1. Characteristics of potential S. gordonii sensor HKs.
    • Table S2. Transcriptome and RT-qPCR fold change in gene expression values for biofilms.
    • Table S3. RT-qPCR fold-change gene expression values for free-growing bacteria.
    • Table S4. Oligonucleotides used in this study.
    • Table S5. Bacterial strains used in this study.
    • Table S6. Plasmids used in this study.
    • Legends for movies S1 and S2
    • Reference (67)

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Colocalization of C-pep in the S. gordonii cell membrane.
    • Movie S2 (.avi format). Colocalization of C-pep in the S. gordonii cell membrane at high magnification.

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