Structural basis for the role of serine-rich repeat proteins from Lactobacillus reuteri in gut microbe-host interactions

Abstract

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP_100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP_100-23 and SRRP₅₃₆₀₈ from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP₅₃₆₀₈-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP₅₃₆₀₈-BR with PGA. Long molecular dynamics simulations showed that SRRP₅₃₆₀₈-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host-microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.

Keywords: Lactobacillus reuteri; SRRP; adhesin; biofilm; mucin.

Fig. 1.

Schematic showing domain organization of precursor SRRPs from L. reuteri. The two proteins are drawn to scale. Domains are labeled as follows: A, cell wall anchor including LPXTG motif; N1, nonrepeat region 1; N2 (BR), nonrepeat region 2 (putative binding region); N3, nonrepeat region 3; S, secretion signal sequence; SRR-1, serine-rich region 1; SRR-2, serine-rich region 2. The beginning amino acid position is indicated below each domain. Regions of the BR that were resolved by crystallography are shaded gray and span amino acids 257–623 for SRRP_100-23 and amino acids 262–571 for SRRP₅₃₆₀₈.

Fig. 2.

Crystal structures of SRRP₅₃₆₀₈-BR_262–571 and SRRP_100-23-BR_257–623. (A) Longitudinal view of SRRP₅₃₆₀₈-BR_262–571. The β-strands in putative binding regions PB1, PB2, and PB3 are shown in deep blue, magenta, and red, respectively; α-helices are dark green, and β-strands of the loop are yellow. (B) Longitudinal view of SRRP_100-23-BR_257–623. PB1, PB2, and PB3 β-strands are in light green, cyan, and pink, respectively. The α-helix is in brown, and loop β-strands are in beige. The black spheres indicate the gaps in the model between amino acids 413–421 and 568–583. (C and D) Cross-sections of SRRP₅₃₆₀₈-BR_262–571 (C) and SRRP_100-23-BR_257–623 (D) β-solenoid superhelices along the helical axis from the N to the C terminal. In both, β1 and α2 are omitted for clarity. The black arrow shows the direction in which the polypeptide chains fold around the helical axis, yielding a right-handed superhelix. The helical twist down each β-sheet is indicated by yellow arrows. Along the helical axis, the β-strands in each parallel β-sheet increasingly twist toward the left with respect to each other.

Fig. 3.

Comparison of the TM-Pel–binding pocket with the PuBS of SRRP₅₃₆₀₈-BR and SRRP_100-23-BR. (A) Crystal structure of TM-Pel (Protein Data Bank ID code 3ZSC) (orange) in complex with TGA (cyan). (B) A close-up view of the TM-Pel–binding pocket, with residues involved in TGA binding represented as sticks. (C) Superposition of SRRP₅₃₆₀₈-BR_262–571 (light pink) and TM-Pel (orange) structures, with an rmsd of 2.63 over 210 residues, shows that the PuBS of the former overlaps with that of TM-Pel. (D) Surface-exposed aromatic and charged residues on PB3 in SRRP₅₃₆₀₈-BR_262–571 PuBS (light pink). This includes the aromatic residue triad W375, Y425, and W450 and the basic residue triad K485, R512, and R543. (E) Solvent-exposed residues of PuBS in the overlaid structures of SRRP₅₃₆₀₈-BR (light pink) and SRRP_100-3-BR (green), with an rmsd of 0.912 over 293 residues, showing that several solvent-exposed residues in both binding sites are conserved. Residues in bold are from SRRP₅₃₆₀₈-BR, and those in italic font are from SRRP_100-23-BR. (F) Residues mutated for functional analysis of SRRP₅₃₆₀₈-BR. Single mutants were created by substituting the residues in red (K377 or R512) with alanine. Residues in blue from F411 to T422 in the lower loop were deleted to generate the ΔF411–T422 deletion mutant.

Fig. 4.

The pH-dependent conformational change affects the PuBS, as predicted by MD simulations. (A) Combined surface and cartoon models of SRRP₅₃₆₀₈BR_262–571 (pink) and SRRP_100-23BR_257–623 (green). (B) Surface representation of SRRP₅₃₆₀₈-BR_262–571 at pH 7.4 and pH 4.0 in the same orientation as in A, showing surface electrostatics (Upper) and surface-exposed putative binding residues (Lower, green). At pH 4.0, PuBS exhibits a more positive electrostatic potential as well as an open conformation that exposes a greater number of putative binding residues to the solvent. Coordinates were obtained from representative frames of each respective MD trajectory (Methods). Surface electrostatics were calculated in PyMOL and are color-coded as blue (positive), white (neutral), and red (negative).

Fig. 5.

Adhesion of SRRP₅₃₆₀₈-BR to GI tissue. (A and B) Immunostaining pattern for SRRP₅₃₆₀₈-BR on cryosections of mouse colon (A) and stomach (B) correlates with WGA lectin staining. (C) SRRP₅₃₆₀₈-BR binding to mouse colonic sections following sodium periodate treatment at pH 4.5 is significantly reduced. (D and E) SRRP₅₃₆₀₈-BR binds to epithelial cells HT29 (D) and mucus-producing HT29-MTX cells (E). The immunostaining pattern for SRRP₅₃₆₀₈-BR on HT29-MTX cells correlates with WGA lectin staining and partly with anti-MUC5AC staining. Cell nuclei of tissue sections were counterstained with DAPI. (Scale bars: 50 µm.)

Fig. 6.

AFM force spectroscopy histograms of SRRP₅₃₆₀₈-BR interacting with mucin or PGA. Insets show examples of force curves for each assay. (A) Quantification of adhesion values to mucin in buffers of neutral and acidic pH values or following PGA addition. (B) Quantification of variations in the mucin adhesion event distances. (C) Quantification of adhesion values to PGA in buffers of neutral and acidic pH values.

Fig. 7.

AFM force spectroscopy histograms of SRRP₅₃₆₀₈-BR interacting with DNA. Quantification of adhesion values of SRRP₅₃₆₀₈-BR interacting with DNA in 137 mM NaCl or 1 M NaCl PBS (A) or after the addition of free DNA (B) or free nucleotides dGTP (C), dATP (D), dCTP (E), all four nucleotides (dGTP, dATP, dTTP, and dCTP) (F), or two nucleotides (dGTP and dTTP) (Inset in F).