PlyNJ3: a three-domain endolysin with broad-spectrum anti-streptococcal activity and synergistic efficacy against multidrug-resistant Streptococcus suis

Abstract

Background

The global spread of multidrug-resistant (MDR) bacteria prompts the exploration of innovative antimicrobial strategies. Phage lysins—peptidoglycan hydrolases known for species-specific activity and low resistance potential—offer promising alternatives to conventional antibiotics. However, their typically narrow spectrum limits broad therapeutic application. To overcome this limitation, we leveraged the wide distribution of streptococcal mobilizable prophage (SMphage) families across Streptococcus species as a strategy to discover lysins with inherent broad-spectrum potential. Within these conserved prophages, we expressed PlyNJ3, an SMphage-derived endolysin homolog identified in S. suis, and evaluated its therapeutic efficacy in infection models.

Results

PlyNJ3 exhibited potent lytic activity against a broad panel of streptococci, including diverse S. suis clinical isolates (15 serotypes), 83.3% of S. uberis, and key strains of S. agalactiae, S. dysgalactiae, and S. pyogenes. Optimal bacteriolytic activity occurred at physiological temperature (37 °C) with dose-dependent efficacy. Electron microscopy revealed PlyNJ3-mediated cell wall perforation, cytoplasmic content release, and structural disintegration. In a murine invasive S. suis infection model, high-dose intraperitoneal PlyNJ3 (0.9 mg/head) conferred 87.5% survival, while synergism with ampicillin prolonged survival. Intramuscular administration in a murine thigh infection model enhanced bacterial clearance compared to intraperitoneal delivery. Phylogenetic and structural analysis confirmed conservation across SMphage-derived lysins, underpinning their broad streptococcal target potential.

Conclusions

Our findings establish SMphage-derived lysins as a conserved, therapeutically valuable class of antimicrobials with inherent broad-spectrum potential across diverse streptococcal pathogens. Building on the previously demonstrated in vivo efficacy of PlySK1249 against S. dysgalactiae and S. agalactiae, we show that its structural homolog, PlyNJ3, exhibits robust therapeutic activity against S. suis infections. Together, these results validate the SMphage lysin platform for generating precision-targeted agents against specific streptococcal infections.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-025-04975-7.

Background

The widespread use of antibiotics in livestock industries and clinical settings has accelerated the emergence of multidrug-resistant (MDR) bacteria, precipitating a global public health crisis [1, 2]. Among these threats, Gram-positive pathogens exhibit escalating resistance to conventional antibiotics, yet the pipeline for antibiotics targeting MDR strains remains critically constrained. This dual challenge underscores the urgent need for innovative antimicrobial strategies [3].

Bacteriophage endolysins—enzymes that degrade bacterial peptidoglycan (PG)—have emerged as promising alternatives to traditional antibiotics [4–6]. Their therapeutic potential stems from three key attributes: (1) a bacteriolytic mechanism associated with low resistance induction; (2) species/strain specificity that minimizes collateral damage to host microbiota; and (3) rapid bactericidal activity through localized PG hydrolysis [7]. Structurally, lysins targeting Gram-positive bacteria typically adopt a modular architecture, comprising an N-terminal enzymatic active domain (EAD) and a C-terminal cell wall-binding domain (CBD) [8]. EADs are classified by catalytic specificity (glycosidases, amidases, endopeptidases) [9], while CBDs confer species or strain specificity by recognizing unique PG motifs or associated surface structures, enabling targeted lysis [8]. In contrast, lysins targeting Gram-negative bacteria often adopt simpler, globular structures to facilitate outer membrane penetration [10–12].

Preclinical studies demonstrate lysins’ therapeutic efficacy against diverse antibiotic-resistant pathogens. For Gram-positive species, notable examples include S. suis (PlySs2) [13], S. pneumoniae (Cpl-1) [14], S. pyogenes (PlyC) [15], S. dysgalactiae (PlySK1249) [16], Bacillus anthracis (PlyG, PlyL) [17], and methicillin-resistant Staphylococcus aureus (MRSA) (LysGH15, LysK) [18, 19], while Gram-negative pathogens like Pseudomonas aeruginosa (PlyPa03) [20, 21] remain challenging due to their protective outer membrane [22, 23]. While clinical lysin development has focused predominantly on Staphylococcus (e.g., P128, SAL200, CF-301, Staphefekt SA.100) [24–27], the high clinical burden of MDR streptococci [28–30]—demands urgent attention. However, a major translational bottleneck for lysins is their typically narrow spectrum, restricting activity to specific species or strains and limiting utility against diverse streptococcal populations. This highlights the critical need for strategies to develop broad-spectrum lysins against these high-impact pathogens.

