RitSun mycobacteriophage was isolated from a soil sample collected from Gurgaon, Haryana, India. The genomic characterisation of RitSun revealed its genome size is 55,975 bp nucleotide, with 60.8% GC content, and the phage belongs to the F2 sub-cluster. Endolysins are the lytic enzymes synthesized in the later stages of the phage infection cycle. They target bonds present in the PG and the linkage between arabinogalactan and mycolic acid, impairing the mycobacterial host’s cell wall, causing phage progeny to release and infect the neighbouring bacterial cells. The characterization and administration of purified recombinant lytic enzymes have been effectively tested in different animal models of human illnesses29,32,33,34,35, and their commercial use has also been approved in some cases21,36. Hence, characterising and developing lysin proteins as effective antimicrobials is gaining considerable importance.In silico characterization of RitSun LysinBRitSun lysis cassetteThe lytic cassette of RitSun consists of LysinA, LysinB and Holin-encoding genes and a hypothetical gene (Fig. 1A). In this report, we have characterised LysinB, which showed an intrinsic activity against M. smegmatis.Figure 1(A) Lytic cassette of RitSun mycobacteriophage.The lytic cassette of RitSun comprises LysinA, LysinB, Holin and a hypothetical protein (HP), located between LysinB and Holin. (B) Domain organization of RitSun LysinB. InterProScan tool predicted two domains in RitSun LysinB: i) an Alpha/Beta hydrolase fold (93–279 aa) and ii) a C-linker domain (280–340 aa).RitSun LysinB domain organisationRitSun LysinB, comprised of 356 aa, is identified as a serine esterase based on the InterProScan predicted motifs and domains (Fig. 1B). The predicted motifs, which are characteristic of LysinB, are G-X-S-X-G pentapeptide (whereX1 = F and X2 = R) and GXP (X = N), a highly conserved motif found in lipases37. The predicted domains include the characteristic Alpha/Beta hydrolase fold and a C-linker domain. InterProScan analysis of LysinB enzymes, reported from other mycobacteriophages, also shows the presence of Alpha/Beta hydrolase fold and a C-terminal linker domain. In addition to these domains, D29 LysinB is reported to have a cutinase/acetyl xylan esterase domain embedded in the Alpha/Beta hydrolase fold (Table 1).
Table1 Domain architecture of LysinB from different mycobacteriophages.Sequence alignment of RitSun LysinB protein sequenceThe enzymatic activity of D29 LysinB has been demonstrated to depend on the presence of the catalytic triad Ser82-Asp166-His240, with serine being a constituent of the pentapeptide. Fraga et al.29 reported Ser82Ala substitution to abolish its activity, indicating serine as a key residue for the enzymatic activity. Also, while Ser82 and Asp166 are highly conserved residues in the catalytic triad, Histidine shows a shift either by one position towards the N-terminal or three positions towards the C-terminal13,40. In Fig. 2,we have demonstrated an alignment of RitSun LysinB protein sequence with Ms6 LysinB (F1 sub-cluster), D29 LysinB (A2 sub-cluster), PDRPxv LysinB (B1 sub-cluster), Che9d LysinB (F2 sub-cluster) and Yoshi LysinB (F2 sub-cluster). Yoshi LysinB shows the highest identity with RitSun LysinB, and Che9d shares the same clade as RitSun LysinB and is observed to originate from a common ancestor. Hence, they are taken here as representative protein sequences from the F2 sub-cluster. In RitSun, Che9d and Yoshi LysinB, His residue is shifted by two positions towards the N-terminal (marked with a circle); Ms6 LysinB has a shift of single position, whereas PDRPxv and D29 LysinB do not show a shift in His residue and shows an alignment with each other.Figure 2Sequence Alignment of LysinB protein sequences. Sequence Alignment of LysinB from RitSun, Yoshi, Che9d, Ms6, D29, and PDRPxv mycobacteriophages was done using ClustalW (https://www.genome.jp/tools-bin/clustalw). The conserved GXSXG and GXP motifs are highlighted with a rectangular box. Ser and Asp of the catalytic triad are present at positions 186 and 270, respectively and marked with a red arrow aligned with that of D29 LysinB, whereas His (339) is shifted by two positions towards N terminal in RitSun LysinB. Symbols ‘*’, ‘:’, and ‘.’ denote identical amino acids, conserved substitutions, and semi-conserved substitutions, respectively.Comparative analysis of RitSun LysinB with phages from other F sub-clustersThe F cluster is divided into five sub-clusters: F1 to F5, and the F2 sub-cluster has seven mycobacteriophages reported to date (excluding RitSun)39. We used BLAST to compare the RitSun LysinB sequence with LysinB from the other F sub-clusters phages. Out of seven sub-cluster phages, RitSun LysinB revealed maximum identity (91.29%) with LysinB from mycobacteriophage Yoshi and a minimum identity (83.71%) with LysinB from mycobacteriophage Soul22.F1 is the most populated sub-cluster, with 221 members. We extracted the LysinB protein sequence from 150 F1 sub-cluster phages for a comparative analysis. We found RitSun LysinB to show > 83% identity (100% query cover) with seven F1 phages: EleanorGeorge, Enby, Girr, Kingsley, Lorde, MisterCuddles and Ovechkin and 84.73% identity, with a query cover of 97%, with Hamulus LysinB. All the other phages from the F1 sub-cluster showed less than 30% identity when the query cover was ≥ 95%. The F3 sub-cluster has only one phage reported, and in its genome, a Lysin gene has not been predicted39. The F4 sub-cluster has four phages, out of which LysinB from phages ThetaBob and TChen showed 88.52% and 88.24% sequence identity, respectively at 100% query cover. The F5 sub-cluster has only one reported phage. Its LysinB showed 28.65% identity (with a 99% query cover) with RitSun LysinB.Using InterProScan, we also performed a comparative domain analysis of RitSun LysinB with the other F sub-cluster phage LysinB. From the F2, F4, and F5 sub-clusters, all the LysinB protein sequences were analysed, while from the F1 sub-cluster, three phages, namely EleanorGeorge, Hamulus, and Flathead, were compared. We observed that LysinB sequences showing a high identity with RitSun LysinB (> 83%) display a similar architecture: an alpha/beta hydrolase fold followed by a linker domain. Interestingly, those showing low sequence identity (< 30%) with RitSun LysinB were predicted to have a C-terminal linker domain embedded within the Alpha/Beta hydrolase fold. A comparison of the domain organisation of RitSun LysinB with low sequence identity LysinBs from F1 sub-cluster (Flathead), F4 sub-cluster (Renaud18) and F5 sub-cluster (Cornie) phages is given in Supplementary Fig. 1.Purification of RitSun LysinB and Western Blot analysisThe His-tagged RitSun LysinB was overexpressed in E. coli BL21 (DE3) and purified to 95% homogeneity by Ni–NTA affinity chromatography (Fig. 3A).Western blotting with anti-His antibodies confirmed the presence of the purified recombinant LysinB (Fig. 3B).Figure 3Analysis of His-tagged recombinant RitSun LysinB. (A) SDS-PAGE (12%) analysis shows His-tagged recombinant RitSun LysinB purified to 95% homogeneity, visualized using Coomassie Brilliant Blue stain. (B) Western blot analysis of His-tagged recombinant RitSun LysinB was performed using anti-His antibodies. The blot was developed using 3,3-diaminobenzidine (DAB) as the chromophore and H2O2 as the substrate.In vitro assay (pNPB) of RitSun LysinBUsing pNPB as the substrate, the esterase activity of LysinB was estimated at different enzyme concentrations and incubation temperatures. The release of pNP was measured at 410 nm (Fig. 4A), and the specific activity was calculated.While examining the concentration effect of RitSun LysinB (0.5–10 μM) on the enzyme activity, the pNP release plateaued at 5 μM concentration (Fig. 4A). Hence, for