Structural and biochemical insights into the bacteriophage PlyGRCS endolysin targeting methicillin‐resistant Staphylococcus aureus (MRSA) and serendipitous discovery of its interaction with a cold shock protein C (CspC)

Abstract

Methicillin‐resistant Staphylococcus aureus (MRSA) causes life‐threatening human infections. Bacteriophage‐encoded endolysins degrade the cell walls of Gram‐positive bacteria by selectively hydrolyzing the peptidoglycan layer and thus are promising candidates to combat bacterial infections. PlyGRCS, the S. aureus‐specific bacteriophage endolysin, contains a catalytic CHAP domain and a cell‐wall binding SH3_5 domain connected by a linker. Here, we show the crystal structure of full‐length PlyGRCS refined to 2.1 Å resolution. In addition, a serendipitous finding revealed that PlyGRCS binds to cold‐shock protein C (CspC) by interacting with its CHAP and SH3_5 domains. CspC is an RNA chaperone that plays regulatory roles by conferring bacterial adaptability to various stress conditions. PlyGRCS has substantial lytic activity against S. aureus and showed only minimal change in its lytic activity in the presence of CspC. Whereas the PlyGRCS‐CspC complex greatly reduced CspC‐nucleic acid binding, the aforesaid complex may downregulate the CspC function during bacterial infection. Overall, the crystal structure and biochemical results of PlyGRCS provide a molecular basis for the bacteriolytic activity of PlyGRCS against S. aureus.

1. INTRODUCTION

The emergence of antibiotic‐resistant bacterial strains has become problematic in modern medicine. About 70% of the bacteria that cause hospital‐acquired infections are resistant to several antibiotics (Giamarellou, 2011). The Gram‐positive Staphylococcus aureus is a major cause of nosocomial infections, ranging from skin infections to life‐threatening diseases such as pneumonia, endocarditis, and septicemia. Methicillin‐resistant S. aureus (MRSA) is one of the most common antibiotic‐resistant bacterial pathogens and is resistant to the β‐lactam class of antibiotics (Pastagia et al., 2011). Hospital‐acquired (HA) and community‐acquired (CA) infections caused by S. aureus via nasal carriage are common. Serious and potentially fatal infections like those at surgical sites, intensive care units, in‐home wards, and in the bloodstream are caused by HA‐MRSA. CA‐MRSA causes severe dermatological diseases (Chambers & Deleo, 2009; Larsen et al., ²⁰²²; Reusch et al., ²⁰⁰⁸). The World Health Organization now considers MRSA to be an important threat to human health (Tacconelli et al., 2018).

Employing enzybiotics (bacteriophage‐encoded endolysin) therapy is one of the better strategies to overcome multi‐drug resistance to S. aureus infections (Nelson et al., 2012). The exogenous application of endolysins to Gram‐positive pathogens leads to rapid and effective “lysis from without” activity, which makes endolysins potent antimicrobial agents (Abedon, 2011; Linden et al., ²⁰¹⁵). The endolysin, PlyGRCS from the GRCS bacteriophage, was isolated from sewers in India (Sunagar et al., 2010). It has recently been reported that PlyGRCS possesses significant lytic activity against S. aureus, including MRSA. Since PlyGRCS activity has been shown in the physiological range, Linden et al. have suggested that it can be used as an antimicrobial agent (Linden et al., 2015).

Based on the sequence, PlyGRCS endolysin (hereafter, PlyGRCS) comprises an N‐terminal catalytic domain (aa: 1‐141) and a C‐terminal cell‐wall binding domain (aa: 156‐250), connected by a linker (Figure 1a). The catalytic domain belongs to the histidine‐dependent amido‐hydrolase/peptidase (CHAP) family, while the cell‐wall binding domain belongs to the src‐homology 3_5 (SH3_5) family. It has been reported that though PlyGRCS has a single catalytic domain, PlyGRCS possesses both N‐acetylmuramoyl‐L‐alanine amidase and D‐alanyl‐glycyl endopeptidase hydrolytic activities (Linden et al., 2015). For the amidase activity, PlyGRCS cleaves the amide bond between MurNAc and Ala; for endopeptidase activity, it cleaves the bond between D‐Ala and Gly or one of the Gly‐Gly bonds.

FIGURE 1.

The crystal structure of the PlyGRCS – Ec‐CspC complex. (a) A schematic representation of the domain architecture of PlyGRCS, showing the CHAP (Cysteine, Histidine‐dependent amidohydrolase peptidase) domain (1‐141aa), linker region (142–155), and the SH3_5 (src‐homology 3) domain (156–250). (b) SDS‐PAGE analysis of Ni‐NTA purified PlyGRCS protein. The “peak 1” fraction contains both PlyGRCS and Ec‐CspC, while the “peak 2” fraction contains PlyGRCS only. (c) A representative 2 m|Fo|‐D|Fc| map contoured at 1.0σ level corresponding to the loop, Gly57–Pro61 (shown in the sticks) of Ec‐CspC connecting the β4–β5 strands. The map is shown as blue mesh. (d) A cartoon diagram of PlyGRCS–Ec‐CspC complex. The N‐terminal region of the CHAP_GRCS domain (yellow) is connected with the C‐terminal SH3_5_GRCS domain (green) by a linker (cyan). A Ca²⁺ ion binds in CHAP_GRCS and is shown as a gray sphere. The MES buffer molecule binds to SH3_5_{GRCS and} is shown as pink sticks. Ec‐CspC (red) binds to PlyGRCS at the interface produced by CHAP_GRCS and SH3_5_GRCS. Structural figures were generated with PyMol (www.pymol.org).

There is little information in the literature about PlyGRCS, although endolysins from many other bacterial strains have been well‐studied. To understand the molecular mechanism of PlyGRCS endolysin, we have determined the tertiary structure of the full‐length of PlyGRCS to 2.1 Å resolution. Moreover, our serendipitous findings revealed that PlyGRCS binds to a cold‐shock protein C (CspC) from the host strain Escherichia Coli (E. coli). CspC, an RNA/ssDNA chaperone, is a major stress responder to various environmental triggers (cold, acid, and oxidative stress) (Cardoza & Singh, 2022). The crystal structure and biochemical results revealed that PlyGRCS may be an effective biotherapeutic molecule for treating MRSA infections.