Our discovery strategy targets conserved endolysins from widely distributed streptococcal mobilizable prophage (SMphage) families across Streptococcus species. This approach is validated by PlySK1249—an SMphage-derived lysin with demonstrated in vivo efficacy against key pathogens like S. dysgalactiae and S. agalactiae [16]. Critically, this strategy holds significant promise for combating zoonotic threats, particularly S. suis—a major animal-derived pathogen causing severe human and livestock infections with rising antimicrobial resistance.

Applying this rationale, we characterized PlyNJ3, a highly homologous SMphage lysin (97% identity to PlySK1249) encoded within a S. suis prophage. We expressed recombinant PlyNJ3 and systematically evaluated its: (1) in vitro lytic activity against diverse streptococci (including multiple S. suis serotypes, S. uberis, S. agalactiae, S. dysgalactiae, and S. pyogenes); (2) its bacteriolytic dynamics under varying conditions, including dose-response, temperature sensitivity, time-kill kinetics, and subinhibitory effects, coupled with ultrastructural characterization via electron microscopy; and (3) its therapeutic efficacy in murine models of both invasive and localized S. suis infections. Additionally, we assessed structural features through domain modeling, evolutionary conservation via phylogenetic analysis, and potential synergy with β-lactam antibiotics. Our results establish PlyNJ3 as a potent therapeutic against its native S. suis and cross-reactive streptococci, directly validating the prophage distribution-guided strategy for discovering broad-spectrum lysins. This positions PlyNJ3, alongside PlySK1249, as exemplars of SMphage-derived lysins for precision therapy against MDR streptococcal infections within One Health frameworks.

Methods

Bacteria, plasmids, and growth conditions

Clinical streptococcal isolates obtained from large-scale farms of China during the National Antimicrobial Resistance Surveillance Program between 2016 and 2022 were used in this study [31]. All strains were cryopreserved at −80 °C. Escherichia coli DH5α and BL21(DE3) competent cells (Vazyme Biotech) and plasmid pET32a(+) were used for cloning and expression. Streptococcus strains were cultured in Todd-Hewitt Broth (THB) or on Todd-Hewitt Agar (THA) supplemented with 5% (v/v) calf serum at 37 °C. E. coli strains were grown in Luria-Bertani (LB) medium under standard conditions.

Genomic analysis and structural characterization of PlyNJ3

The S. suis NJ3 genome was sequenced using Illumina HiSeq 2500 and PacBio RS II platforms, and annotated using NCBI PGAP. The genomic DNA sequences of S. suis NJ3 has been deposited in GenBank database (GenBank: NZ_CP082203.1), Prophage-associated endolysin PlyNJ3 (protein_id: WP_222760833.1) was identified through BLASTp homology analysis. Domain architecture of PlyNJ3 was predicted via NCBI Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/cdd/). The amino acid sequence of PlyNJ3 was aligned with reported streptococcal phage lysins using ClustalW, and a phylogenetic tree was constructed via the neighbor-joining method in MEGA software (v11.0) with 1,000 bootstrap replicates. The three-dimensional homology model of PlyNJ3, generated via SWISS-MODE, was visualized and analyzed using PyMOL Molecular Graphics System.

