Screening of putative peptidoglycan hydrolases from the A. baumannii proteome.

(a) Workflow for the development of an integrated system for peptidoglycan hydrolase screening. (b) Phylogenetic analysis of putative peptidoglycan hydrolases from the A. baumannii proteome database. Multiple sequence alignments are performed using MEGAX software with ClustalW algorithm, and the Neighbor-joining method is used to construct a phylogenetic tree. Classified clades are labeled in different colors and representative peptidoglycan hydrolases from each clade are pointed by colored circles.

PHAb10 and PHAb11 are highly thermostable peptidoglycan hydrolases with a broad-spectrum of action.

(a) Heat map showing the halo size formed by different concentrations of peptidoglycan hydrolases on different bacterial lawns. (b) Bactericidal activity of five peptidoglycan hydrolases (50 μg/ml) in 20 mM Tris-HCl (pH 7.4) at 37°C for 1 hour against different exponential bacteria. (c) Time-dependent bactericidal activity of 50 μg/ml PHAb10 or PHAb11 against exponential A. baumannii in 20 mM Tris-HCl (pH 7.4). (d) Bactericidal activity of 50 μg/ml PHAb10 or PHAb11 in 20 mM Tris-HCl (pH 7.4) at 37°C for 1 hour against different bacteria in exponential and stationary phases. (e-f) Dose-dependent bactericidal activity of PHAb10 and PHAb11 against multiple exponential bacteria in 20 mM Tris-HCl (pH 7.4) at 37°C for 1 hour. (g-h) Susceptibility of various Gram-negative (g) and Gram-positive (h) bacterial strains to PHAb10 and PHAb11. Exponential cultures of each bacterium are treated with 50 μg/ml PHAb10 or PHAb11 for 1 hour at 37°C and residual viable bacterial cells are counted by plating serial dilutions onto agar plates. For E. faecalis, 10 μg/ml of each peptidoglycan hydrolase is used. (i) Thermal stability of PHAb10 and PHAb11. Each enzyme is stored at different temperatures for 1 hour, cooled to room temperature, and then incubated with exponential A. baumannii 3437 in 20 mM Tris-HCl (pH 7.4) at a final concentration of 50 μg/ml at 37°C for 1 hour. Viable bacteria are counted after each treatment by plating serial dilutions on LB agar. Dash lines represent the limit of detection and data below the limit of detection is not shown.

Mechanism of action of PHAb10 and PHAb11 on Gram-negative and Gram-positive bacteria.

(a) Schematic representation of PHAb10 and PHAb11 domains. (b) Bactericidal activity of peptides derived from PHAb10 and PHAb11. Bacterial cells are treated with 50 μg/ml P10-CP, P11-NP, or P11-CP in 20 mM Tris-HCl at 37°C for 1 hour. (c-d) Bactericidal activity of different truncations of PHAb10 and PHAb11. Exponential A. baumannii 3437 cells are incubated with 50 μg/ml of each truncated fragment in 20 mM Tris-HCl for 1 hour at 37°C. Dash lines represent the limit of detection and data below the limit of detection is not shown. (e) Peptidoglycan hydrolytic activity of different truncations of PHAb10 and PHAb11. 0.1 μg of each truncated fragment is dropped onto autoclaved lawns of A. baumannii 3437 and S. aureus N315 and incubated at 37°C for 4 h. The groups treated with an equal volume of 20 mM Tris-HCl served as controls. (f) Schematic bactericidal mechanisms of PHAb10 against Gram-negative and Gram-positive bacteria.

PHAb10 and PHAb11 are thermostable dimers.

