Molecular basis of the final step of cell division in Streptococcus pneumoniae

Abstract

Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molecular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci.

Keywords: CP: Microbiology; LytB; SAXS; StkP; Streptococcus pneumoniae; bacterial division; cell wall; crystallography; molecular dynamics; peptidoglycan; teichoic acid.

Figure 1.. Substrate recognition by the catalytic module of LytB

(A) Schematic representation of the modular nature of LytB is shown. The 18 repeats (R1–R18) composing the choline-binding module of LytB are labeled. The position of the catalytic residue E585 is indicated by a triangle. (B) Apo structure of the complete catalytic module of LytB in its closed conformation. The three domains building the catalytic module are colored differently and labeled. The catalytic E585 residue is represented as capped sticks and labeled. The calcium ion found attached to the SH3b domain is represented as a red sphere and coordinating residues as capped sticks. (C) Detailed view of the differences in the catalytic loop between the closed (salmon) and the open (gray) conformations in the apo state. Some relevant residues are represented as capped sticks and labeled. Polar contacts are represented as dotted lines. (D) Three-dimensional structure of the LytB_cat:NAG₄ complex in its open conformation, with NAG₄ depicted in capped sticks colored by atom type (green for the carbons). Sites occupied by the ligand are labeled. (E) Detailed view of substrate recognition by LytB as observed in the LytB_cat-E585Q:(NAG-NAM)₂ complex. Substrate spanning from site −3 to +2 is depicted as capped sticks colored by atom type (green for carbon). Relevant active-site residues are given in capped sticks (colored white for carbons) and labeled. Hydrogen-bond interactions are represented as dotted lines. (F) LytB_cat:PG fragment complex model in its closed conformation. Peptide stems and glycan chains are colored by atom type with yellow and dark green for carbon atoms, respectively. (G) Phase-contrast microscopy images of WT, lytB-GH73-Y₆₃₅A, lytB-SH3b-K₄₂₆E, lytB-GH73-2Mut (Y₆₀₆A/D₆₀₇K), lytB-GH73-3Mut (Y₆₅₄A/S₆₅₆A/D₆₅₇K), lytB-GH73-E₅₈₅A, lytB-GH73-E₅₈₅Q, lytB-WW-5Mut (Y₄₇₇A/E₄₇₉K/Y₄₈₆A/Y₄₈₈A/Y₅₁₁A), and ΔlytB cells. Scale bar, 2 μm. (H) Percentage of cells with a chaining phenotype (minimum four cells per chain), and n indicates the number of cells scored from three independent experiments. The error bar and the data points overlapping the histogram (mean of three experiments) represent the SEM and the mean of each experiment, respectively. Statistical comparison was done with one-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001 and ns, not significant, p > 0.05.

Figure 2.. Structure and role of the choline-binding module of LytB

(A) The molecular surface representation of the complete LytB_CBM with each subdomain colored differently is given: N subdomain is colored in yellow, M subdomain is colored in cyan, and C subdomain is in blue. Choline molecules bound to LytB_CBM are represented as spheres. The hinge regions (located around K99 and K160 residues) are depicted in orange cartoon with side chains in ball-and-stick representation. Lys residues at the hinge regions are labeled. (B) Three-dimensional structure of a canonical choline-binding site (C2) in LytB. (C) Structure of a GYMA choline-binding site (GYMA 2) in LytB. (D) Structure of a hinge site (hinge 2) in LytB. (E) Structure of a starting choline-binding site (S1) in LytB. Aromatic residues involved in the cation-π interactions with choline and other relevant residues are represented as capped sticks and labeled. Choline molecule are shown as spheres colored by atom type with carbons in white. (F) Phase-contrast microscopy images of WT, lytB-ΔN, lytB-ΔM, lytB-ΔNΔM, lytB-DC, and ΔlytB cells; scale bar, 2 μm. (G) Percentage of cells with a chaining phenotype (minimum four cells per chain), with n indicating the number of cells scored from three independent experiments. The error bar and the data points overlapping the histogram (mean of three experiments) represent the SEM and the mean of each experiment, respectively. Statistical comparison was done with one-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001, *p < 0.05, and ns, not significant, p > 0.05. (H–J) Impact of exogenously added LytB or derivatives on ΔlytB cell chaining. (H) ΔlytB cells were treated with LytB or LytB_cat or LytB_NM-cat or LytB_C-cat and then imaged. Phase-contrast images. Scale bar, 2 μm. (I) Percentage of cells with a chaining phenotype (minimum four cells per chain). n indicates the number of cells scored from three independent experiments. The error bar and the data points overlapping the histogram (mean of three experiments) represent the SEM and the mean of each experiment, respectively. Statistical comparison was done with one-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001, ***p < 0.001, and ns, not significant, p > 0.05. (J) Total fluorescence of GFP-LytB, GFP-LytB_cat, GFP-LytB_NM-cat, and GFP-LytB_C-cat bound to ΔlytB cells. A super-violin plot with data from three independent experiments in yellow, green, and blue is shown. The error bar, the data points, and the black horizontal line represent the SEM, the median of each experiment, and the mean of the three experiments, respectively. Data obtained with GFP-LytB_cat, GFP-LytB_NM-cat, and GFP-LytB_C-cat were normalized to data with GFP-LytB taken as 1. Statistical comparison was done using t test. ****p < 0.0001 and ns, not significant.