2. RESULTS

2.1. The architecture of PlyGRCS‐ Ec‐CspC complex

During Ni‐NTA affinity purification, the full‐length PlyGRCS protein was eluted in two peaks (Figure 1b). In peak 1, PlyGRCS co‐eluted with an unknown small‐size protein of ~7.5 kDa, and peak 2 has only one band corresponding to the PlyGRCS protein. We have attempted to crystallize both peak 1 and peak 2 independently and got crystals for peak 1 only. During tracing the full‐length of PlyGRCS (Figure 1a) on the 2m|Fo|‐D|Fc| and the difference Fourier m|Fo|‐D|Fc| maps, surprisingly, an additional electron density consistently appeared corresponding to a polypeptide fragment near the interface between the CHAP_GRCS and SH3_5_GRCS domains. The final tracing of the polypeptide chain revealed that it is a cold shock protein CspC from the host bacteria, E. coli. To further confirm whether the bound protein with PlyGRCS is indeed Ec‐CspC, we performed in‐gel digestion for mass spectrometry analysis using an Orbitrap Fusion Tribrid mass spectrometer. The mass‐spectrometry derived raw data were searched against the E. coli protein database from NCBI using the Sequest search engine in Proteome Discoverer 2.1 (Sunitha et al., 2016). The results obtained from this study further reiterated that the peptide sequence (Figure S1) was specific to that of Ec‐CspC. In the final refined structure, PlyGRCS complexed with Ec‐CspC (Figure 1c,d and Table 1). The crystal structure of PlyGRCS contains the CHAP_GRCS and SH3_5_GRCS domains connected by a linker, 142‐155aa. The electron density corresponding to a long flexible region (Asn141–Asn156), which connects the CHAP_GRCS and SH3_5_GRCS domain, is unambiguously clear (Figure 2a). The region from Phe137 to Leu145 interacts with the CHAP_GRCS domain, while the region from Asn151 to Asn155 interacts with the SH3_5_GRCS domain. The head region of the long hairpin loop, which connects β7 and β8 strands in SH3_5_GRCS, grabs the C‐terminal region of the linker region, thereby stabilizing the structure in the linker region.

TABLE 1.

Data collection and final refinement parameters for the crystal structure of the PlyGRCS–Ec‐CspC complex.

Data collection parameters
Space group	P212121
Cell dimensions a b c (Å)	50.67, 55.29, 99.31
Wavelength (Å)	0.97373
Resolution (Å)	55.29–2.10 (2.16–2.10)
Unique reflections a	16,495 (1319)
Rmerge b	0.137 (0.540)
⟨I/σ⟩	7.4 (3.6)
Completeness (%)	98 (98)
Redundancy	4.2 (4.2)
CC1/2 e	0.97 (0.37)
Refinement
Resolution (Å)	49.65–2.10 (2.23–2.10)
No. of reflections	16458 (2659)
c Rwork/ d Rfree (%)	0.188 (0.239)/0.255 (0.318)
Total no. of atoms Protein atoms Water molecules MES molecule Ca2+‐ion	2763 2523 224 1 1
Average B‐factor (Å2)	22.9
RMSD bonds (Å)	0.007
RMSD angles (°)	0.941

^a Numbers in parentheses are values in the highest resolution shell.

^b ${\mathrm{R}}_{\text{merge}}=\sum _{\mathrm{hkl}}\sum _{\mathrm{i}}\left|{\mathrm{I}}_{\mathrm{i}}\left(\mathrm{hkl}\right)-⟨\mathrm{I}\left(\mathrm{hkl}\right)⟩\right|/{\sum }_{\mathrm{hkl}}{\sum }_{\mathrm{i}}{\mathrm{I}}_{\mathrm{i}}\left(\mathrm{hkl}\right)$, where I _i (hkl) is the intensity of the ith measurement and ⟨I(hkl)⟩ is the mean intensity for that reflection.

^c ${\mathrm{R}}_{\text{work}}=\sum _{\mathrm{hkl}}\left|\left|{\mathrm{F}}_{\mathrm{obs}}\right|-\left|{\mathrm{F}}_{\text{calc}}\right|\right|/{\sum }_{\mathrm{hkl}}\left|{\mathrm{F}}_{\mathrm{obs}}\right|$, where |F _obs| and |F _calc| are the observed and calculated structure‐factor amplitudes, respectively.

^d R _free was calculated with 5.0% of reflections in the test set.

^e Values correspond to the highest resolution shell.

FIGURE 2.

Structure of PlyGRCS. (a) A representative 2m|Fo| ‐ D|Fc| map contoured at 1.0 ϭ level corresponding to the linker region (shown in cyan sticks). A long flexible region (N141 ‐ N154) connects the CHAP_GRCS (yellow) and SH3_5_GRCS (green) domains. The map is shown as brown mesh. (b) Near the Ca²⁺ binding region. The Ca²⁺ ion is coordinated with Asp20, Asp22, Ala24, Gly26, and Asp31. (c) Near the catalytic site region. Cys29, His89, and Glu105, the conserved catalytic residues, are shown as sticks. (d) Near the interface between CHAP_GRCS and SH3_5_GRCS. The residues involved in the intermolecular interactions are shown as yellow and green sticks, respectively, corresponding to the CHAP_GRCS and SH3_5_GRCS regions.

2.2. The structure description of PlyGRCS‐Ec‐CspC complex

2.2.1. The CHAP domain of PlyGRCS

The CHAP_GRCS domain lies in the N‐terminal region of PlyGRCS and possesses the endopeptidase fold (CATH superfamily) (Figure 1a,d). The CHAP_GRCS domain (aa: 1‐141) comprises a central six‐stranded antiparallel β‐sheet arranged in the topology of the β1β6β2β3β4β5 (Figure 1d). Two long helices (α1 and α2) from the N‐terminal region of the CHAP_GRCS domain have been positioned on one side of the β‐sheet. The long flexible loop that connects α1 and α2 harbors a Ca²⁺ ion. The secondary structure elements in the CHAP_GRCS domain are connected by seven long loops: the loop L1 comprising His14–Cys29 connects α1 and α2; L2 (Thr41–Ala63) containing a 3₁₀ helix which connects α2 and β1; L3 (Glu67–Tyr82) connects β1 and β2; L4 (Tyr82–His89) connects β2 and β3; L5 (Gly96–Tyr101) connects β3 and β4; L6 (Glu105–Thr12) connects β4 and β5; and L7 (His124–Thr130) connects β5 and β6.

A Ca²⁺ ion is found in the L1 loop, which connects α1 and α2 (Figure 2b). The Ca²⁺ ion is coordinated with the carbonyl groups of Asp22, Ala24, and Gly26 and the side chain of Asp20 and Asp31. The coordination distance is ~2.32 to 2.55 Å. The experimental metal ion‐oxygen distances are 2.3–2.5 Å for the calcium (II) coordination (Harding, 2006), consistent with the observed values. The substrate binding pocket, which is adjacent to the Ca²⁺ binding region, contains the catalytic triad, Cys29, His89, and Glu105. The pocket is formed by the loops connecting β2 & β3, β4 & β5, and β5 & β6 as a wall, and the base by the strands β3‐β5 (Figure 2c).