the subsequent analysis of the temperature effect on enzyme activity, the assays were performed using 5 μM enzyme concentration.Figure 4In-vitro esterase activity of RitSun LysinB using pNPB assay. (A) Effect of enzyme concentration on esterase activity. The enzyme concentration effect was measured using different concentrations of RitSun LysinB with 10 mM pNPB (in 25 mM Tris buffer, pH 7.2). The Absorbance410 was measured after incubation at 37 °C for 15 min. The red arrow indicates the concentration used to study temperature effects on enzyme activity. (B) Effect of temperature on enzyme activity. The activity of RitSun LysinB at different incubation temperatures (37 °C, 45 °C, 55 °C) was analysed. All the enzyme reactions were performed twice in duplicate sets (n = 4).We found our enzyme activity was stable up to 55 °C (Fig. 4B). Other researchers, who examined the thermostability of LysinB enzymes by measuring their melting temperatures (Tm) using NanoDifferential Scanning Fluorimetry (NanoDSF), found the values in the 45–60 °C range. Examples include LysinB from mycobacteriophage Omega, which has a Tm of 57.7 °C, while D29’s Tm is 54.7 °C. Obama12 and Saal showed lower Tm values of 47.9 °C and 45.7 °C, respectively40.Notably, the specific activity (1.36 U/mg) of this novel LysinB is higher- five to ten times more than the reported LysinBs: PDRPxv (0.56 U/mg)13, D29 (0.72 U/mg)14, and Ms6 (0.12 U/mg)38.This may be attributed to differences in motifs, domain organization or conserved residues essential for enzyme activity. Our preliminary analysis of RitSun LysinB sequence alignment with other LysinBs (Fig. 2) reveals a distinctive feature: a shift in the position of Histidine residue by two positions towards the N-terminal in the catalytic triad of RitSun, Che9d and Yoshi LysinB, compared to a single-position shift in Ms6 LysinB, and no shift in PDRPxv and D29 LysinBs. Although the in vitro activity of Che9d and Yoshi LysinB has not been documented, their belonging to the same sub-cluster (F2), RitSun LysinB’s highest sequence identity with Yoshi LysinB, and sharing a clade with Che9d  LysinB suggest that the shifted position of Histidine residue may have a role in the higher activity of RitSun LysinB. However, this hypothesis requires further validation through structural and mutational analysis.Antibacterial effect of RitSun LysinB on M. smegmatis Mc2 155Spot test, turbidity reduction method (TRM) and growth inhibitory effectThe initial evidence of endolysins’ potential as antimicrobial agents came from their intrinsic bacteriolytic activity on Gram-positive bacteria, confirmed by their capacity to lyse bacteria on exogenous application20,41. Enzymatic activity can be detected by measuring the clearance of bacterial suspension as a zone of clearance and a reduction in viable bacterial cells. A spot test with a fixed amount of 100 μg (15 μM) of RitSun LysinB (in the presence of 0.05% Tween-80) showed a clear zone on M. smegmatis lawn after 48 h of incubation at 37 °C (Fig. 5A), demonstrating its cell lytic activity from without. The turbidometric reduction method (TRM) and plate dilutions of treated cells were used to study the growth inhibitory effect of RitSun LysinB. TRM analysis (Fig. 5B) showed a 45.6% ± 2.5% reduction in M. smegmatis Mc2 155 growth after 24 h of treatment with 15 µM enzyme concentration. Untreated cells showed a 232.1% ± 9.5% increase in growth after 24 h compared to 0 h. Dilutions of enzyme-treated M. smegmatis cells were spotted on 7H10 plates, and at 10−2 dilution, no bacterial colonies were observed after 24 h of incubation (Fig. 5C).Figure 5Anti-bacterial activity of RitSun LysinB. (A) Spot test of RitSun LysinB on M. smegmatis Mc2 155 growth in 7H10 media containing Tween-80 (0.05%). (B) Turbidity Reduction Method (TRM). Antimycobacterial activity of RitSun LysinB (measured at OD600) on M. smegmatis Mc2 155 at 0 