Figure 3.. Interplay between the LytB NM domain and the StkP-PASTA4 repeat

(A and B) ΔlytB cells were treated with GFP–LytB (A) or GFP-LytB_C-Cat (B) and then imaged. Phase contrast (PC, left), GFP fluorescent signal (middle), and overlays (right) are shown; scale bar, 1 μm. The corresponding heat maps representing the localization patterns of GFP-LytB and GFP-LytB_C-cat are shown on the right. The n value represents the number of cells analyzed in a single representative experiment made in triplicate. (C) Same as (A) and (B) with ΔlytB-stkP-ΔPASTA4 cells treated with GFP-LytB. (D) Same as (A) and (B) with ΔlytB cells treated with GFP-LytB_Nmut. (E) Phase-contrast microscopy images of WT, stkP-ΔPASTA4, lytB-Nmut, ΔlytB, and ΔlytB-stkP-ΔPASTA4 cells. Scale bar, 2 μm. (F) Percentage of cells with a chaining phenotype (minimum four cells per chain) from three independent experiments. The error bar and the data points overlapping the histogram (mean of three experiments) represent the SEM and the mean of each experiment, respectively. Statistical comparison was done with one-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001, ***p < 0.001, and *p < 0.05. (G) Microscale thermophoresis binding assays of labeled LytB_NM (green dots) or LytB_C (purple dots) domains to increasing concentrations of the StkP-PASTA4 repeat. The fraction bound is plotted against the ligand concentration.. Measurements are represented by dots (mean of three independent experiments) and the fitted curve by a line. The error bar represents the standard deviation.

Figure 4.. Teichoic acid and StkP recognition by LytB

(A) Zoom view of the interaction interface between StkP-PASTA4 (green) and subdomain N (dark yellow) of LytB, displaying its key interacting residues in sticks. (B) Microscale thermophoresis binding assays of labeled LytB_N (blue dots) or LytB_Nmut (red dots) domains to increasing concentrations of the StkP-PASTA4 repeat. The fraction bound is plotted against the ligand concentration. Measurements are represented by dots and the fitted curve by a line. The error bar represents the standard deviation. (C) Total fluorescence of GFP-LytB bound to ΔlytB, ΔlytBΔtacL, and ΔlytBΔlytR cells. A super-violin plot with data from three independent experiments in yellow, green, and blue is shown. The error bar, the data points, and the black horizontal line represent the SEM, the median of each experiment, and the mean of the three experiments, respectively. Data from ΔlytBΔtacL and ΔlytBΔlytR cells were normalized to ΔlytB data taken as 1. Statistical comparison was done using t test. *p < 0.05 and ns, not significant, p > 0.05. (D) ΔlytBΔtacL cells were treated with GFP-LytB and then imaged. Phase contrast (PC, left), GFP fluorescent signal (middle), and overlays (right) are shown; scale bar, 1 μm. The corresponding heatmaps representing the localization patterns of GFP-LytB are shown on the bottom. The n value represents the number of cells analyzed in a single representative experiment made in triplicate. (E) Total fluorescence of GFP-LytB bound to ΔlytB cells or protoplasts. A super-violin plot with data from three independent experiments in yellow, green, and blue is shown. The error bar, the data points, and the black horizontal line represent the SEM, the median of each experiment, and the mean of the three experiments, respectively. Data obtained with protoplasts were normalized to data with cells taken as 1. Statistical comparison was done using t test. ***p < 0.001. The phase-contrast image shows the pneumococcal protoplasts generated upon treatment with lysozyme and mutanolysin; scale bar, 2 μm. (F) Zoom view of canonical choline-binding site C7 of the subdomain C (slate) represented in cartoon and displaying its key interactions with teichoic acids (carbons colored in white) depicted in sticks. (G) Zoom view of GYMA choline-binding site G2 of the subdomain C (slate) represented in cartoon and displaying its key interactions with teichoic acids (carbons colored in white) depicted in sticks.

Figure 5.. SAXS analysis of full-length LytB in solution

(A) Experimental scattering curve (dots) and theoretical scattering curve computed for the model of LytB (smooth) at 4 mg mL⁻¹ concentration. (B) The plot shows the normalized pair-distance distribution function P(r) for LytB (blue graph). a.u., arbitrary units. (C) Overlaying of the ab initio determined SAXS envelope for LytB with the model based on the crystal structures reported here. The different regions of the generated model are displayed following the Figure 1A coloring code, and the envelope is colored in light orange.

Figure 6.. Model of StkP-LytB interaction and control of the final cell division step in streptococci

(A) Proposed model of LytB interaction with teichoic acids and StkP. While the C subdomain ensures the binding of LytB to the cell wall by winding around the wall teichoic acids decorated with phosphorylcholine, the NM subdomains drive the localization at the division septum through the interaction with the distal PASTA4 domain of StkP. With this organization, the catalytic domain of LytB can be sequestered up to 400 Å from the membrane surface. The StkP model was generated using AlphaFold2. (B) A model of PG turnover performed by LytB and StkP at the final step of cell division is shown in the cartoon. Upon the export of LytB, the NM subdomains interact with the distal PASTA4 of StkP to position LytB at the division septum (step 1). Concomitantly, the C domain of LytB is wrapped by the wall teichoic acids protruding from the peptidoglycan layer (step 2). These interactions, together with the flexible nature of both the LytB_CBM and the extracellular domain of StkP, allow the LytB catalytic domain to be erected across and toward the surface of the peptidoglycan layer. The linker between the catalytic domain and the CBM of LytB allows its positioning in different orientations to allow appropriate hydrolysis of the peptidoglycan.