2.2.2. The SH3_5 domain of PlyGRCS

The SH3_5_GRCS domain of PlyGRCS comprises eight β‐strands (β1‐β8) (Figure 1d). The overall structure can be divided into two β‐sheets nearly right‐angle to each other. The first sheet is formed by strands β5‐β7 and the N‐terminus of β2, while the second sheet is produced by strands β3, β4, β8, and the C‐terminal part of β2. The strands β3 and β4 are connected by the long RT loop (as per the nomenclature of the lysostaphin and Ale‐1 SH3b domains) (Gonzalez‐Delgado et al., 2020; Holm & Sander, ¹⁹⁹³)_. The β1, β3, and β4 strands are unique to prokaryotic SH3b domains in PlyGRCS, lysostaphin, and Ale‐1; however, the other strands are present both in eukaryotic and bacterial SH3b domains. An extra‐long loop (Gln231–Trp246) connects β7 and β8 strands. The MES buffer molecule binds in a shallow pocket produced by the β‐sheet of β2, β5‐7 strands. The sulfonic moiety of MES is exposed to the solvent region, while the morpholine group is surrounded by Thr163, Tyr165, Pro186, Glu206, and Tyr226 (not shown).

2.2.3. The interacting region between CHAP_GRCS and SH3_5_GRCS domains

In the ternary structure of the PlyGRCS‐Ec‐CspC complex, the N‐terminal region of the CHAP_GRCS domain moderately interacts with the C‐terminal SH3_5_GRCS domain (Figures 1d and 2d). The Ca²⁺–binding loop and the long flexible loop connecting α2 and β1 of the CHAP_GRCS domain make contact with the loops connecting β2 & β3, and β7 & β8 in SH3_5_GRCS. The carbonyl group of Phe21 forms hydrogen bonds with the NE1 atom of Trp246 and with the side chain of Gln231. Leu244 hydrophobically interacts with the side chains of Phe21, Val34, Ala35, Tyr38, and Arg46. The residue Tyr38 makes a weak salt bridge with Arg230. A water‐mediated interaction is also observed between Tyr38 and the carbonyl group of Ile242. The bulky side chain of Trp48 is surrounded by the backbone atoms in the loop that connects β2 and β3 strands and with the N‐terminal region of the β8 strands in SH3_5_GRCS. The residue Phe176 in SH3_5_GRCS, which lies in the loop connecting β2 and β3 strands, also hydrophobically interacts with Gly49. The NE atom of Arg46 hydrogen bonds with the carbonyl group of Pro245.

2.2.4. PlyGRCS‐Ec‐CspC complex

In the ternary complex, Ec‐CspC binds in the interface of the CHAP_GRCS and SH3_5_GRCS domain of PlyGRCS (Figures 1d, 3 and S2). The tertiary structure of Ec‐CspC in the complex consists of five antiparallel β‐strands to form a closed β‐barrel (Figure 1d). The β‐barrel is created by strands β1‐β3 on one side while strands β4 and β5 are on the other side. The β‐strands are connected by variable loops and turns. A long loop comprising about 16 amino acids connects strands β3 and β4.

FIGURE 3.

Interaction between PlyGRCS and Ec‐CspC. (a) Electrostatic surface potential diagram of PlyGRCS (left) and Ec‐CspC (right). The surface is colored red and blue for potential values below −5 k_BT and above +5 k_BT, respectively, where k_B is the Boltzmann constant, and T is room temperature. (b) The loop connecting the β4–β5 strands in Ec‐CspC projects into the PlyGRCS interface contributes to hydrophilic interactions with PlyGRCS. (c) Intermolecular hydrophobic interactions between SH3_5_GRCS and Ec‐CspC. The residues involved in intermolecular interactions are shown as sticks. The hydrogen bonds are depicted by dotted lines.

A long loop between the α‐helices α1 and α2 in the N‐terminal region of the CHAP domain makes substantial intermolecular interactions with Ec‐CspC (Figure 3b). The side chain Gln58 in Ec‐CspC contributes hydrogen bonds with Asp22 in CHAP and Q231 in SH3_b. The residue Asp22 falls in the disallowed region of the Ramachandran map as it tends to interact with Ec‐CspC and contribute an ionic interaction with Ca²⁺‐ion simultaneously (Figure 3b). Based on the structure, we speculate that the geometry constraints may be relaxed in this region in the absence of Ec‐CspC. Gln231 (SH3_5_GRCS region) contributes a hydrogen bond with Gln58 of Ec‐CspC. A direct backbone interaction occurs between the amino group of Phe180 (SH3_5_GRCS region) and the carbonyl group of Lys59 in Ec‐CspC. The guanidium group of Arg182 (SH3_5_GRCS region) forms a weak hydrogen bond with Asp56 in Ec‐CspC. A water‐mediated interaction is observed between the carbonyl group of Asp211 (SH3_5_GRCS) and Gln58 of Ec‐CspC. A π‐anionic interaction between Trp246 (SH3_5_GRCS region) and Lys59 of Ec‐CspC is observed. Intriguingly, a hairpin loop (Asp56–Pro61) connecting β4 and β5 in Ec‐CspC, which is critical to Csp's interaction with ssDNA/RNA, makes extensive interactions with both domains of PlyGRCS.

A substantial hydrophobic interface is formed by the residues, Trp10, Lys15 (aliphatic region), Phe17, Phe19, Lys27 (aliphatic region), Phe30, His32, and Pro61 in Ec‐CspC, and the residues Phe176, Leu177, Pro178, Phe180, Leu188, Pro191, Trp195, and Pro198 in PlyGRCS (Figure 3c). As shown in Figure 3c, the intermolecular hydrophobic interactions are mainly mediated through the SH3_5_GRCS domain. The residue, Ile54, which lies in strand β4 in Ec‐CspC, contributes to symmetry‐related interactions with PlyGRCS (not shown). A water‐mediated interaction is produced between the carbonyl group of Ile54 in Ec‐CspC and the amino group of symmetry‐related residue, Tyr125’ (CHAP_GRCS). Moreover, the side chain of Ile54 (Ec‐CspC) hydrophobically interacts with the side chain of the symmetry‐related residue, Tyr125’ (CHAP_GRCS). The side chain of Glu53 (Ec‐CspC) contributes symmetry‐related interactions with the carbonyl group of Tyr A126’ (CHAP_GRCS).

2.3. Microscale thermophoresis

Based on the PlyGRCS‐Ec‐CspC complex structure, a quantitative binding assay was performed between Ec‐CspC and PlyGRCS (wild‐type and mutants) to evaluate their binding efficiency. Site‐specific mutations were carried out in PlyGRCS for the critical residues involved in intermolecular interactions with Ec‐CspC. The specific residues mutated in the PlyGRCS are D22A, Q231A, and D22A+Q231A. The binding affinity of PlyGRCS with Ec‐CspC showed a K_D value of 145 nM, while that for the mutants D22A, Q231A, and double mutant D22A+Q231A yielded K_D values of 167 nM, 1.89 μM, and 887 nM, respectively (Figure 4). The observed higher binding affinity for the wild‐type compared to the PlyGRCS mutants suggests that the binding of Ec‐CspC with PlyGRCS is sequence‐specific. Next, to understand whether S. aureus CspC also interacts with PlyGRCS, similar to Ec‐CspC, we carried out a quantitative binding assay between Sa‐CspC and PlyGRCS. The binding affinity of Sa‐CspC with the PlyGRCS was determined to be 152 nM (Figure 4e), like that observed in the Ec‐CspC–PlyGRCS complex.