Recombinant PlyNJ3 expression and purification

The PlyNJ3 gene was amplified from S. suis NJ3 genomic DNA using primers BamHI-PlyNJ3F: 5′-AGGGATCCGGAAAACATCTAGTCATTTGTG-3′ and SalI-PlyNJ3R: 5′-AGGTCGACTTATGATATTCTAAACCAACCTACAAC-3′. The PCR product was cloned into the expression vector pET-32a via BamHI/SalI restriction sites. The recombinant plasmid was transformed into E. coli DH5α competent cells and confirmed by sequencing. For protein overexpression, the verified plasmid was introduced into E. coli BL21 cells cultured in LB medium supplemented with 50 mg/L ampicillin. Protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at an OD₆₀₀ nm of 0.7, followed by 10-hour incubation at 16 °C with agitation (150 rpm). Cells were harvested by centrifugation, washed with phosphate-buffered saline (PBS), and lysed via ultrasonication. The soluble lysate was subjected to Ni²⁺-NTA affinity chromatography for recombinant protein purification. The imidazole concentrations in the lysis (20 mM), wash (50 mM), and elution (250 mM) buffers were optimized to reduce nonspecific binding. Aliquots (15 µL) of protein samples collected at each purification stage were mixed with SDS-PAGE loading buffer, denatured at 95 °C for 10 min, and resolved on a 10% SDS-polyacrylamide gel. Proteins were visualized by Coomassie Brilliant Blue staining. The purity of recombinant PlyNJ3 was assessed via SDS-PAGE (see Supplementary Fig. S1).

Lytic spectrum analysis

The lytic spectrum of PlyNJ3 was tested against 102 clinical isolates, including 67 S. suis, 11 S. agalactiae, 9 S. dysgalactiae, 6 S. uberis, 1 S. pyogenes, 1 E. faecalis, 1 E. faecium, 4 E. coli, 1 Salmonella enteritidis and 1 Klebsiella pneumoniae, using turbidity reduction (OD₆₀₀ nm). Bacterial suspensions (OD₆₀₀ nm = 1.0) were incubated with 100 mg/L PlyNJ3 at 37 °C for 30 min, with PBS-treated controls. Strains exhibiting > 50% reduction in OD₆₀₀ nm were categorized as susceptible and selected for validation via viable count assays.

For viable count validation, cultures of susceptible isolates (OD₆₀₀ nm = 1.0) were treated with 100 mg/L PlyNJ3 for 30 min. Parallel PBS-treated samples under identical conditions served as negative controls. Treated and untreated samples were serially diluted (10-fold), and 10 µL aliquots were spotted onto THA plates. After 18-hour incubation at 37 °C, viable colonies were enumerated. Bactericidal activity was quantified as the reduction in CFU/mL relative to controls.

Dose- and temperature-dependent activity

To assess the dose-dependent bactericidal activity, stationary-phase cultures (OD₆₀₀ = 1.0) of two multidrug-resistant S. suis clinical isolates - SC070731 and GMJ14 (both demonstrating resistance to macrolides, tetracyclines, and β-lactam antibiotics) were treated with PlyNJ3 at graded concentrations (100, 50, 25, 12.5, and 6.25 mg/L) for 1 h at 37 °C. Optical density measurements (OD₆₀₀ nm) were recorded at 5-minute intervals using a microplate spectrophotometer (Synergy H1, BioTek). Parallel controls were treated with equal volumes of PBS under the same conditions.

To evaluate the temperature-dependent lytic efficacy, stationary-phase suspensions of S. suis SC070731 and GMJ14 (OD₆₀₀ nm = 1.0) were exposed to 100 mg/L PlyNJ3 at 20, 28, 37, and 45 °C for 30 min. Initial (t = 0 min) and final (t = 30 min) OD₆₀₀ nm values were measured to calculate the percentage turbidity reduction. Control assays were performed with PBS at each temperature.

Time-kill kinetics

To characterize the time-kill kinetics of PlyNJ3 against S. suis isolates SC070731 and GMJ14, logarithmic-phase bacterial cultures (OD₆₀₀ nm = 0.6) were treated with 100 mg/L PlyNJ3 at 37 °C. Aliquots (100 µL) were collected at 2, 5, 10, 30, 60, and 120 min post-treatment, serially diluted (10-fold in sterile saline), and plated on THA. Plates were incubated at 37 °C for 24 h, after which colony-forming units (CFU/mL) were enumerated. Parallel controls were treated with equal volumes of PBS under the same conditions. Bactericidal activity was quantified as the reduction in CFU/mL relative to untreated controls.

