Biological characterization of the phage lysin AVPL and its efficiency against Aerococcus viridans-induced mastitis in a murine model

ABSTRACT

Aerococcus viridans (A. viridans) is an important opportunistic zoonotic pathogen that poses a potential threat to the animal husbandry industry, such as cow mastitis, due to the widespread development of multidrug-resistant strains. Phage lysins have emerged as a promising alternative antibiotic treatment strategy. However, no lysins have been reported to treat A. viridans infections. In this study, the critical active domain and key active sites of the first A. viridans phage lysin AVPL were revealed. AVPL consists of an N-terminal N-acetylmuramoyl-L-alanine amidase catalytic domain and a C-terminal binding domain comprising two conserved LysM. H40, N44, E52, W68, H147, T157, F60, F64, I77, N92, Q97, H159, V160, D161, and S42 were identified as key sites for maintaining the activity of the catalytic domain. The LysM motif plays a crucial role in binding AVPL to bacterial cell wall peptidoglycan. AVPL maintains stable activity in the temperature range of 4–45°C and pH range of 4–10, and its activity is independent of the presence of metal ions. In vitro, the bactericidal effect of AVPL showed efficient bactericidal activity in milk samples, with 2 µg/mL of AVPL reducing A. viridans by approximately 2 Log₁₀ in 1 h. Furthermore, a single dose (25 µg) of lysin AVPL significantly reduces bacterial load (approximately 2 Log₁₀₎ in the mammary gland of mice, improves mastitis pathology, and reduces the concentration of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in mammary tissue. Overall, this work provides a novel alternative therapeutic drug for mastitis induced by multidrug-resistant A. viridans.

IMPORTANCE

A. viridans is a zoonotic pathogen known to cause various diseases, including mastitis in dairy cows. In recent years, there has been an increase in antibiotic-resistant or multidrug-resistant strains of this pathogen. Phage lysins are an effective approach to treating infections caused by multidrug-resistant strains. This study revealed the biological properties and key active sites of the first A. viridans phage lysin named AVPL. AVPL can effectively kill multidrug-resistant A. viridans in pasteurized whole milk. Importantly, 25 μg AVPL significantly alleviates the symptoms of mouse mastitis induced by A. viridans. Overall, our results demonstrate the potential of lysin AVPL as an antimicrobial agent for the treatment of mastitis caused by A. viridans.

INTRODUCTION

As a zoonotic pathogen, Aerococcus viridans is associated with urinary tract infections, arthritis, and endocarditis in humans (1). It is also responsible for bovine mastitis, swine pneumonia, swine meningitis, and bacteremia in clinical veterinary practice (2). Especially, in recent years, A. viridans has been successively isolated from raw milk samples of clinical and subclinical mastitic cows worldwide, causing serious economic losses as an emerging agent of dairy mastitis (3–7). Previously, A. viridans was found to be susceptible to commonly used antibiotics. However, with the widespread use of antibiotics, A. viridans has developed resistance not only to β-lactam antibiotics such as penicillin and ampicillin but also to many types of antibiotics including aminoglycosides, macrolides, lincomycins, and tetracyclines (8, 9). The development of antibiotic resistance has posed a serious threat to veterinary clinical practice and even human health (10). Although antibiotic therapy is still considered the preferred drug for combating A. viridans infections, it is necessary to find a potential alternative antibiotic that can effectively combat multidrug-resistant A. viridans.

Phage-derived lysin is an alternative or adjuvant to conventional antibiotics (11). Lysins lyse bacterial cell walls by direct contact without being affected by conventional antibiotic resistance, thus providing new perspectives for the prevention and treatment of disease (12). Most endolysins found in phages infected with Gram-positive bacteria are modular, consisting of a combination of enzymatically active structural domains (EADs) and cell wall binding domains (CBDs) (13). The investigation into these functional molecular details will not only enhance our understanding of the properties of this specific lysin but also contribute to the broader comprehension of this type of lysin. The efficacy of phage lysins against infections caused by various pathogenic bacteria has been evaluated in vitro, in mouse models, and in clinical practice. However, there are no reports on the A. viridans phage lysin bactericidal mechanism and treatment of A. viridans infections with phage lysin (13).

In a previous study, we isolated the first A. viridans phage vB_AviM_AVP, which has good bactericidal activity in vitro and in vivo (14, 15). We also found that the lysin AVPL encoded by vB_AviM_AVP has a cross-generic bactericidal efficacy against Streptococcus suis (S. suis) infections (16). However, the biological characteristics, antibacterial mechanism, and anti-A. viridans effect of AVPL in vivo are still unclear. Here, recombinant lysin AVPL was expressed and characterized, which efficiently lysed A. viridans in vitro, especially in milk. In addition, the predicted 3D structure, critical active domains, and key active sites of AVPL were revealed. Furthermore, the therapeutic potential of lysin AVPL was evaluated in vivo against mastitis induced by multidrug-resistant A. viridans in mice. This study indicates that the lysin AVPL may become a novel antibacterial drug against A. viridans infections.