FIGURE 4.

MST analysis of Ec‐CspC interactions with PlyGRCS (wild‐type (Wt) and mutants) proteins. Binding curves for the interactions of fluorescently labeled Ec‐CspC with (a) Wt‐PlyGRCS, (b) D22A PlyGRCS. (c) Q231A PlyGRCS. (d) D22A+Q231A PlyGRCS, and the interaction of fluorescently labeled Sa‐CspC with (e) Wt PlyGRCS were shown as the change in normalized fluorescence (ΔFnorm). The K_D values are summarized in the Table. The K_D values were obtained after fitting the binding curves between ΔF_norm plotted as a function of the ligand concentration. The values are plotted on normalized fluorescence (y‐axis) against increasing concentrations (x‐axis). Error bars represent SEM (n = 3).

CspC is known to interact with RNA/ssDNA for its chaperone activity by binding and stabilizing the structure of the RNA/ssDNA (Cardoza & Singh, 2022; Heinemann & Roske, ²⁰²¹). To validate whether PlyGRCS inhibits the nucleic‐acid binding to CspC, a quantitative binding assay was again carried out for Sa‐CspC and Ec‐CspC with ssDNA. For this study, we have custom‐synthesized 7mer ssDNA (TTTTTTT) based on the previous report on the Bacillus subtilis CspB‐ssDNA interaction (Max et al., 2006). The binding affinity of Sa‐CspC and Ec‐CspC with the ssDNA was 714 nM and 677 nM, respectively. Both Sa‐CspC and Ec‐CspC showed a similar binding affinity for ssDNA. These results suggest that the binding affinity of CspC with PlyGRCS is higher compared to that with ssDNA (5‐fold) (Figure 5a,b). Next, to understand whether PlyGRCS‐CspC interaction blocks the nucleic‐acid binding of CspC, we carried out a quantitative binding assay of the above complex with ssDNA. For this study, Sa‐CspC was mixed with an increasing concentration of PlyGRCS (1:1 and 1:2 ratio), then the complex was studied for its interaction with ssDNA. The binding affinity of ssDNA with Sa‐CspC in the presence of PlyGRCS (1:1 and 1:2 ratio) yielded a K_D value of 4.73 μM and weak/no binding, respectively. The above study clearly revealed that PlyGRCS significantly blocks the binding of CspC with ssDNA (Figure 5c,d).

FIGURE 5.

MST analysis of Sa‐CspC, Ec‐CspC, and PlyGRCS interactions with ssDNA. Binding curves for the interactions of fluorescently labeled (a) Sa‐CspC and (b) Ec‐CspC, respectively, with ssDNA were shown as the change in normalized fluorescence (ΔFnorm). Binding curves for the interactions of fluorescently labeled (c) Sa‐CspC+PlyGRCS complex (1:1 ratio) and (d) Sa‐CspC+PlyGRCS complex (1:2 ratio), respectively, with ssDNA were shown as the change in normalized fluorescence (ΔFnorm). The K_D values were obtained after fitting the binding curves between ΔF_norm plotted as a function of the ligand concentration. The values are plotted on normalized fluorescence (y‐axis) against increasing concentrations (x‐axis). Error bars represent SEM (n = 3).

2.4. Turbidity reduction assay (lytic activity)

Turbidity reduction assays were performed to evaluate the lytic activity of PlyGRCS against ATCC 29213 S. aureus, MRSA, and the nonspecific Gram‐positive S. saprophyticus and Gram‐negative E. coli bacterial strains as controls (Figure 6). The lytic activity was measured based on turbidity reduction by using a spectrophotometer. The PlyGRCS showed significant lytic activity against ATCC 29213 S. aureus at varying concentrations from 1.25 to 80 μg/mL (Figure 6a). Significant lytic activity was also observed for MRSA at the standardized minimal and maximal concentrations (10 and 80 μg/mL) of PlyGRCS (Figure 6b). The lytic activity in the controls was nearly absent (Figure 6c,d), suggesting that PlyGRCS is specific to S. aureus isolates.

FIGURE 6.

Lytic activity of PlyGRCS against S. aureus and controls by turbidity reduction assay. Log phase cells of the S. aureus strain were collected, and lysis was observed by monitoring OD measurements (OD_600nm) spectrophotometrically. PlyGRCS was treated in a concentration‐dependent manner from 1.25 μg/mL to 80 μg/mL against (a) S. aureus and with 10 μg/mL and 80 μg/mL concentrations against (b) MRSA, (c) S. saprophyticus, and (d) E. coli. All curves represent mean ± standard deviation from three independent readings.

2.5. Turbidity reduction assay (lytic activity) in the presence of CspC

As we observed that PlyGRCS significantly inhibits the CspC binding from ssDNA, we further checked whether CspC affects the lytic activity of PlyGRCS in S. aureus. For that, we have incubated PlyGRCS with increasing concentrations of Ec‐CspC or Sa‐CspC individually (1:1, 1:3, and 1:5 ratio) as well as for the peak 1 corresponding to the PlyGRCS‐Ec‐CspC complex and carried out lytic assay in S. aureus. Both CspCs moderately reduced the lytic activity of PlyGRCS (Figure 7a,b), as observed in the PlyGRCS‐Ec‐CspC complex. These findings suggest that Sa‐CspC can protect the S. aureus strain from PlyGRCS moderately.

FIGURE 7.

Lytic activity of PlyGRCS and its effect with Ec‐CspC and Sa‐CspC against S. aureus by turbidity reduction assay. Log phase cells of S. aureus were collected, and lysis was observed by monitoring OD measurements (OD_600nm) spectrophotometrically. The lytic activity of (a) PlyGRCS (80 μg/mL, positive control), PlyGRCS‐Ec‐CspC (80 μg/mL) (peak1), and different ratios of PlyGRCS with Ec‐CspC (1:1, 1:3, and 1:5) against S. aureus and (b) PlyGRCS (80 μg/mL, positive control), PlyGRCS‐Sa‐CspC (80 μg/mL), and different ratios of PlyGRCS with Sa‐CspC (1:1, 1:3, and 1:5) against S. aureus. The untreated S. aureus cells, Ec‐CspC, and Sa‐CspC were used as controls. All curves represent mean ± standard deviation from three independent readings.

