Structure of pneumococcal peptidoglycan hydrolase LytB reveals insights into the bacterial cell wall remodeling and pathogenesis

Abstract

Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-β-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375-Asp-658) of LytB (termed LytBCAT), excluding the choline binding domain. LytBCAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a "T-shaped" pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases.

Keywords: Bacterial Pathogenesis; Cell Wall Remodeling; Enzyme Structure; Peptidoglycan; Streptococcus; Structural Biology.

FIGURE 1.

Domain organization and overall structure of LytB. A, domain organization of the full-length LytB. SP, signal peptide; SH3b, bacterial SH3b module; WW, WW domain-like module; GH73, the catalytic module of GH73. B, schematic representation of LytB_CAT overall structure. The SH3b, WW, and GH73 modules are colored in yellow, magenta, and green, respectively.

FIGURE 2.

Structural comparison of the GH73, SH3b, and WW modules. Superposition of LytB_GH73 to the GH73 domain of L. monocytogenes (A) autolysin Auto and Sphingomonas sp. flagellar protein FlgJ (B). The GH73 domains of LytB, Auto, and FlgJ are colored in green, red, and pink, respectively. RBD, rod binding domain. The domain organizations of Auto and FlgJ are shown above the corresponding superimposed structures. Structural superposition of the LytB_SH3b module (yellow) against the SH3b domain of A. variabilis AvPCP (cyan) and S. capitis ALE-1 (gray) (C) and the WW module of LytB (magenta) against the chitin binding domain (blue) of ChiB from S. marcescens (D).

FIGURE 3.

A simulated model of LytB_CAT binding to the putative substrate TSPP. A, surface representation of LytB_CAT with TSPP in the substrate binding pocket. B, interactions between LytB_CAT and TSPP. TSPP is shown in cyan sticks. Residues involved in hydrogen bonds and van der Waals interactions with TSPP are assigned using the PISA server (64) and are shown as sticks and colored in yellow, magenta, and green. Hydrogen bonds are indicated with dashed lines. C, multiple-sequence alignment of LytB_CAT and homologs. Sequences of proteins are downloaded from the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov) with accession numbers: Streptococcus pseudopneumoniae, ZP_09990767.1; S. mitis, ZP_13525443.1; Streptococcus sp. oral, ZP_07458768.1; Streptococcus oralis, ZP_12441552.1; Streptococcus sanguinis, ZP_07887886.1; Streptococcus tigurinus, ZP_23320820.1; Streptococcus infantis, ZP_08523398.1; Streptococcus peroris, ZP_08065421.1; Gemella bergeriae, WP_021753068.1; Gemella haemolysans, WP_003144609.1; Gemella morbillorum, WP_004633969.1; Gemella sanguinis, WP_016359661.1. The alignment was performed with the programs ESPript. The residues participating in TSPP binding are labeled with blue triangles, and residues involved in catalysis are labeled with blue star.

FIGURE 4.

Hydrolytic activity of LytB and mutants toward purified PGN. The hydrolytic activity toward the immature and mature PGN are shown as solid and open bars, respectively. The enzymes applied to the assays include 1) the full-length LytB (LytB), 2) the catalytic domain (LytB_CAT), 3) the WW and GH73 modules (LytB_WW-GH73), 4) the GH73 module (LytB_GH73), 5) the CBD fused with GH73 (LytB_ΔSH3b-WW), and 6) the single E564Q mutant of the full-length (LytB^E564Q). The hydrolytic activities of all mutants are shown as a percentage to that of the full-length LytB. Data are presented as the means ± S.D. from three independent assays. Two-tailed Student's t test is used for the comparison of statistical significance. The p values of <0.05, 0.01, and 0.001 are indicated with *, **, and ***, respectively.

FIGURE 5.

The chain-dispersing activity of LytB and mutants. The cell morphology of the wild-type S. pneumoniae TIGR4 strain (A) and lytB knock-out strain (B) is shown. Incubation of the lytB knock-out strain with the phosphate buffer (C), the full-length LytB (D), LytB_CAT, the catalytic domain of LytB (E), LytB_WW-GH73 (F), LytB_GH73 (G), and LytB^E564Q (H) mutant. Images of the bacteria dyed by Gram's stain are taken after incubation at 37 °C for 30 min.

FIGURE 6.

Morphology of pneumococci with deletion of the coding region for LytB or module(s). A, the streptomycin-resistant derivative S. pneumoniae strain ST002 (Table 1). B, the lytB knock-out streptomycin-resistant derivative S. pneumoniae strain ST003; complement of coding region for lytB_ΔSH3b-WW (C), lytB_ΔSH3b (D), lytB^E564Q (E), and wild-type lytB (F). Images of bacteria dyed with Gram's stain are taken when the A_{620 nm} reached 0.3; G, PCR validation of the deletion and complement of various strains.

FIGURE 7.

Efficiency of adhesion to and invasion into human lung epithelial cells by S. pneumoniae TIGR4 and its isogenic lytB mutants. Quantification of bacteria adhesion to (A) and invasion into (B) human lung epithelial A549 cells. C, comparison of the wild-type and various lytB-mutant strains of pneumococci adhering to A549 cells by fluorescence microscopy. Cells grown on coverslips were incubated with wild-type S. pneumoniae and its isogenic lytB mutants. The strains used are listed in Table 1. WT, wild-type S. pneumoniae ST002 was used as the positive control; KO, the lytB knock-out strain ST003; ΔSH3b, the lytB_ΔSH3b strain ST005; ΔSH3b-WW, the lytB_ΔSH3b-WW strain ST004; E564Q, the lytB^E564Q mutant strain ST008. Bacteria were label with fluorescein isothiocyanate. A549 cells were labeled with propidium iodide. The adhesion rates are shown as the percentage of wild-type pneumococci. Data are presented as the means ± S.D. for three independent experiments. One-way analysis of variance with a post hoc The Dunnett test was used for the comparison of statistical significance. The p values of <0.05, 0.01, and 0.001 are indicated with *, **, and ***, respectively.

FIGURE 8.

The modular organization of SH3b-WW-GH73 in S. mitis group and Gemella species. The term and boundaries of each choline binding repeat are defined according to the PROSITE database and/or sequence alignment.