RESULTS

Identification and purification of AVPL and 3D structure prediction of its truncated fragments

A search for putative conserved domains using the Position-Specific Iterated Basic Local Alignment Search Tool (PSI-BLAST) revealed that AVPL does not share identity with the published endolysin sequences. However, the N-terminal of AVPL (residues 32–162) has 59.17% identity over 48% coverage with N-acetylmuramoyl-L-alanine amidase, and the C-terminal (residues 186–428) contains two putative LysM domains (Fig. S1A), which are consistent with the modular structural composition of common lysins consisting of a catalytic structural domain and a cell wall-binding structural domain. Furthermore, genome annotation showed that AVPL is adjacent to the gene encoding holin. Based on the above findings, we hypothesized that AVPL, consisting of 594 amino acids, may encode the lysin of phage vB_AviM_AVP. AVPL, AVPL-C-EGFP, AVPL-B1-EGFP, AVPL-B2-EGFP, and AVPL-B1B2-EGFP were successfully expressed in E. coli BL-21(DE3), and the corresponding band sizes of the purified proteins on SDS-PAGE were approximately 60 kDa, 44 kDa, 40 kDa, 50 kDa, and 56 kDa, respectively(Fig. S1B) (16).

The 3D structures of the catalytic active domain and the two cell wall-binding active domains of AVPL were successfully modeled with Phyre2. The amidase-2 domain of AVPL (residues 1–185) was modeled with 100.0% confidence and 97% coverage by the single highest scoring template (PDB: 3lat), which is the crystal structure of Staphylococcus peptidoglycan hydrolase AmiE. However, the amino acid sequence identity between the two structural domains was only 39% (Fig. 1A). The modeling results revealed that the amidase-2 structural domain exhibited an αβαβββαβααα topology, consisting of five-stranded central β-sheets surrounded by six α-helices to a globular structure (Fig. 1D). These five β-sheets and six α-helices align well with AmiE, with a root mean square deviation (RMSD) of 1.70 Å. Nevertheless, at the positions of L5 and L1, amidase-2 of AVPL had one less α-helix and one less β-sheet than AmiE, respectively (Fig. 1E).

Fig 1.

Functional domain analysis and expression of AVPL. (A–C) The sequences alignment of amidase-2 (A), lysM1 (B), and lysM2 (C) domains with homologous proteins (c3latB: peptidoglycan hydrolase AmiE of staphylococcus; c4ivvA: lytic amidase LytA of Streptococcus pneumoniae; c4olsA: amidase-2 domain of lysin lysGH15; c4s3jc: spore cortex-lytic enzyme2 slel of B. cereus; c4uz2D: LysM domains from the putative2 nlpc/p60 d,l endopeptidase of Thermus thermophilus; and c4s3kA: spore cortex-2 lytic enzyme slel of B. megaterium). All alignments were generated using CLUSTAL W (http://www.genome.jp/tools-bin/clustalw). The figure was generated using ESPript (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). A schematic representation of the secondary structure elements of the corresponding structures is shown above the sequences. Key active sites are indicated by blue squares. (D) The overall structure of the amidase-2 domain (residues 1–185). α-helices and β-sheets are numbered. (E) Structural comparison of the amidase-2 domain of AVPL (green) and the staphylococcal peptidoglycan hydrolase AmiE (gray). (F) The structural model of the LysM1 domain. (G) The structural model of the LysM2 domain. The 3D structural model was established using PHYRE2 (17).

The LysM1 domain (residues 180–290) of AVPL was modeled with 99.3% confidence and 86% coverage using Bacillus cereus spore cortex-lytic enzyme slel (PDB:4S3J) as the highest scoring template, with only 32% identity of the two proteins (Fig. 1B). The 3D structure of the LysM1 domain contains three α-helices and two β-sheets (Fig. 1F). The predicted 3D structure of the LysM2 domain of AVPL (residues 291–495) was modeled using the T. thermophilus NlpC/P60 endopeptidase containing LysM (PDB:4XCM) as a template with 99.6% confidence and 50% coverage, which shares only approximately 27% sequence identity (Fig. 1C), and LysM2 consists of six α-helices and two β-sheets (Fig. 1G).

Enzymatic characteristics and the lytic activity of AVPL

To determine the drug resistance of the Aerococcus used in this study, drug susceptibility assays were performed. The results showed that all the strains had varying degrees of resistance (Table S2), with the highest rate of resistance to Methotrexate-sulfamethoxazole (14/15) and meager resistance to Vancomycin, Ciprofloxacin, and Ampicillin. P_1-1F₂, Xueru, Ezhong, and N15 resisted three or more antibiotics. In addition, all strains were susceptible to Meropenem. Multidrug-resistant A. viridans strain P_1-1F₂ was used in an ex vivo activity assay of AVPL.