2.6. Disc diffusion assay

To further evaluate the antibacterial activity of the purified proteins, we carried out a Disc‐diffusion assay on S. aureus and MRSA (Figure 8a,b) and also PlyGRCS‐Ec‐CspC complex (peak 1) against S. aureus (Figure 8c). Protein spots (80, 40, 20, and 10 μg) were applied, and the strength of lytic zones was evaluated qualitatively. The lytic behavior of the disc diffusion assay correlates well with the turbidity reduction assay, which validates the lytic activity of the PlyGRCS. Both assays were remarkably able to show the antibacterial property of PlyGRCS against its target host, S. aureus. In contrast, PlyGRCS is host specific as evidenced by the lack of lytic activity in nonspecific hosts S. saprophyticus and E. coli (Figure 8d,e).

FIGURE 8.

Disc diffusion assay of PlyGRCS against S. aureus isolates. PlyGRCS was treated against (a) S. aureus with amounts of (2) 10 μg, (3) 20 μg, (4) 40 μg, and (5) 80 μg. (b) MRSA with amounts of (2) 10 μg, and (3) 80 μg. (c) PlyGRCS‐Ec‐CspC (peak1) was treated against S. aureus with an amount of (2) 10 and (3) 80 μg. PlyGRCS was treated against (d) S. saprophyticus with amounts of (2) 10 μg and (3) 80 μg, and (e) E. coli with amounts of (2) 10 μg and (3) 80 μg. In all panels (a)–(e), #1 represents the control (protein buffer only).

3. DISCUSSION

The Gram‐positive Staphylococcus aureus is a major infection and disease‐causing pathogen. Methicillin‐resistant S. aureus (MRSA), the most common antibiotic‐resistant bacterial pathogen, is becoming resistant to last‐resort antibiotics like vancomycin, teicoplanin, linezolid, tigecycline, and daptomycin (Rybak et al., 2020). Therefore, alternate solutions for developing an effective therapy are urgently needed. Endolysins are promising biotherapeutic targets as they are highly efficient in lysing the bacterial cell wall by hydrolyzing the peptidoglycan layer, resulting in a sudden drop in turgor pressure and osmotic lysis to cause bacterial cell death (Haddad Kashani et al., 2018). In this study, a high‐resolution crystal structure of the full‐length PlyGRCS endolysin and functional studies provide molecular mechanisms of PlyGRCS in bacterial lytic activity. The crystal structure of the full‐length PlyGRCS clearly showed the N‐terminal CHAP domain and the C‐terminal SH3_5 domain connected by a long linker. The Ca²⁺ coordination in the CHAP_GRCS domain is necessary for its catalytic activity. The Ca²⁺ deficiency may alter the conformation in the loop region, leading to a loss of enzyme activity.

3.1. Comparison with CHAP domains

A DALI (Holm & Sander, 1993) search of the CHAP_GRCS domain structure of PlyGRCS was performed against the RCSB PDB database. The search revealed that though the sequence similarity of the solved structure is low with the known CHAP domain structures, CHAP_GRCS is closely similar to CHAP_K of LysK from S. aureus (PDB Id: 4CSH, Z‐score: 20.6, 42% identity) (Sanz‐Gaitero et al., 2014) and CHAP domain of LysGH15 from Staphylococcus phage G15 (PDB Id: 4OLK, Z‐score: 20.5, 42% identity) (Gu et al., 2014) with an rmsd value of ~1.4 Å for 136 Cα‐atoms. As shown in Figure S3b, the overall structures of these domains are similar despite substantial structural variations found in the loop region. In the Ca²⁺ binding region, a long‐extended loop insertion is found in LysK. However, the position of conserved catalytic residues (Cys29, His89, and Glu105) are identical. Based on the Ca²⁺ coordination and the catalytic site region, we speculate that the catalytic function in both the CHAP_GRCS domain and LysK may be similar.

The DALI search also identified the PlyPy endolysin structure, which contains the CHAP domain and the SH3b domain (PDB Ids: 5UDM and 5UDN; unpublished data). PlyPy endolysin from Streptococcus pyogens has a typical two‐domain structure with an N‐terminal catalytic CHAP domain and a C‐terminal binding (SH3) domain (Lood et al., 2014). In the PlyPy endolysin structure, several regions are absent, including the linker region connecting the CHAP domain and SH3b domain. The sequence identity corresponding to the CHAP domains of PlyGRCS and PlyPy is 22%. By superposition of these two CHAP‐domain structures, it showed the rmsd value of 2.2 Å for 117 Cα‐atoms with a Z‐score value of 14.9 (Figure S3b). Though the secondary structural elements are moderately superimposed, the loops connecting the secondary elements are quite different. In the catalytic triad region, the positions of Glu and His are nearly identical except for Cys (Figure S3b).

3.2. Comparison with lysostaphin SH3b

The crystal structures of lysostaphin SH3b complexes from Staphylococcus simulans with a pentaglycine peptide and peptidoglycan (P4‐G5), respectively, were recently reported (Gonzalez‐Delgado et al., 2020; Mitkowski et al., ²⁰¹⁹). The S. simulans lysostaphin is composed of a catalytic domain and a SH3b domain (Sabala et al., 2014). The Zn²⁺‐binding catalytic domain of lysostaphin belongs to the M23 family peptidases (Bochtler et al., 2004). In the S. simulans SH3b—pentaglycine complex (PDB Id: 5LEO, unpublished data), the pentaglycine peptide, which represents the pentaglycine cross‐bridge, binds to S. simulans SH3b at a groove formed between β1 and β2 strands. In the S. simulans SH3b–P4‐G5 complex (PDB Id: 6RK4) (Gonzalez‐Delgado et al., 2020), the pentaglycine cross‐bridge and the peptide‐stem bind to S. simulans SH3b at two different binding sites. The mode of pentaglycine moiety binding in this complex is the same as in the previous structure; however, the cross‐bridge link (K to G5) and the stem peptide (γQKA) in the S. simulans SH3b complex bind to a pocket positioned on the opposite side of the symmetry‐related SH3b molecule.

Based on the Ss. SH3b–PG‐G5 complex, Gonzalez‐Delgado et al. have proposed a model of cooperative binding for a ligand at two sites based on a flexible linker connecting pentaglycine and stem‐peptide of PG (Gonzalez‐Delgado et al., 2020). By comparing with this structure (Figure S4), we speculate that PlyGRCS may recognize two PG molecules simultaneously: (i) a pentaglycine moiety of PG binds to the pentaglycine binding site in SH3_5_GRCS of PlyGRCS while the stem‐peptide region of the same PG molecule may bind to SH3_5_GRCS of the other PlyGRCS molecule, (ii) the stem‐peptide moiety of other peptidoglycan binds at the stem‐peptide region in SH3_5_GRCS while the pentaglycine cross‐link of the same peptidoglycan binds in such a way at the catalytic groove in CHAP_GRCS for its catalytic activity. The conformation of P4‐G5 in the S. simulans SH3b is a nonspecific flexible structure suggesting the peptidoglycan may adopt a different flexible conformation upon binding to these two domains simultaneously. With the predicted two binding sites and the presence of dual enzyme activities of amidase and endopeptidase, we speculate, based on the structure, that PlyGRCS may possess synergetic catalytic function on the two peptidoglycan molecules simultaneously. However, further studies of the PlyGRCS complex with the peptidoglycan may be required to understand better how the PG molecule(s) bind to the PlyGRCS for their catalytic activity.