Minimum inhibitory concentration (MIC) assays

The MICs of PlyNJ3 was determined using a broth microdilution-based growth curve. Briefly, overnight cultures of S. suis clinical isolates were diluted to ~ 1 × 10⁵ CFU/mL in MH broth. Bacterial suspensions were added to 96-well microplates containing two-fold serial dilutions of PlyNJ3 (ranging from 2 to 1024 mg/L). Plates were incubated at 37 °C in a microplate reader (Synergy H1, BioTek), and OD₆₀₀ was continuously monitored at 1 h intervals over 24 h. Growth curves were plotted and compared to untreated controls. MIC is defined as the minimum inhibitory concentration.

Sublytic effects

To evaluate the growth-modulatory effects of PlyNJ3 on S. suis SC070731 and GMJ14, lag-phase bacterial cultures (OD₆₀₀ nm = 0.1) were treated with PlyNJ3 at subinhibitory to inhibitory concentrations (1/8×MIC, 1/4×MIC, 1/2×MIC, and 1×MIC) for 12 h at 37 °C. Optical density (OD₆₀₀ nm) was monitored at 30-minute intervals using a spectrophotometer (Synergy H1, BioTek), and growth curves were generated to assess bacteriostatic or bacteriolytic activity. Parallel control groups received equivalent volumes of phosphate-buffered saline under identical conditions.

Electron microscopy (SEM and TEM)

Logarithmic-phase S. suis SC070731 cultures (OD₆₀₀ nm = 0.4) were harvested by centrifugation (3,000 × g, 10 min, 4 °C) and washed three times with sterile phosphate-buffered saline (PBS, pH 7.4). Bacterial pellets were resuspended in PBS and divided into experimental and control groups. The experimental group was treated with 100 mg/L PlyNJ3 for 30 min at 37 °C, while the control group received an equivalent volume of PBS under identical conditions. Following incubation, both groups were centrifuged (3,000 × g, 3 min), and pellets were washed three times with PBS to remove residual lysin. Final pellets were fixed with 1 mL of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 24 h at 4 °C. The processed samples were analyzed using scanning electron microscopy (SEM; Hitachi SU8010) and transmission electron microscopy (TEM; Hitachi HT-7800) at Zhuanbo Testing Technology Co., Ltd.

Murine invasive S. suis infection model

Female SPF-grade BALB/c mice (4–6 weeks old) were intraperitoneally injected with graded doses of the highly virulent serotype 2 S. suis SC070731 (5 × 10⁶, 1 × 10⁷, 5 × 10⁸, and 1 × 10⁹ CFU). Observations were conducted every 12 h for 5 days. A control group received phosphate-buffered saline (PBS), while a blank group remained untreated.

The therapeutic efficacy of PlyNJ3 was evaluated in a mouse invasive S. suis infection model induced by intraperitoneal (i.p.) injection of 1 × 10⁸ CFU of the S. suis SC070731. One hour post-infection, mice were treated with varying concentrations of PlyNJ3 and ampicillin as the reference antibiotic:

• Group 1: Untreated control.

• Groups 2–4: PlyNJ3 (0.1, 0.5, 0.9 mg/head).

• Group 5: Ampicillin (0.9 mg/head))

• Group 6: PlyNJ3 (0.5 mg/head)) + ampicillin (0.9 mg/head))

Survival was monitored for 5 days (n = 8/group).

Murine thigh infection model

For the S. suis strain GMJ14, which exhibited low virulence, bacterial load quantification in thigh muscle tissue was used to evaluate the therapeutic efficacy of PlyNJ3 in vivo. A murine thigh infection model was established using either intramuscular (i.m.) or intraperitoneal (i.p.) injection of S. suis GMJ14 (1 × 10⁶ CFU). Two hours post-infection, mice received PlyNJ3 or ampicillin treatment via the same route as the infection, allowing route-matched evaluation of therapeutic efficacy, with daily treatments administered for 3 days. Female SPF-grade BALB/c mice (4–6 weeks old) were randomly assigned to five groups (n = 5 per group):

• Group 7: Untreated control.

• Group 8: 0.5 mg lysin/mouse (i.m.).

• Group 9: 0.5 mg lysin/mouse (i.p.).

• Group 10: 0.9 mg ampicillin/mouse (i.m.).

• Group 11: 0.9 mg ampicillin/mouse (i.p.).

For bacterial burden analysis, mice were euthanized and thigh muscle tissues were aseptically excised. Approximately 100 mg of tissue was placed in sterile prefilled homogenization tubes containing stainless steel beads and 1 mL phosphate-buffered saline (PBS) at 6.0 m/s for 60 s. Homogenates were serially diluted and plated on THA for CFU quantification 3 days post-treatment (n = 5/group).