AVPL at a final concentration of 50 µg/mL was used for the enzymatic characteristics assay. The AVPL maintains a stable high activity (>90% reduction in CFU/mL) in the range of 4°C–45°C, with a considerable drop (about 50%) in activity after heat treatment at 55°C and retention of 33% of the activity after 15 min of heat treatment at 75°C (Fig. 2A). The lytic activity was optimal at pH 6.0, with a 2.2 Log₁₀ reduction in viable cells compared with the control (no AVPL addition) (Fig. 2B). The NaCl concentration only slightly affected the lytic activity of AVPL, which maintained a relative activity of >80% in 800 mM NaCl (Fig. 2C). In addition, consistent with amidase-2 of the S. suis phage lysin Ply1228, a high concentration of ethylenediaminetetraacetic acid (EDTA) (100 mM) did not affect AVPL activity (Fig. 2D) (18).

Fig 2.

Lytic activity of AVPL. The effects of temperature from 4°C to 75°C (A), pH from 2 to 10 (B), NaCl concentration from 0 to 800 mM (C), and EDTA (D) on the activity of AVPL were detected. N.D., not detected. The log₁₀ CFU/mL maximum reduction was regarded as the highest activity, and the ratio of the maximum reduction to the initial bacterial load was defined as 100% activity. The percentage of activity observed under each condition was compared with the highest activity to generate values (% Rel. Activity) shown. (E) Lytic activity of AVPL (final concentration, 30 µg/mL) against different Aerococcus strains in vitro over 30 min. (F) Time-killing curve of different concentrations of AVPL on A. viridans P_1-1F₂. (G) Susceptibility of different generations of AVPL-treated P_1-1F₂ strains to AVPL. (H) The lytic activity of AVPL on A. viridans P_1-1F₂ in pasteurized whole milk [limit of detection (LOD) = 30 CFU/mL]. All experiments were conducted in the sterile phosphate buffer. P-value: * <0.05, ** <0.01, and *** <0.001 compared with the control group, ns, no significance. The values represent the mean ± SEM (n = 3).

A lysis test revealed that 30 µg/mL of AVPL was highly active against all the strains used in the study, including 14 strains of A. viridans and one strain of Aerococcus urinae. (Fig. 2E). The lysis curves indicated that the bactericidal effect had a time- and dose-dependent response, and killing efficacies increased with increasing enzyme concentrations, with CFU decreasing 2 Log₁₀ in 60 min after treatment with 50 µg/mL AVPL, whereas 300 µg/mL AVPL resulted in a reduction of approximately 4 Log₁₀ in 60 min (Fig. 2F). The AVPL exhibited similar bactericidal activity against each generation of strains exposed to AVPL, indicating that A. viridans P_1-1F₂ does not develop resistance to AVPL within 12 generations (Fig. 2G).

The AVPL activity was not affected by the composition in milk, and 12.5 µg/mL of AVPL was sufficient to reduce 2 × 10⁶ CFU/mL P_1-1F₂ in milk to undetectable within 3 h (Fig. 2H). In addition, 2 µg/mL of AVPL reduced the number of colonies by 3 Log₁₀ within 4 h.

Analysis of the activity of individual truncated fragments of AVPL

Individual domains of AVPL were fused with EGFP, and the involvement of each EGFP truncated domain in host cell wall binding was observed by spectrofluorophotometer and laser scanning confocal microscopy (LSCM). Measurement of fluorescence intensity revealed that the peak was detected at 510 nm for all samples after AVPL-B1-EGFP, AVPL-B2-EGFP, or AVPL-B1B2-EGFP treatment (Fig. S2). In contrast, no peak was observed after AVPL-C-EGFP treatment, indicating that AVPL-C-EGFP had no binding activity. The binding activity of the fusion proteins was further observed with LSCM. The green fluorescence of EGFP was detected at 488 nm in A. viridans cells treated with AVPL-B1-EGFP, AVPL-B2-EGFP, and AVPL-B1B2-EGFP, indicating the binding ability of AVPL-B1 and AVPL-B2 (Fig. 3A). In contrast, AVPL-C-EGFP was unable to bind to the cell surface.

Fig 3.

Functional analysis of the truncated fragments of AVPL. (A) Binding activity of truncated fragments of AVPL. Before visualization with LSCM, strains were stained with 20 µM Hoechst No. 33342 for 10 min at 37°C and incubated with the EGFP truncated domains for 20 min. Blue: localization at 405 nm and emitted by Hoechst No. 33342; green: localization at 488 nm and emitted by EGFP; and bright: normal light. The merge of blue and green indicates the colocalization of each recombinant protein with A. viridans P_1-1F₂. Scale bar, 20 µm. Protein profiles of the AVPL-C (B), AVPL-B1 (C), and AVPL-B2 (D) domains of AVPL were detected by SDS-PAGE. The lanes were loaded as follows: lane 1, molecular mass marker (Thermo Scientific, Waltham, MA, America, Cat: 26617); lane 2, crude extract of induced (1 mM IPTG at 16°C) E. coli BL21 cells; lane 3, supernatant after sonication; and lane 4, purified protein fraction eluted from Ni-NTA His•Bind slurry. (E) Lytic activity of truncated fragments of AVPL. (F) Location of residues at the periphery of the groove on the surface of the amidase-2 structure. The amidase-2 domain of AVPL was modeled based on AmiE (PDB: 3lat) as a template. Red: key residues; Green: non-key residues. (G) The bactericidal activity of AVPL containing different mutations against A. viridans P_1-1F₂. 100% activity = 1.9 Log₁₀. The values represent n = 3 biological replicates and mean ± SEM of triplicates.