3.3. Identification of CspC

Our crystal structure, mass spectrometry, and extensive quantitative binding studies unambiguously confirm the serendipitous binding of CspC with PlyGRCS. The Csps' possess a conserved nucleic acid (ssDNA/RNA) binding domain, namely the cold‐shock domain (CSD) (Graumann & Marahiel, 1996). CSD contains two ribonucleotide protein (RNP) motifs which are essential to facilitate the nucleic acid binding (Heinemann & Roske, 2021; Schindelin et al., ¹⁹⁹⁴). Ec‐CspC shares 81% sequence identity with Ec‐CspA and Ec‐CspB for 67 residues. On the other hand, it shares 67% and 61% sequence identity with B. subtilis CspB and S. aureus CspC, respectively, for 64 residues (Figure S5). The surface of Ec‐CspC, as in the known Csp isoform structures, possesses hydrophobic and basic patches (Figure S2). The ribonucleotide protein (RNP) motifs, RNP1 and RNP2, positioned next to each other, are produced by the conserved hydrophobic and basic residues. In the Bs‐CspB complexes with ssDNA (PDB Id: 2ES2 (Max et al., 2006) and ssRNA (PDB Id: 3PF5; Sachs et al., ²⁰¹²), the nucleic acid bindings are nearly similar: the single strand binds in a groove composed of a positively charged protein surface with exposed aromatic side chains. The bases of the nucleic acid fragments are oriented toward the protein and form a limited number of hydrogen bonds with the protein backbone or side chains. In contrast, the backbone of the nucleic acid fragments faces the exposed solved region and is not in contact with the protein (Max et al., 2006). The key hydrophilic residues, Gln58 and Lys59, unique to Ec‐CspC, contribute to critical intermolecular interactions with PlyGRCS. Interestingly, S. aureus CspC also possesses similar‐charged residues (Asp55 and Arg56) in this region. The hairpin loop containing these residues is one of the hotspots for the interaction with ssDNA/RNA. The superposition of the PlyGRCS‐Ec‐CspC complex onto the Bs‐CspB‐ssDNA complex revealed that Ec‐CspC is unlikely to bind to PlyGRCS and nucleic acids simultaneously (Figure 9). As predicted based on the structural comparison, the biochemical assay also confirmed a substantial inhibition of ssDNA binding to CspC by PlyGRCS.

FIGURE 9.

Superposition of cold shock proteins. Superposition of Ec‐CspC (red) onto the CspA from E. coli (green, PDB: 1MJC) and CspB from B. subtilis in complex with a DNA fragment (yellow, PDB: 2ES2). The DNA molecule is shown as blue sticks.

Our antimicrobial assay demonstrated that PlyGRCS has significant lytic activity in ATCC 29213 S. aureus and MRSA strains and correlates well with reported results in various MRSA strains (Linden et al., 2015). Moreover, the study also revealed that CspC moderately inhibits the lytic activity of PlyGRCS. In the late phase of the lytic cycle, the phage progeny is released with the help of phage lysin by lysing the cell wall of host bacteria. This is mediated through phage‐derived holin protein, which channels the phage lysin protein to cleave peptidoglycan and release newly produced phages. In our experiment, PlyGRCS was added to S. aureus, which acts externally to kill the S. aureus cells; but when Ec‐CspC or Sa‐CspC was preincubated with PlyGRCS, there was a decrease in the lytic activity as compared to PlyGRCS alone. Hence, based on the above studies, we speculate that PlyGRCS may negatively regulate the CspC function, thereby arresting the production of stress proteins required for cell survival under environmental insults.

Taken together, we hypothesize how PlyGRCS may kill the bacteria through two simultaneous distinct pathways such as (i) indigenous lytic activity of bacterial cell wall and (ii) negatively regulating the downstream pathway of arresting stress‐related proteins synthesis through inhibiting the CspC chaperone activity (Figure 10). However, further biological studies may shed light on why PlyGRCS and CspC interact with each other and whether they have synergetic activities to regulate cellular function.

FIGURE 10.

PlyGRCS plays a prominent role in killing bacteria simultaneously by distinct pathways. (a) PlyGRCS displaying the lytic activity on Staphylococcus aureus. (b) PlyGRCS‐CspC complex displays moderate lytic activity on Staphylococcus aureus, which inhibits the interaction of CspC with RNA/ssDNA and down‐regulates the bacterial cellular process. (c) Interaction of CspC with RNA/ssDNA during stress conditions in the bacteria, which up‐regulates the bacterial cellular process.

4. MATERIAL AND METHODS

4.1. Bacterial strains

Escherichia coli DH5α, BL21 (DE3) codon plus, and Rosetta (DE3) pLysS (Novagen) strains were used for cloning, site‐directed mutagenesis, and protein overexpression. S. aureus (ATCC 29213), S. saprophyticus (ATCC 19701), E. coli (ATCC 25922), and methicillin‐resistance S. aureus (MRSA) were used for microbial assays.

4.2. Cloning of PlyGRCS , CspC, and site‐directed mutagenesis

The PCR amplification of PlyGRCS and E. coli CspC was carried out from the genomic DNA of the GRCS bacteriophage and the genomic DNA of E. coli, respectively. The amplified PCR product of PlyGRCS was inserted between NcoI and XbaI restriction sites, and Ec‐CspC was inserted between NdeI and XhoI restriction sites into the bacterial expression vector pBAD24 and pET28a, respectively. pBAD24 PlyGRCS and pET28a Ec‐CspC clones were confirmed by sequencing. The commercially synthesized Sa‐CspC gene (GeneScript) was inserted between NdeI and XhoI restriction sites into the bacterial expression vector pET28a. pET28a Sa‐CspC clone was confirmed by sequencing. Site‐directed mutagenesis in PlyGRCS was carried out using Phusion HF DNA polymerase (New England Biolabs). The amplified product was subjected to DpnI (Takara Bio Inc.) treatment and transformed into E. coli DH5α and the clones were confirmed by sequencing.

4.3. Expression and purification of PlyGRCS and CspC

The protein expression conditions for PlyGRCS were standardized in Rosetta (DE3) pLysS (Novagen) cells, induced with 0.25% L‐arabinose, and incubated the cells at 16°C for 16 h post‐induction. The protein expression conditions for Ec‐CspC and Sa‐CspC were standardized in BL21 (DE3) codon plus cells with 1 mM IPTG induction and overnight incubation at 37°C.