(a) Overall structure of PHAb8 monomer. (b) Dimeric structure of PHAb10. Chain A is colored in green, Chain B is colored in magenta, and the gray stick denotes the rotation axis. The electrostatic surface of PHAb10 with two potential substrate binding cavities are shown (negative = red; positive = blue). (c) Dimeric structure of PHAb11. Chain A is colored in wheat, Chain B is colored in green-cyan, and the red stick denotes the rotation axis. The electrostatic surface of PHAb11 with two potential substrate binding cavities is also shown (negative = red; positive = blue). (d) Superimposition of monomeric PHAb8, PHAb10, and PHAb11. PHAb8 is shown in light blue, PHAb10 in green, and PHAb11 in wheat. Residues involved in the catalytic triad are shown in sticks. (e-f) Residual bactericidal activity of PHAb10, PHAb11, and their truncation variants. Each truncation variant is treated at 100°C for 1 hour, stored at 25°C for 1 hour, and then tested for bactericidal activity by a log-killing assay. Dash lines represent the limit of detection and data below the limit of detection is not shown. (g) Thermal unfolding curves of PHAb8, PHAb10, and PHAb11 from 20 to 100°C as determined by nanoDSF. Values on the y-axis represent the first derivative of the fluorescence ratio at 350 nm and 330 nm. Peaks represent the transition temperature of each protein. (h-j) Circular dichroism spectra of PHAb8 (h), PHAb10 (i), and PHAb11 (j) at 90°C and temperatures close to their transition temperatures. (k-o) Circular dichroism spectra of PHAb10, PHAb11, and their truncation variants before and after heat treatment. Each domain fragment is treated at 100°C for 1 hour, stored at 25°C for additional hour prior to CD detection.

Folding-refolding thermodynamics of PHAb10.

(a) Intermolecular interactions of PHAb10 dimer. The residues involved are shown as sticks. (b) SDS-PAGE analysis of PHAb10 with or without chemical cross-linker. (c) Native-PAGE analysis of PHAb10 and its variants. Marker: the upper band represents BSA (66.4 kDa), and the lower band represents lysozyme (14 kDa). (d) Bactericidal activity of PHAb10 and its variants before and after heat treatment. Each protein is incubated for 1 hour at 4 or 100°C, stored at 25°C for an additional 1 hour, and then examined for bactericidal activity in 20 mM Tris-HCl (pH 7.4) at a concentration of 50 μg/ml against exponential A. baumannii 3437 cells for 1 hour. Dash lines represent the limit of detection and data below the limit of detection is not shown. Data are analyzed by two-tailed Student’s t-tests. ns: statistically not significant; *: p<0.05. (e-f) Circular dichroism spectra of PHAb10 and its mutants before and after treatment at 100°C for 1 hour. (g) Zipper model showing the thermodynamics of PHAb10 dimer.

PHAb10 shows high efficacy in mouse infection models.

(a) Experimental schema for burn wound infection model. Burn wounds produced by exposing naked skin to 65°C water for 12 s was infected with 10 µl 5×108 cfu/ml of A. baumannii 3437. At 24-hour post-colonization, mice are treated with 5 or 10 µg/mouse PHAb10 (10 µl; n = 5), 4 µg/mouse minocycline (10 µl; n = 5), or an equal volume of Tris buffer (10 µl; n = 5). Four hours post-treatment, viable bacteria on the burn wound skin is collected and counted. (b) Viable A. baumannii collected from burn wound skin after each treatment. The number of viable cells in each group is normalized and compared with the number of viable cells treated with Tris buffer by ANOVA. ns: statistically not significant; *: p<0.05; **: p<0.01. (c) Experimental schema for abscess model. Mice are infected hypodermically with 25 μl 5×108 cfu/ml of A. baumannii 3437 on the right side of the back dorsum. Twenty-four hours after infection, mice are injected hypodermically with 10 µg/mouse PHAb10 (10 µl; n = 6), 4 µg/mouse minocycline (10 µl; n = 7), or an equal volume of Tris buffer (10 µl; n = 8). Each group is injected subcutaneously once a day for 5 consecutive days. Viable bacteria in site of infection are counted 24 hour after the last dose. (d) Viable A. baumannii collected from skin after each treatment. The number of viable cells from each group is normalized and compared with the number of viable cells treated by the Tris buffer by ANOVA. ns: statistically not significant; **: p<0.01. (e) Changes in mice body weight per treatment group.