Statistical analysis

All statistical analyses were conducted using GraphPad Prism v9.0. For comparisons between two independent groups, an unpaired Student’s t-test was used. For comparisons involving three or more groups, one-way analysis of variance (ANOVA) was performed, followed by Tukey’s multiple comparison test to identify significant differences between specific group pairs. Results with p-values less than 0.05 were considered statistically significant.

Results

Structural and evolutionary insights into PlyNJ3

The genomic DNA sequences of S. suis prophage ΦNJ3 identified PlyNJ3, a 490-amino acid endolysin with a unique tripartite architecture (Fig. 1A). Conserved domain analysis revealed that PlyNJ3 comprises an N-terminal N-acetylmuramoyl-L-alanine amidase (MurNAc-LAA) domain (residues 9–170), a central LysM cell wall-binding domain (residues 260–312), and a C-terminal cysteine-histidine-dependent amidohydrolases/peptidase (CHAP) domain (residues 351–433) (Fig. 1A). Homology modeling of the three domains of PlyNJ3 was performed individually using SWISS-MODEL. Domain-specific templates were selected for CHAP, MurNAc-LAA, and LysM based on homology to known structures. For the LysM domain, the crystal structure of a S. suis peptidoglycan-binding domain (PDB: A0A4T2GW72.1.A) was used. The CHAP domain adopted a β-α-β supersecondary structure essential for catalysis, the MurNAc-LAA domain displayed α-helix/β-sheet folds ensuring stability, and the LysM domain formed a β-α-α-β fold enabling dimerization and enhanced substrate binding (Fig. 1B). Phylogenetic analysis of streptococcal phage lysins clustered PlyNJ3 with S. dysgalactiae and S. suis lysins (PlySK1249, Ly7917, PlySs2) from SMphages, suggesting evolutionary conservation among lysins targeting zoonotic streptococci (Fig. 1C).

Fig. 1.

Structural and phylogenetic characterization of PlyNJ3 lysin. A Schematic diagram of the domain organization of the PlyNJ3 lysin. B Structure prediction of the PlyNJ3 lysin using the SWISS-MODE approach. C Phylogenetic analysis of PlyNJ3 lysin. The amino acid of PlyNJ3 lysin was analyzed by MEGA. The protein sequence numbers used in the phylogenetic analysis are as follows: WP_222760833.1, EGL49245.1, AGF87477.1, AGF87539.1, AAM79913.1, WP_000405191.1, Q9Z4J8, ACB88204.1, YP_004306947.1, ABB02702.1, AAN28166.2 and AAR99416.1. The numbers indicate percent amino acid sequence identity between endolysins

Broad-spectrum anti-streptococcal activity

The lytic spectrum of PlyNJ3 was evaluated through spectrophotometric analysis (100 mg/L treatment for 30 min at OD₆₀₀ = 1.0) and Complementary colony-forming unit (CFU) counting assays, demonstrating potent bacteriolytic activity against S. suis (67.16%, 45/67 clinical isolates), including 10 strains with > 90% bactericidal efficacy. Significant bacteriolytic activity was also observed against S. uberis (83.33%, 5/6 isolates; 3 strains > 90% efficacy), S. dysgalactiae (33.33%, 3/9), and S. agalactiae (18.18%, 2/11), while showing limited activity against S. pyogenes (1 susceptible strain) (Fig. 2A). PlyNJ3 exhibited serotype-independent efficacy across diverse S. suis serotypes (types 2, 9, 12, 16, 21, 29, 30, 31 and NCL1, 3, 7, 8, 10, 23, 24;) (Fig. 2B). Notably, no significant bactericidal effects were detected against non-streptococcal Gram-positive bacteria or any tested Gram-negative species, indicating broad anti-streptococcal activity with particularly high efficacy against S. suis and S. uberis.

Fig. 2.