The PlyGRCS‐expressed bacterial culture pellet was resuspended in a lysis buffer containing 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10% glycerol, and 10 mM β‐mercaptoethanol and lysed by sonication. The cell lysate was centrifuged at 14,000 rpm for 30 min at 4°C. The supernatant was collected and loaded into a 5 mL HiTrap Ni‐NTA affinity chromatography column (Protein Ark) in the presence of 10 mM imidazole. PlyGRCS was eluted in the 175–300 mM imidazole range and loaded to 15% SDS‐PAGE. The protein was dialyzed against 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10% glycerol, and 10 mM dithiothreitol (DTT). The protein was aliquoted and stored at −80°C for crystallization and biochemical studies.

The Ec‐CspC and Sa‐CspC bacterial culture pellets were resuspended in a lysis buffer containing 50 mM Tris–HCl pH 7.5, 300 mM NaCl, and 10 mM β‐mercaptoethanol and subjected to sonication. The cell lysates were centrifuged at 14,000 rpm for 30 min at 4°C. The supernatants were collected and loaded on the Ni‐NTA affinity column (G Biosciences, USA). The proteins of interest were eluted in an elution buffer containing 50 mM Tris–HCl pH 7.5, 300 mM NaCl, 10 mM β‐mercaptoethanol, and 250 mM imidazole. The proteins were dialyzed in a buffer containing 50 mM Tris–HCl pH 7.5, 150 mM NaCl, and 10 mM β‐mercaptoethanol and were aliquoted and stored at −80°C for biochemical studies.

4.4. Crystallization, x‐ray data collection, and structure determination

Both peak 1 and 2 fractions were pooled independently and concentrated to ~5 mg/mL each for crystallization screening using Hampton Research and Molecular Dimensions Screening kits. A hanging drop of 3 μL in size containing 1.5 μL of protein with 1.5 μL of the reservoir solution and the drops were equilibrated against 500 μL reservoir solution. The crystallization setup at 20°C yielded thin needle‐shaped crystals for peak 1 (PlyGRCS‐Ec‐CspC complex), which grew as clusters within a month. The crystals appeared in a drop comprising 1.5 μL of protein, 1.5 μL of the reservoir solution (50 mM MES pH 6.0, 5 mM CaCl₂, 35% polyethylene glycol MME 5000) and 1 μL of additive, 4% v/v 2,2,2 trifluoroethanol. X‐ray diffraction data were collected on the beamline ID23 at ESRF, Grenoble, France. The x‐ray data diffracted to 2.1 Å resolution were merged, indexed, and integrated using iMosflm (Powell et al., 2013) and scaled with Aimless from the CCP4 program package (Winn et al., 2011).

The structure determination of the full‐length PlyGRCS by molecular replacement using Phaser‐MR (McCoy et al., 2007) was not straightforward, as a full‐length structure of the model was not available in the database. The sequence identity of CHAP_GRCS and SH3_5_GRCS was about 42%, with the respective domain structures (CHAP and SH3b) available in the RCSB database. Hence, the CHAP (PDB Id: 4CSH; Sanz‐Gaitero et al., ²⁰¹⁴) and SH3b (PDB Id: 5UDM) structures were truncated to alanine wherever the respective residues were mismatched. Initially, the trimmed CHAP domain structure was used as a search model. The oriented structure of the CHAP domain, obtained from the molecular replacement solution, was fixed, and the trimmed SH3b domain structure was subsequently used as a search model. It gave a clear solution for the SH3_5_GRCS domain. The phases obtained from Phaser‐MR were then subjected to Arp/Warp (Langer et al., 2008) for tracing the chain. A few rounds combined with Arp/Warp tracing, manual model building, and refinement gave unambiguous electron density for the full length of PlyGRCS. The crystal structure was refined by using module phenix.refine of the PHENIX package (Adams et al., 2010). The Coot program (Emsley & Cowtan, 2004) was used for tracing the protein chains on the electron density maps.

Surprisingly, during the model building of PlyGRCS on the maps, a distinct electron density map corresponding to a long fragment of an unknown molecule was consistently observed. The polyalanine chain initially fit into this electron density, and subsequent refinement produced clear electron density maps for the side chains like Phe, Trp, Ile, His, and Lys. The fragments containing these residues were subjected to a sequence database search, which led us to confirm a cold shock protein. The residues in the loop connecting the β‐strands of 4 and 5 further confirm the isoform protein Ec‐CspC. Subsequent refinement cycles and the maps for motifs on different chain regions unambiguously showed a small protein (69 aa) of E. coli cold shock protein, Ec‐CspC, which was subsequently confirmed by mass spectroscopy.

The x‐ray data collection, scaling, and refinement statistics are summarized in Table 1. The final structures and refined maps were visualized and illustrated using PyMOL v.1.3r (“CCP4 Newsletter Article: PyMOL: An Open‐Source Molecular Graphics Tool”). The MOLPROBITY program (Chen et al., 2010) was used to assess the stereochemistry of these crystal structures. The structure factors and structural coordinates of the PlyGRCS‐Ec‐CspC complex have been deposited in the RCSB (PDB ID: 8H1l).

4.5. Mass spectrometry

4.5.1. In‐gel digestion

Excised gel bands were destained in 40 mM ammonium bicarbonate and 40% acetonitrile, and the destained bands were then chopped into 1 × 1 mm² pieces and dehydrated with 100% acetonitrile. Protein in‐gel pieces were reduced in 5 mM DTT in 40 mM ammonium bicarbonate prepared freshly and incubated at 60°C for 30 min), followed by alkylation in 20 mM iodoacetamide (IAA) in 40 mM ammonium bicarbonate (freshly prepared and incubated at RT for 10 min). DTT and IAA were completely removed using 100% acetonitrile. Acetonitrile was removed, and Promega sequencing grade trypsin was added in a 1:20 (enzyme: protein ratio) for digestion. Samples with added trypsin were incubated overnight at 37°C. The digestion reaction was quenched by adding 5% formic acid (FA). Peptides were extracted from gel pieces using 5% formic acid in 40% acetonitrile and shaken vigorously in a shaker. Extracted peptides were dried and cleaned using C₁₈ stage tips. Cleaned peptides were dried and stored in a deep freezer until mass spectrometry analysis.