The protein profiles of purified individual catalytic and binding domains are shown in Fig. 3B through D, with sizes of approximately 64 kDa (AVPL-C), 12 kDa (AVPL-B1), and 23 kDa (AVPL-B2), respectively. The results showed that both the catalytic and binding structural domains alone lost their bactericidal activity (Fig. 3E).

On the surface of the 3D structure of amidase-2 of AVPL, there is a deeply recessed region formed with β1, β4, and β2 as the bottom (Fig. 1D and 3F), which is also found in its homologous proteins including Staphylococcus peptidoglycan hydrolase AmiE, lytic amidase LytA of Streptococcus pneumoniae (PDB: 4IVV), and the amidase-2 domain of lysin lysGH15 (PDB: 4OLS). The structure of the groove and its high degree of conservation suggested that this is likely to be the key site for interacting with peptidoglycan. The active site residues of the catalytic center are fully conserved between LytA (residues Glu87 and His147) and AmiE (residues Glu119 and His177); thus, it is suggested that the catalytic mechanism may also apply to amidase-2 of AVPL (19).

In addition to the center of the groove, single-point mutations were performed on the sites around the groove to further explore other potential key active sites of AVPL (Fig. 3F). Mutants H40A, N44A, E52A, W68A, H147A, and T157A retained only 30%–42.5% bactericidal effectiveness compared with the native AVPL, whereas mutations F60A, F64A, I77A, N92A, Q97A, H159A, V160A, and D161A almost abolished the lytic activity of AVPL (Fig. 3G). The results revealed that residues F60, F64, I77, N92, Q97, H159, V160, and D161 are essential for AVPL lytic activity, whereas residues H40, N44, E52, W68, H147, and T157 are also involved. In addition, the activity of S42T decreased by 31.3%, indicating that S42 is also involved in amidase-2 domain activity.

AVPL has therapeutic effect on A. viridans-infected murine mammary glands

The bacterial loads in the mammary glands of the A. viridans-infected group 24 h post-infection was approximately 1.78 × 10⁵ CFU/g, whereas the number of colonies in the mammary glands of mice administered AVPL at 1 h post-infection was significantly lower (P < 0.01), reaching 2.26 × 10⁴ CFU/g (12.5 µg group), 1.89 × 10³ CFU/g (25 µg group), and 154.00 CFU/g (50 µg group), respectively (Fig. 4A). The antibiotic-treated groups also had significantly lower bacterial loads at 24 h post-infection (P < 0.01) but were not significantly different from the 50 µg AVPL-treated group.

Fig 4.

The therapeutic effect of AVPL on mouse mastitis. (A) Bacterial loads of A. viridans in the mammary gland. Mammary glands from different treatment groups were collected aseptically for colony counting 24 h post-infection (n = 6), data below LOD (30 CFU/g) are not shown. Cytokine concentrations of IL-1β (B), TNF-α (C), and IL-6 (D) in the breast tissues were detected. P-value: * <0.05, ** <0.01, and *** <0.001 compared with the buffer-treated group, ns, no significance. Data represent n = 3 biological replicates and mean ± SEM of triplicates. (E) Representative images of pathological observations. Hematoxylin and eosin (H&E) staining of mammary tissues of mice in each group. Tissues treated with AVPL only were used as safety controls. Scale bar = 200 µm.

The results of the concentration of inflammatory cytokines in the mammary gland are consistent with the observed reduction in the number of bacteria. IL-1β, TNF-α, and IL-6 concentrations were significantly elevated in the mammary tissue of the A. viridans-infected group 24 h post-infection (Fig. 4B through D). However, AVPL treatment attenuated mammary gland damage, with significant reductions in inflammatory cytokine concentrations in the 25 and 50 µg/gland groups, and similar effects in the antibiotic-treated groups as in the control group. In addition, cytokine concentrations in mammary tissue of the safety control group tended to be similar to those of the control group.

Pathological histological observations revealed that A. viridans infection resulted in significant thickening of the acinar wall with extensive inflammatory cell infiltration and disruption of acinar integrity (Fig. 4E). In contrast, the disruption of mammary cell acinar structures in the AVPL-treated group was attenuated, and the inflammatory cell infiltration was alleviated in a dose-dependent manner, although a small amount of neutrophil infiltration was still observed in the breast tissue at 24 h after treatment (Fig. 4E). The antibiotic-treated groups also showed significant improvement in pathology. In addition, no typical pathological damage was found in the safety control group, similar to the control group (Fig. 4E).