Range of activity of PlyNJ3 lysin. A PlyNJ3 lysin activity against streptococcal, enterococcal (E. faecalis SF23-1 and E. faecium QF31), and Gram-negative (Salmonella enteritis S1, Klebsiella pneumoniae 9 − 1, and Escherichia coli isolates) bacterial was measured as the rate of decrease in OD. Shaded areas indicate rates of decrease in OD values greater than 50%. B PlyNJ3 activity against streptococcal was measured as the rate of decrease in CFU

In vitro bacteriolytic properties

Dose-dependent bacteriolysis

Turbidity reduction assays determined the optimal lytic concentration of PlyNJ3 against two multidrug-resistant S. suis clinical isolates - SC070731 and GMJ14 (both demonstrating resistance to macrolides, tetracyclines, and β-lactam antibiotics). High-concentration PlyNJ3 treatment demonstrated enhanced lytic capacity, reducing the OD₆₀₀ nm ratio to approximately 0.45 after 60 min, while untreated controls maintained stable turbidity (Fig. 3. AB).

Fig. 3.

Bactericidal efficacy of PlyNJ3 against clinical S. suis. Turbidity reduction assay of PlyNJ3 at different concentrations against S. suis SC070731 (A) and GMJ14 (B). C Effect of temperature on PlyNJ3 lysin activity. The bars indicate the decrease in turbidity of the two representative clinical strains SC070731 and GMJ14 suspensions after incubation with the PlyNJ3 lysin at each of the indicated temperatures. D Time-kill assay of PlyNJ3 against S. suis SC070731 and GMJ14. Growth inhibition of S. suis SC070731 (E) and GMJ14 (F) by PlyNJ3. (Data represent the mean ± SD of three technical replicates from two independent biological experiments.)

Temperature sensitivity

Temperature-dependent lytic activity assays revealed maximal turbidity reduction at 37 °C, indicating optimal PlyNJ3 activity under this condition. High enzyme efficiency was not observed at other tested temperatures, further demonstrating that 37 °C is the optimal temperature for PlyNJ3-mediated bacterial lysis (Fig. 3C).

Time-kill kinetics

Time-kill assays demonstrated rapid bactericidal activity of PlyNJ3. A 2- to 4-fold decrease in CFU/mL was observed within 5 min of exposure, with gradual reduction in bacterial survival at subsequent time points. These reductions exceeded 99% within 30 min, suggesting that PlyNJ3 exhibited bactericidal rather than bacteriostatic activity, consistent with minimum bactericidal concentration (MBC) criteria. These kinetic data were consistent with real-time turbidity reduction curves, confirming the rapid and sustained bacteriolytic activity of PlyNJ3 (Fig. 3D).

Subinhibitory effects

Bacterial growth curves under varying PlyNJ3 concentrations revealed dose-dependent inhibitory effects. The minimum inhibitory concentration (MIC) of PlyNJ3 for S. suis SC070731 and GMJ14 was 256 mg/L. At high concentrations, PlyNJ3 delayed the logarithmic phase by 2–4 h compared to untreated controls, and treated cultures reached stationary phase with significantly reduced biomass accumulation. These findings suggest dual mechanisms of action: direct bacteriolysis at lytic concentrations and bacteriostatic interference with growth kinetics at lower doses (Fig. 3E, F).

Ultrastructural evidence of lysis

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses confirmed that PlyNJ3-induced turbidity reduction resulted from cell lysis and subsequent bacterial death. SEM imaging revealed severe membrane damage in PlyNJ3-treated S. suis cells, characterized by structural collapse, cellular debris, and loss of surface integrity (Fig. 4A, B). In contrast, untreated controls retained their typical diplococcal morphology with smooth, intact surfaces. TEM analysis further demonstrated ultrastructural disruptions in PlyNJ3-treated bacteria, including extensive envelope perforations, cytoplasmic extrusion, and complete dissolution of membrane architecture (Fig. 4C, D). Control cells maintained intact cytoplasmic membranes with preserved peptidoglycan layers and densely packed intracellular contents.

Fig. 4.