4.5.2. LC–MS/MS analysis

Peptides were analyzed on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, Bremen, Germany) interfaced with Easy‐nLC 1000 nanoflow liquid chromatography system (Thermo Scientific, Bremen, Germany). Vacuum dried each fraction reconstituted in 0.1% FA and loaded onto a 2 cm long pre‐column 75 μm × 2 cm, nanoViper, C₁₈, 3 μm particle, and 100 Å pore size (Thermo scientific Acclaim PepMap 100) and analytical column 2 μm, 75 μm × 50 cm, 75 μm × 50 cm, 100 Å pore size (Thermo scientific PepMap TM RSLC C18) using a linear gradient of 5%–30% of solvent B (0.1% formic acid in 95% acetonitrile) over 100 min and flow rate of 300 nL/min. The total run time was set to 120 min. The mass spectrometer was operated in a data‐dependent acquisition mode. A precursor survey full scan MS (from m/z 350–1600) was acquired in the Orbitrap at a resolution of 120,000 at 200 m/z. The AGC target for MS1 was set as 4 × 10⁵, and the ion filling time was set at 50 ms. The most intense ions with a charge state ≥2 were isolated and fragmented using HCD fragmentation with 34% normalized collision energy and detected at a mass resolution of 30,000 at 200 m/z. The AGC target for MS/MS was 1 × 10⁵, and the ion filling time was set at 100 ms. The isolation width was used as 1.6 m/z.

4.6. Microscale thermophoresis (MST) assay

The fluorescent tris‐NTA dye NT‐647 (NT‐647 His‐tag labeling kit, NanoTemper Technologies, Germany) was used to label recombinant protein Ec‐CspC and Sa‐CspC. The Ec‐CspC and Sa‐CspC proteins concentration were adjusted to 200 nM in 100 μL using the assay buffer. The fluorescent dye concentration was adjusted to 100 nM in 100 μL using the assay buffer. The protein–dye mixture (1:1 ratio) was incubated for 30 min in the dark at room temperature. The protein–dye mixture was centrifuged at 15,000 g for 10 min at 4°C. A pre‐test was performed to check the fluorescence intensity count of the labeled protein at 650 and 280 nm. The purified wild‐type & mutant PlyGRCS proteins and synthesized ssDNA 7mer (TTTTTTT–dT7) (ligands) were used for the interaction studies. The reaction mixture of the experiment consisted of a 16‐serial dilution series of ligands in assay buffer and the labeled protein in a 1:1 ratio. The ligand concentration range in the reaction mixture was used from 2 μM to as low as 61 pM for wild & mutant PlyGRCS and 50 μM to 763 pm for ssDNA. The final labeled protein concentration in the reaction mixture was 50 nM. The samples were loaded into the Monolith standard capillaries, and thermophoresis was measured on a Monolith NT.115 equipment (NanoTemper Technologies, Germany). All the experiments were carried out in triplicate. The acquired data were analyzed by the MO Affinity Analysis 2.2.7 software. The binding constants (K_D) were calculated by fitting the data with a 1:1 stoichiometry according to the law of mass action.

4.7. Turbidity reduction assay

As previously described, the lytic activity of PlyGRCS was performed by turbidity reduction assay (Nelson et al., 2001). To evaluate against S. aureus ATCC 29213 by measuring the reduction in optical density at 600 nm by using UV‐Spectrophotometry. Varying concentration of PlyGRCS was treated against S. aureus (test control), MRSA, S. saprophyticus, and E. coli (negative controls). All the strains used for the experiment were grown at 37°C till the OD₆₀₀ reached 1.0. Cells were pelleted at 4000 rpm for 5 mins and resuspended in 1× phosphate‐buffered saline to an OD₆₀₀ of 1.0. Different concentrations of PlyGRCS ranging from 80 μg/mL to 1.25 μg/mL and PlyGRCS‐Ec‐CspC complex of 80 μg/mL and 10 μg/mL were treated against S. aureus and MRSA. A total of 80–10 μg/mL of PlyGRCS concentration was used for negative controls. The reduction in cells' optical density (OD_600nm) was assessed by taking the readings for every 20 s for 20 min at 37°C. The experiment was performed in triplicate.

For PlyGRCS and individual Csps', that is, Ec‐CspC and Sa‐CspC were taken in molar ratios 1:1, 1:3, and 1:5 of 80 μg/mL (2.73 μM) and 10 μg/mL (0.34 μM) for both and incubated for 1 h, which further treated against S. aureus at 1.0 OD. As a control, 2.73 μM of Ec‐CspC and Sa‐CspC were taken. The OD was measured against S. aureus for 15 min with the same condition as mentioned above.

4.8. Disc diffusion assay

As previously described (Schmelcher et al., 2012), lytic activity was performed based on the disc diffusion assay. The assays were performed to assess the lytic activity of PlyGRCS against S. aureus, MRSA, and negative controls. The cells targeted for the assay were grown at 37°C until the OD₆₀₀ reached 1.0. Cells were pelleted at 4000 rpm for 5 min and resuspended in 1× phosphate buffered saline to an OD₆₀₀ of 1.0. The concentrations of purified PlyGRCS at 80, 40, 20, and 10 μg were treated against 10⁸ CFU cells of different bacterial strains. The plates were kept at 37°C for overnight incubation. The experiment was performed in triplicate.

4.9. Bioinformatics analysis

Nucleotide, Protein sequence similarity searches and identification of conserved domains were carried out with BLAST, CLUSTAL Omega, and CDD tools, which are resources of the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi), Conway Institute (https://clustal.org/omega) and (http://ccd.rhpc.nki.nl). Protein secondary structures, disordered regions, and domain prediction were analyzed with PSIPRED 4.0, DISOPRED3, and DomPRED (http://bioinf.cs.ucl.ac.uk/psipred/).

4.10. Statistical analysis

All data were obtained from repeated assays, with values representing the mean ± standard deviation from three independent experiments. Statistical significance was evaluated with a One‐way ANOVA test in GraphPad Prism 9.3.1.

AUTHOR CONTRIBUTIONS

Balasundaram Padmanabhan designed the research. Gopinatha Krishnappa, Mitali Mandal, Saranya Ganesan, and Sudhagar Babu performed molecular biology, biochemical assays, and protein crystallization. Balasundaram Padmanabhan performed x‐ray data processing, structure determination, and analysis. Veena Kumari Haradara Bahubali provided microbial strains and assisted with experimental design and data interpretation for microbiology work. Sivaraman Padavattan provided scientific inputs. Balasundaram Padmanabhan, Sivaraman Padavattan, Gopinatha Krishnappa, Mitali Mandal, and Saranya Ganesan wrote the original draft. All authors contributed to the final version of the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Data S1: Supporting Inforamtion.

Krishnappa G, Mandal M, Ganesan S, Babu S, Padavattan S, Haradara Bahubali VK, et al. Structural and biochemical insights into the bacteriophage PlyGRCS endolysin targeting methicillin‐resistant Staphylococcus aureus (MRSA) and serendipitous discovery of its interaction with a cold shock protein C (CspC). Protein Science. 2023;32(9):e4737. 10.1002/pro.4737

DATA AVAILABILITY STATEMENT

Structural data are available in the RCSB‐PDB database under the accession number, 8H1I.