DISCUSSION

Although researchers have extensively studied the pathogenic bacteria that cause mastitis in dairy cows, attention has focused primarily on traditional pathogens or their therapeutic investigations. However, more potential mastitis-causing bacteria have been identified including A. viridans (20). Previous studies have rarely reported the impact of A. viridans in cases of mastitis. This may be related to the misidentification of A. viridans due to its morphological characteristics and biochemical properties similar to those of Staphylococci or Streptococci, leading to an underestimation of its pathogenicity (21). However, it is worth noting that data from milk samples reported from six commercial dairy farms in the United States in 2013 and one commercial dairy farm in China in 2017 revealed isolation rates of Aerococcus spp. second only to coagulase-negative staphylococci (20, 22).

There has been a lack of relevant testing and statistics in recent years, but some strains of A. viridans isolated from bovine clinical or subclinical mastitis cases have developed antibiotic resistance (4, 5, 7). Importantly, A. viridans strains have been demonstrated to adhere to and invade bovine mammary epithelial cells, and dairy cows infected with A. viridans have higher somatic cell counts (SCC) compared with healthy cows, with severe effects on milk yield and composition (3, 20). Multiple strains of A. viridans used in this study were isolated from the milk of cows with clinical mastitis, all of which exhibited varying degrees of resistance. Thus, A. viridans is an important pathogen that causes cow mastitis, which needs to be paid attention to by researchers and dairy cattle breeding-related personnel. Clearly, there is a need to develop new backup drugs to deal with multidrug-resistant A. viridans.

In a previous study, we successfully isolated the first A. viridans phage that demonstrated promising therapeutic effects against mastitis (15). In general, the phage-encoded lysins directly act on the pathogen-exposed peptidoglycan layer from the inside or the outside to exhibit rapid and effective bactericidal activity (23). Therefore, it is crucial to develop and investigate the potential applications of A. viridans phage lysins for treating A. viridans infections. This study represents the effort to identify and biochemically characterize the A. viridans phage-encoded lysin, as well as evaluate its efficacy in a mouse model of intramammary A. viridans infection.

The low amino acid identity (59.17%) of the lysin AVPL with other published sequences suggests that it is a novel phage lysin. The activity of A. viridans lysin AVPL as a peptidoglycan hydrolase was demonstrated in this study. The OD₆₀₀ of purified A. viridans peptidoglycan after exposure to AVPL (5 and 50 µg/mL) decreased rapidly from 0.9 to 0.2–0.3 by 1 h (Fig. S3). Consistent with other studies of phage lysin, the structural integrity of AVPL is indispensable for maintaining its bactericidal activity (24). Previous studies have shown that each of the six LysM modules in the Enterococcus faecalis autolysin AtlA binds peptidoglycan individually, and the affinity increases with the number of LysMs (25). AVPL encodes one catalytic domain and two binding domains; each of the two LysMs that comprise the CBD of AVPL can individually exert binding activity. Thus, the two LysM modules of AVPL may contribute to maintaining binding activity. Notably, compared with the phage vB_AviM_AVP, which encodes AVPL, lysin AVPL broadened the lysis spectrum against A. viridans and exhibited cross-generic bactericidal activity against S. sui (16). This may related to the function of the structural domains of the lysin AVPL, whose two CBD bind not only to A.viridnas but also to S. suis.

Although the low amino acids sequence identity with other reported lysins, the modeled structure of the amidase-2 domain of AVPL matches that of Staphylococcus peptidoglycan hydrolase AmiE, indicating that the same class of amidases from different phage lysins is conserved (26, 27). Metal ions are constituents of many metalloproteins, which exert catalytic or structural functions. The structure of AmiE from Staphylococcus peptidoglycan hydrolase reveals a zinc-binding site near the active site groove (Fig. S4), which is conserved in both the amidase LytA (PDB: 4IVV) and the amidase-2 domain of lysin lysGH15 (PDB: 4OLS). However, considering that the activity was not eliminated after EDTA treatment, the activity of AVPL is not dependent on Zn²⁺. Consistent with this, PGRP-Iα, which is homologous to LytA, does not bind Zn²⁺, whereas amidase-2 of AVPL exactly belongs to the PGRP superfamily (19). Mutational analysis identified multiple key amino acid sites located at the periphery of the active center groove and also affect amidase activity, suggesting that efficient binding between amidase-2 and peptidoglycan requires multiple polysaccharide residues for broad interaction (19). Collectively, these detailed investigations of the active domain of AVPL will also contribute to developing more potent lysins.