Physicochemical properties of putative antimicrobial peptides in PHAb10 and PHAb11

Data collection and refinement statistics of PHAb8, PHAb10, and PHAb11 structures

T4L-like lysozymes with characterized structures

Intermolecular interactions of PHAb10 dimer

PHAb10 variants

Bacterial strains used in this work.

Primers used in this study.

Workflow of an integrated system for the exploitation of peptidoglycan hydrolases.

Three distinct phases of the workflow were highlighted in different colors: (Ⅰ) Screening of peptidoglycan hydrolases using cationic peptides P307 as templates (yellow). (Ⅱ) Expression and purification of protein (blue). (Ⅲ) Biochemical characterization (green).

Selection of five putative peptidoglycan hydrolases.

(a) Phylogenetic tree of A. baumannii peptidoglycan hydrolases homolog to P307. (b) Multiple sequence alignment of PHAb7, PHAb8, PHAb9, PHAb10, PHAb11, PlyF307, and AcLys using MEGAX software and ClustalW algorithm. Secondary structures were annotated by Espript 3.0 website.

Testing of bactericidal activity of screened putative peptidoglycan hydrolases.

(a) Purified proteins were analyzed on a 12% SDS-PAGE gel. M: standard protein marker. (b) Halos of five PHAbs on A. baumannii 3437 lawns. The concentrations of each peptidoglycan hydrolase were 2.5 μg/ml, 5 μg/ml, and 10 μg/ml. (c) pH stability of PHAb10 and PHAb11. Log-phase A. baumannii 3437 cells were incubated with 50 μg/ml peptidoglycan hydrolases at the indicated pH conditions for 1 hour at 37°C. pH buffer: 20 mM Boric acid mixed with 20 mM phosphoric acid in equal volumes and adjusted to different pH values with HCl. (d) Log-phase A. baumannii 3437 cells incubated with 50 μg/ml of each peptidoglycan hydrolase at different concentrations of NaCl (left) and urea (right) for 1 hour at 37°C in 20 mM Tris-HCl (pH 7.4). (e) Relative activity of five PHAbs against A. baumannii 3437 after 1 hour storage at different temperatures. In all cases, viable bacteria were counted by serial dilution and plating assays. Experiments were carried out in triplicate; error bars represent standard deviation.

Structural analysis of PHAb8, PHAb10, and PHAb11.

(a-c) Structures of monomeric PHAb8 (a), PHAb10 (b), and PHAb11 (c). (d) Structural superposition of PHAb8, PHAb10, PHAb11 and five classical T4L-like lysozymes that share high similarity from Dali search. PHAb8 is shown in light blue, PHAb10 in green, PHAb11 in wheat, AcLys in red, LysF1 in blue, SpmX-Mur-Ae in yellow, P22 in purple, and R21 in orange. (e-h) Structural alignment of PHAb8, PHAb10, PHAb11 with various lysozymes including HEWL (e, cyan), GEWL (f, deep salmon), phage lambda lytic transglycosylase (g, yellow) and T4 lysozyme (h, orange). Residues in the conservative catalytic triad are shown as sticks. (j) Scattering profiles of PHAb8, PHAb10, and PHAb11 at different temperatures determined by NanoDSF.

Prophage analysis of PHAbs.

PHAb8, PHAb10, and PHAb11 encoding sequences were retrieved from A. baumannii strain 9201, A. baumannii strain LWSS-03-02-11B, and A. baumannii strain ABUH797 and analyzed by PhageBoost tool. Results revealed that all three genes are indeed located within prophage regions.

Relative viability of HepG2 cells exposed to different concentrations of PHAb10.

Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin for 24 h. After the cells were exposed to a series of concentrations of PHAb10 (0, 6.25, 12.5, 25, 50, and 100 μg/ml) for another 24 h, the relative viability of the cells after each treatment was determined by the CCK-8 assay.