and 24 h of treatment. The percentage decrease in growth was calculated by taking growth at 0 h as 100%. The enzyme concentration was 15 µM, and the experiment was performed twice in duplicate sets (n = 4). (C) Growth inhibitory effect of RitSun LysinB on M. smegmatis Mc2155, performed at 50 μg and 100 μg (corresponds to 7.5 µM and 15 µM concentrations) after 24 h of incubation at 37 °C. 1–Untreated cells; 2–Treatment with 7.5 µM RitSun LysinB; 3–Treatment with 15 µM RitSun LysinB: ‘a’ represents growth at dilution 10−1 and ‘b’ at 10−2. Untreated cells do not contain RitSun LysinB. (D) FESEM images of M. smegmatis Mc2 155 cells treated with 7.5 μM RitSun LysinB for 4 h at 37 °C. The images were acquired at 15,000 × magnification. The assays were performed twice in duplicate sets (n = 4).Based on a comparable or higher specific activity of RitSun LysinB with the reported LysinB from other mycobacteriophages and its antibacterial effect, the potential of this enzyme as an antimycobacterial protein is promising. Moreover, given the complex and hydrophobic structure of the mycobacterial cell envelope42, it is significant that RitSun LysinB can lyse M. smegmatis cells on exogenous application without the aid of an outer membrane permeabilizing agent (OMP). To test the enzyme’s permeability to other mycobacterial species, RitSun LysinB’s lytic ability was tested on M. fortuitum and M. tuberculosis (H37Rv). Although the enzyme was not active against these pathogenic species of mycobacteria (data not shown), we believe its further characterization, particularly identifying features enabling it to cross the complex mycobacterial cell envelope and those that make it host-specific, could provide valuable insights.FESEM analysis of RitSun LysinB treated planktonic cellsTo observe the effect of the enzyme on cell morphology, M. smegmatis cells were treated with LysinB (7.5 μM) for 4 h at 37 °C and analysed by FESEM (Fig. 5D). Compared to the untreated cells, RitSun LysinB-treated cells showed morphological disruption, indicating the cell-damaging effect of LysinB. Structural damage to the cell wall compromises bacterial cell integrity, leading to cell death10. LysinB targets mycobacteria due to its activity as a mycolylarabinogalactan esterase, hydrolyzing the ester bond between the mycolic acid-rich outer membrane andarabinogalactan. This activity likely weakens and disrupts the bacterial cell wall structure29. The ability of our endolysin to cause mycobacterial cell (M. smegmatis) lysis on external application, without the aid of a permeabilizing agent, is significant and can hold potential for clinical applications.Inhibitory effect of RitSun LysinB on M. smegmatis Mc2 155 biofilm formationBiofilms are notorious for causing recalcitrant infections and contributing to drug resistance43. Endolysins, with their ability to damage bacterial cell walls without requiring metabolically active bacteria, can target persistent bacteria and biofilms that lead to chronic infections44. We used the CV staining method to assess the impact of RitSun LysinB on M. smegmatis biofilm formation and noted an inhibitory effect of 79.18% ± 2.20% (Fig. 6). The anti-biofilm property of a chemical drug molecule or a biologic makes them relevant for clinical applications. Since the role of endolysins on mycobacterial biofilm formation hasn’t yet been reported, the inhibitory effect of RitSun LysinB noted in this study further supports the efficacy of this novel enzyme as an enzybiotic candidate.Figure 6Inhibitory effect of RitSun LysinB on M. smegmatis Mc2 155 biofilm formation. Control: M. smegmatis Mc2 155 biofilm formation after 96 h incubation at 37 °C; Treatment group: LysinB added to M. smegmatis Mc2 155 before biofilm formation. The assay was performed twice in duplicate sets (n = 4).
Discovery and characterization of a novel LysinB from F2 sub-cluster mycobacteriophage RitSun