During clinical mastitis therapy, milk components may interfere with enzyme activity (28). As far as we know, few phage lysins have been developed that show strong antimicrobial activity during milk processing (29). However, it was encouraging that AVPL exhibited efficient lytic activity against A. viridans in pasteurized whole milk, representing potential clinical applications. Normally, milk is stored and transported at 1°C–4°C, and AVPL maintains good bactericidal activity at 1°C–4°C. Therefore, lysin AVPL can be used to kill A. viridans in milk during storage and transportation. All these excellent properties of AVPL are beneficial for the treatment of mastitis in dairy cows.

At present, although there have been some studies concerning the treatment of mastitis in dairy cows with lysins, most have been limited to in vitro sterilization and little attention has been paid to the role of A. viridans in mastitis (30). In this study, we successfully constructed a mouse mastitis model with multidrug-resistant A. viridans. Bacterial loads and inflammatory factor concentrations in the mammary glands of mice receiving single treatments with different doses of AVPL decreased. The attenuation of pathological damage and the therapeutic effects of the high-dose AVPL treatment group were also similar to those of the antibiotic-treated groups. These results were consistent with previous reports and again demonstrate that phage lysin is a promising therapy in the treatment of mastitis. Certainly, utilizing the unique advantages of phage lysins to generate chimeric lysins with a broader range of lytic activity seems promising and is also worthy of further research. Overall, however, the phage lysin AVPL holds promise as an effective tool for combating drug-resistant A. viridans infections.

MATERIALS AND METHODS

Animals

Female BALB/c pregnant mice (Specific-pathogen-free, 18–20 days of pregnancy) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). During the experimental period, the animal room was temperature-controlled with light-dark cycles. Feed and fresh water were available ad libitum.

Bacterial strains and culture conditions

All strains used in this study were laboratory preserved, with five A. viridans (P_1-1F₂, N14, M6-1, M13-1, and M19-1) isolated from fresh milk samples of cows with clinical mastitis (15). Specifically, a total of 81 milk samples were collected from 81 cows in four commercial dairy farms in Changchun, Jilin Province, China. The isolation methods and steps are briefly described below. Cows with clinical signs observed as stiffness and swelling of one or more udder quarters, gray-white milk with clots, and reduced milk production were selected to isolate causative organisms. The above tests and judgments were performed by veterinarians with management training and experience. After sterilizing the teats, the first three milk jets were discarded, and only one diseased udder quarter of each cow was selected for sampling. Milk samples (10 µL) were inoculated on Brain Heart Infusion (BHI) (Becton, Dickinson and Company, USA) agar containing 5% defibrinated sheep blood and incubated for 24 h at 37°C. A. viridans was initially screened based on colony morphology, α hemolytic reaction, and Gram staining (31). All suspected isolates were amplified and sequenced [Sangon Biotech (Shanghai) Co., Ltd. China] using 16 s rRNA primer pairs 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′), as well as specific primer pairs F (5′-GTGCTTGCACTTCTGACGTTAGC-3′) and R (5′-TGAGCCGTGGGCTTTCACAT-3′) (32). A. viridans were eventually isolated from five milk samples (each A. viridans was from a separate milk sample). Strains identified as A. viridans were stored at −80°C in glycerol (3:1, vol/vol).

All strains were incubated in BHI broth and shaken at 180 rpm for 12 h at 37°C. The solid culture medium was obtained by adding 1.5% agar as required. Aerococcus strains were tested for susceptibility to antibiotics using a Mérieux bacteria identification instrument (VITEK 2 Compact, BioMérieux, France) according to Clinical and Laboratory Standard Institute (CLSI) guideline (33).

Constructs, protein expression, and purification

The primer information for those used in this experiment is available in Table S1. A polymerase chain reaction amplified the full-length gene of lysin in phage vB_AviM_AVP (GenBank accession: MH729379) and was cloned into the pMCSG7 vector as previously reported (34). The amplified truncated catalytic domain AVPL-C (residues 30–170) was cloned into the pCold TF vector, and the binding domains AVPL-B1 (residues 180–290) and AVPL-B2 (residues 291–495) were cloned into the pET-15b vector. Positive cloned plasmids (containing 6 × His tag) were screened out. Construction of fusion genes consisting of truncated catalytic domain AVPL-C and binding domains AVPL-B1, AVPL-B2, and AVPL-B1B2 (residues 180–429) fragments with an enhanced green fluorescent protein (EGFP) was carried out, respectively, as described previously (16). The above plasmids containing the target genes were transformed into E. coli BL21 (DE3). AVPL and all recombinant truncated protein purification were performed as previously described (16). SDS-PAGE was used to verify the purity and size of the proteins.