Electron microscope images of S. suis SC070731. Observations were made by scanning electron microscopy (SEM). SEM/TEM imaging was performed after treating S. suis with PlyNJ3 at 100 mg/L, a concentration corresponding to bactericidal levels based on MIC and time-kill results. A, B and transmission electron microscopy (TEM) (C, D) for the untreated group (A, C) and plyNJ3-treated group (B, D), respectively

In vivo therapeutic efficacy

Invasive S. suis infection model

To evaluate the protective efficacy of PlyNJ3 in vivo, a murine infection model was established via intraperitoneal (i.p.) injection of the hypervirulent S. suis strain SC070731 (serotype 2) (Fig. 5A). Dose optimization experiments revealed that a challenge dose of 1 × 10⁸ CFU induced severe clinical signs (e.g., hunched posture, piloerection, hypothermia) and 100% mortality within 12 h post-infection (Fig. 5C).

Fig. 5.

A, B In vivo therapeutic efficacy of PlyNJ3 in a mouse model of S. suis infection. C Survival curves of mice injected intraperitoneally with different doses of S. suis SC070731. D Therapeutic efficacy of PlyNJ3 and ampicillin administration. G1: Negative control, G2: 0.1 mg lysin/mouse, G3: 0.5 mg lysin/mouse, G4: 0.9 mg lysin/mouse, G5: 0.9 mg ampicillin/mouse, G6: 0.5 mg lysin + 0.9 mg ampicillin/mouse; Each group contained eight mice (n=8/group). E Bacterial clearance of PlyNJ3 and ampicillin thigh muscle tissue. G7: Negative control, G8: 0.5 mg lysin/mouse (i.m.), G9: 0.5 mg lysin/mouse (i.p.), G10: 0.9 mg ampicillin/mouse (i.m.), G11: 0.9 mg ampicillin/mouse (i.p.); Each group contained five mice (n=5/group). Statistical comparisons were performed using one-way ANOVA followed by Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001

Mice infected with 1 × 10⁸ CFU of S. suis SC070731 were treated with PlyNJ3 via i.p. injection at four distinct doses. High-dose (0.9 mg/head) PlyNJ3 treatment significantly improved survival rates to 87.5%, whereas low-dose regimens delayed mortality onset by 8–12 h and moderately increased survival by 30% compared to untreated controls. Notably, coadministration of PlyNJ3 with ampicillin demonstrated synergistic enhancement of therapeutic outcomes, achieving 87.5% survival—a 12.5% improvement over ampicillin monotherapy. This combinatorial efficacy highlights the potential for integrating bacteriophage-derived lysins with conventional antibiotics to overcome bacterial resistance and enhance antimicrobial potency (Fig. 5D).

Thigh infection model

Quantification of bacterial load in thigh muscle tissue post-euthanasia revealed that PlyNJ3 treatment significantly reduced S. suis GMJ14 colonization, a low-virulence but multidrug-resistant strain (showing resistance to macrolides, tetracyclines, and β-lactam antibiotics including penicillins) (Fig. 5B). Route-matched intramuscular injection of PlyNJ3 showed greater efficacy than the corresponding intraperitoneal injection, confirming that the delivery route critically influences therapeutic outcome. (Fig. 5E). These results demonstrate that localized intramuscular delivery optimizes bactericidal activity and tissue penetration (Fig. 5B, E).

The results demonstrate that PlyNJ3 exerts a route-dependent protective effect against S. suis infection, with localized intramuscular delivery maximizing bactericidal activity and enhancing tissue penetration.

Discussion

The global AMR crisis has underscored the urgent need for innovative therapeutic strategies to combat multidrug-resistant bacterial pathogens [7]. Phage endolysins, distinguished by their unique capacity to hydrolyze peptidoglycan bonds while minimizing resistance development, represent a significant advancement in antimicrobial therapy [32]. This study validates a platform strategy that utilizes conserved lysins from broadly distributed SMphages to overcome the narrow-spectrum limitation that has impeded the therapeutic use of lysins.

PlyNJ3 exemplifies this SMphage lysins family, sharing 97% identity and an identical tripartite architectural structure (comprising CHAP, MurNAc-LAA, and LysM domains) with PlySK1249, a lysin proven effective against S. dysgalactiae and S. agalactiae [16]. This conservation across species reflects evolutionary selection for a robust lytic mechanism with innate broad-spectrum potential. Our functional extension to S. suis—a significant zoonotic pathogen—illustrates the platform’s utility across streptococcal infections. The tripartite domains enable a synergistic “dual-key” mechanism (simultaneous cleavage of D-Ala-Gly cross-bridges and MurNAc-L-Ala bonds; Fig. 1B) [33–35]. This dual action not only broadens the lytic spectrum but also reduces the likelihood of resistance emergence—a key advantage over traditional antibiotics and single-domain lysins [36]. Consistently, PlyNJ3 showed remarkable activity against S. suis isolates across multiple serotypes and was comparably effective against S. uberis, S. agalactiae, and other streptococcal pathogens (Fig. 2). This serotype-independent activity is attributed to its targeting of evolutionarily conserved peptidoglycan motifs, particularly the cross-bridge structure shared among streptococci [37].