Enzymatic characteristics and host range

A. viridans strains P_1-1F₂ were resuspended in phosphate buffer with various concentrations of NaCl (0, 50, 100, 150, 300, 500, and 750 mM) or different pH (2–10). All bacterial suspensions were exposed to 50 µg/mL AVPL and then incubated at 37°C for 30 min before colony counting. AVPL was incubated at different temperatures (4°C–75°C) for 15 min or treated with 100 mM EDTA followed by ultrafiltration (MWCO of 10 kd, 3,500 × g, 15 min) to remove the EDTA. The bacterial suspension was mixed with different temperature- or EDTA-treated AVPL at a final concentration of 50 µg/mL for 30 min at 37°C, and the colonies of surviving bacteria were counted on BHI agar plates. Controls were all treated with equal volumes of phosphate solution. All assays were repeated three times. The values represent n = 3 biological replicates and mean ± standard error of the means (SEM) of triplicates. Relative activity was expressed as a percentage of the maximal bactericidal activity.

The turbidity reduction assay was used to determine the lytic activity of AVPL against different A. strains. Bacteria were incubated to an OD_{600 nm} of 0.6 to 0.8, washed, and resuspended in phosphate buffer. The bacterial suspension was exposed to a final concentration of 30 µg/mL of AVPL and incubated at 37°C for 30 min before recording spectrophotometric reading. The bacteria were treated with phosphate buffer under the same conditions as a negative control. The experiment was performed in triplicate.

Determination of AVPL bactericidal assays

To evaluate the effect of concentration and time on the bactericidal effect of lysin AVPL, different concentrations (0, 50, 100, 200, and 300 µg/mL) of AVPL were incubated with cell suspensions of A.viridans strains P_1-1F₂ at 37°C for 1 h, and samples were taken at 10 min intervals for serial dilution in PBS solution for determination of viable counts. As a negative control, bacteria were treated with sterile phosphate buffer under the same conditions. Experiments were performed in triplicate.

Peptidoglycan hydrolysis assay

Peptidoglycan from A. viridans was extracted as described previously (35). The hydrolysis of peptidoglycan by different concentrations of AVPL was determined. Purified peptidoglycan with an OD₆₀₀ of about 0.9 was incubated with various concentrations (5 and 50 µg/mL) of AVPL at 37°C, and the absorbance at 600 nm was measured at regular time intervals.

3D structure model prediction and key amino acid analysis of AVPL

BLASTP and HMMER were used to analyze the protein sequence of AVPL (Genbank accession number MH729379.1) to identify the individual active domains of the lysin. The 3D structure of AVPL was modeled by inputting the protein sequence into the bioinformatics software Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2).

The potential distribution regions of key amino acid sites important for AVPL enzyme activity were initially identified by 3D structure prediction. The predicted sites in the active region were mutated to alanine to further confirm the critical amino acids. The primers used to generate the mutants are listed in Table S1. The correct mutant plasmids were transformed into E. coli BL21 (DE3), and the AVPL mutants were expressed and purified as described above.

An equal final concentration (30 µg/mL) of each mutant was added to 4 × 10⁸ CFU/mL of bacterial suspension and incubated at 37°C for 1 h for the determination of bactericidal activity. Serial dilutions of 10-fold cell suspensions were taken for colony counting. For the blank control, the same volume of phosphate buffer was added. All experiments were performed in triplicate.

Binding and lytic activity of truncated domains of AVPL

To determine the binding domain of AVPL, the logarithmic phase of A. viridans was incubated with 100 µg/mL of AVPL-C-EGFP, AVPL-B1-EGFP, AVPL-B2-EGFP, or AVPL-B1B2-EGFP at 37°C for 20 min. The bacteria with free protein removed were resuspended in 100 µL PBS buffer, and the fluorescence intensity at an excitation wavelength of 488 nm and emission wavelength of 510 nm was recorded by a spectrofluorophotometer (Shimadzu, RF-5301PC, Japan).

To determine the binding of AVPL to bacteria more visually, logarithmic phase A. viridans was treated with 20 µM Hoechst 33342(Cat: B2261, Sigma-Aldrich) fluorescent dye for 10 min at 37°C and washed five times with PBS buffer. The bacteria resuspended in 100 µL PBS buffer were incubated with 100 µg/mL AVPL-C-EGFP, AVPL-B1-EGFP, AVPL-B2-EGFP, or AVPL-B1B2-EGFP at 37°C for 20 min, the cells were collected by centrifugation (8,000 × g, 4°C, 5 min), and then washed to remove unbound proteins before suspending in 100 µL PBS buffer. Cell fluorescence was detected by LSCM at 488 nm or 510 nm wavelengths.

The turbidity reduction assay was used to determine the lytic activity of truncated domains of AVPL. Truncated proteins at a final concentration of 30 µg/mL were added to bacterial suspensions with OD_600nm of 0.8–0.9 and statically incubated at 37°C for 30 min, followed by recording spectrophotometer readings. The experiments were performed in triplicate biological replicates.

Determination of bacterial resistance to AVPL

The potential resistance of A. viridans P_1-1F₂ to AVPL was determined by repeated exposure to plate lysis assays with some modifications based on previous methods (36). Serial 2-fold dilutions of AVPL (concentrations ranged from 1000 μg/mL to 15.6 μg/mL) were dropped on the plated lawn of A. viridans and incubated at 37°C for 24 h. Bacteria obtained from the edge of the zone of inhibition were re-cultured to the log phase to generate a new lawn for plate lysis analysis. The above operation was repeated for 12 cycles, and the sensitivity of cells to AVPL (30 µg/mL) was determined for each generation.

Lytic activity in milk

Pasteurized whole milk (Inner Mongolia Yili Industrial Group Co., Ltd. China) was inoculated with 2 × 10⁶ CFU/mL of A. viridans P_1-1F₂, and purified AVPL (2, 12.5, or 50 µg/mL) or phosphate buffer was added immediately. The mixture was incubated at 37°C without shaking and sampled at 1 h intervals up to 4 h for colony counting. All experiments were performed in triplicate.

The therapeutic effect of AVPL on murine mastitis

A mouse mastitis model of multidrug-resistant A. viridans P_1-1F₂ was constructed according to a previous method with some modifications (37). The pups were separated from 48 lactating mice (7–10 days of lactation) 1–2 h in advance, and the mice were anesthetized intraperitoneally with a mixture of ketamine (87 mg/kg) and xylazine (13 mg/kg). To facilitate inoculation, the distal ends of the L4 and R4 (the left and right teats of the fourth pair of mammary glands) teats of the mice were snipped off by 0.5 mm. Fifty microliters of A. viridans P_1-1F₂ suspension (5 × 10⁴ CFU/gland) were delivered through the teat canal into the mammary glands using a 32-gauge blunt microsyringe (inserted about 4 mm) to induce mastitis. Mastitis development was determined after 24 h by inflammatory reactions such as edema, congestion, and damage to the mammary tissue.

The efficacy of AVPL in a murine model of bovine mastitis was evaluated as described previously with some modifications (28). A total of 42 lactating mice, 6 mice per group, were randomly divided into the following groups: control, A. viridans-infected, different doses of AVPL-treated (A. viridans + AVPL), antibiotic-treated group, and safety control groups. A. viridans-infected and AVPL-treated mice were given 50 µL/gland of sterile phosphate buffer and an equal volume of 12.5, 25, or 50 µg/gland of AVPL, respectively, after 1 h of infection. The antibiotic-treated groups were administered 100 mg/kg ampicillin at 1 h post-infection. The control group was not treated, and the safety control group was infused with 50 µg AVPL/ gland only.

Mice in each group were euthanized by cervical dislocation at 24 h after infection, and the L4 and R4 mammary glands were aseptically removed. A portion of each mammary tissue was weighed and homogenized in sterile PBS (100 mg/mL), serially diluted 10-fold, and plated on BHI agar plates to determine the load of A. viridans in the mammary gland. The LOD was 30 CFU/g. The remainder of the gland is for histopathological analysis, as well as for inflammatory cytokine detection.

Histopathological evaluation

The murine mastitis histopathological was evaluated according to a previous description, with minor modifications (38). A portion of mammary tissue from mice of different treatment groups was fixed (48 h) with 4% paraformaldehyde. The dehydrated tissues were placed into paraffin embedding and further sectioned (3–5 µm thickness). The tissue slices were dried at 37°C for 24 h and then stained with H&E. The pathological damage to the mammary tissue was evaluated using light microscopy.

Analysis of inflammatory cytokine concentrations in mammary tissue

TNF-α, IL-1β, and IL-6 concentrations were evaluated using ELISA kits (BioLegend, San Diego, CA, USA, Cat: 430904, 432604, and 431304) according to the manufacturer’s instructions. Briefly, mammary tissue was homogenized in PBS (0.2 g/mL), centrifuged at 12,000 × g for 20 min at 4°C, and the supernatant was collected for the assays.

Statistical analysis

SPSS version 19.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 9.0 (Graph Pad Software, San Diego, CA) were used for statistical analysis. Data were analyzed using a one-way analysis of variance (ANOVA) followed by least significant difference (LSD) analysis. *P < 0.05, **P < 0.01, and ***P < 0.001 were significant. Error bars represent the means ± SEM.

Conclusion

In this study, the functional domain and key active site of A. viridans phage lysin AVPL were revealed. AVPL has good temperature and pH stability, and efficient bactericidal activity on A. viridans in pasteurized whole milk. Furthermore, in a mouse model of mastitis induced by A. viridans, the lysin AVPL achieved a decrease in bacterial load in the mammary gland and a significant improvement in the pathologic changes of mastitis. Thus, AVPL potentially represents a good candidate as a non-antibiotic therapy drug for mastitis induced by multidrug-resistant A. viridans.

DATA AVAILABILITY

The data sets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article.

ETHICS APPROVAL

All animal experiments were approved (no. SY202211010) by the Animal Welfare and Research Ethics Committee at Jilin University.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.00461-24.

Supplemental material. aem.00461-24-s0001.docx.

Figures S1 to S4; Tables S1 and S2.

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