However, it is important to note that reduced susceptibility in some S. suis strains suggests that variations in capsule composition, surface charge, or peptidoglycan modifications may impede domain accessibility. These variations warrant further investigation. Unlike typical lysins constrained by species specificity [38], PlyNJ3’s broad streptococcal coverage positions it as a versatile therapeutic option for both localized and systemic infections. Its optimal activity at 37 °C also enhances its clinical relevance at sites where streptococci commonly colonize.

Ultrastructural evidence from SEM/TEM imaging provides mechanistic clarity: PlyNJ3 induces rapid cell wall perforation, cytoplasmic leakage, and complete structural collapse (Fig. 4A–D). These observations correlate precisely with enzymatic activity assays, confirming that bacteriolysis—not bacteriostasis—drives its antimicrobial effects. Notably, subinhibitory concentrations delayed bacterial growth by 2–4 h (Fig. 3E, F), suggesting potential applications in infection prevention or combination therapies.

In vivo studies revealed two critical therapeutic advantages. First, PlyNJ3 monotherapy achieved 87.5% survival in lethal invasive S. suis infection model—a performance surpassing ampicillin alone (Fig. 5D). Second, its synergy with β-lactams reduced antibiotic doses while extending survival duration. The route-dependent efficacy (3.8-fold higher bacterial clearance via intramuscular vs. intraperitoneal delivery (Fig. 5E) highlights the importance of administration strategies for optimizing tissue penetration—a consideration often overlooked in lysin development.

While previous streptococcal lysins like PlySs2 (Phase III trials for S. aureus [39, 40]) and PlyC (broad anti-streptococcal activity [41, 42]) have advanced the field, PlyNJ3 addresses a translational gap for zoonotic streptococci. Its conserved multi-domain architecture may contribute to its broad activity spectrum, contrasting with the typically narrow coverage of single-domain lysins such as Pal and Cpl-1 [43–47]. The modular architecture of PlyNJ3 and PlySK1249—featuring multiple conserved catalytic and binding domains—may underlie their ability to target diverse streptococcal species. Their validated efficacy against zoonotic pathogens such as S. suis, S. dysgalactiae, and S. agalactiae, highlights its potential as a flexible antimicrobial solution where cross-species adaptation is a concern.

In conclusion, we establish SMphage lysins as a promising therapeutic platform against streptococcal infections. PlyNJ3 exemplifies how conserved prophage lysins can achieve functional expansion to S. suis, robust in vivo efficacy, and β-lactam synergy—positioning it for precision antimicrobial therapy. These findings highlight the translational potential of SMphage-derived lysins against streptococcal infections within One Health frameworks.

Authors’ contributions

J.H. conceived and designed the experiments. J.Z. and J.W, performed bioinformatics and statistical analyses. J.Z. P.H., S.G. and C.Y. generated strains and plasmids and other experiments. J.Z. and H.J. performed the animal experiments. J.Z. wrote the original draft. J.H., L.W., and X.W. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Ningxia Hui Autonomous Region (No. 2024AAC05098), the National Natural Science Foundation of China (32172917), the Fundamental Research Funds for the Central Universities (KYCXJC2025001), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Data availability

The datasets generated and/or analysed during the current study are available in the GenBank database. The complete genome sequence of strains S. suis NJ3 and the protein sequence accession numbers of PlyNJ3 are NZ_CP082203.1 and WP_222760833.1, respectively.

Declarations

Ethics approval and consent to participate

Animal experiments were carried out at the Laboratory Animal Center of Nanjing Agricultural University, according to the guidelines of Experimental Animal Management Measures of Jiangsu Province and were approved by the Laboratory Animal Monitoring Committee of Jiangsu Province, China [Permit number: SYXK (Su) 2021-0086].

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.