Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin

Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively.


Abbreviations (alphabetical order) 27
Cell wall-binding domain (CBD), enzymatically active domain (EAD), Glucose (Glc), glycerol 28 phosphate (GroP), Glycosyl Hydrolase (GlyH), Group  Endolysins are bacteriophage-encoded PG hydrolases that normally function from within the cell 57 to lyse the bacterial host, releasing progeny phage and completing the phage lifecycle [1]. 58 However, the lytic activity of endolysins can be harnessed for antimicrobial use due to their ability 59 to equally lyse bacteria when applied exogenously, without infection by a parental phage. Due to 60 their direct lytic action on target PG, endolysins are not affected by efflux pumps, alterations in 61 metabolism, or other mechanisms of antibiotic resistance, making them ideal candidates for 62 development against multi-drug resistant organisms [2][3][4]. Notably, at least three endolysins, 63 some of which are active against methicillin-resistant Staphylococcus aureus, are currently being 64 evaluated in human clinical trials for their antimicrobial activity (reviewed in [5]). 65 66 Most endolysins, and in particular those from phage that infect Gram-positive bacterial hosts, are 67 comprised of modular domains. An enzymatically active domain (EAD) is generally found in the 68 N-terminal region, while a cell wall-binding domain (CBD) is located in the C-terminal region [6]. 69 As the name implies, the EAD is a catalytic domain that is responsible for cleaving specific bonds 70 in the PG, the nature of which is dependent on the mechanistic class of the EAD. Occasionally, 71 endolysins contain two EADs, although both are not necessarily active. The CBD binds at high 72 affinity [7] to a cell wall-specific epitope and was suggested to dictate genus, species and serovar-73 specificity of the endolysin. The CBD targets may be surface carbohydrates, wall teichoic acids 74 linked to the Gram-positive bacterial cell wall, or the PG itself [8]. 75

76
The endolysin now known as PlyC is one of the first described endolysins and remains one of the 77 most studied. In 1934, Alice Evans noted a "nascent lysis" activity derived from streptococcal 78 phage lysates on streptococcal strains that were not sensitive to the phage itself [9]. By 1957, 79 Krause had determined that the phage used by Evans was specific for Group C Streptococci 80 (GCS), but an "enzyme" produced by the phage could lyse Groups A, A-variant, and C 81 medium supplemented with either 50 µg/ml kanamycin, 100 µg/ml ampicillin or 35 µg/ml 133 chloramphenicol as needed. 134 135 Bacterial strains E. coli CS2775 were transformed with pRGP1 plasmid [28], gacABCDEFG [23] 136 to produce pRha or empty plasmid control (pHD0131). The bacterial cells were grown overnight 137 in LB containing erythromycin (150 g/ml) at 37°C and used next day for whole cell Western blots 138 and FACS and microscopy analysis. 139 140

Recombinant expression and purification of PlyCB WT and PlyCB R66E 141
PlyCB WT (GenBank ID: NC_004814.1:7517-7735) was expressed from a pBAD24 vector and 142 purified from BL21 cells as previously described [12]. In brief, the culture was grown in LB and 143 induced with 0.25% L-arabinose at OD 600 ~1.2-1.4 (Alfa Aesar, cat no. A11921). Cultures were 144 grown at 37°C with shaking at 180 RPM for 3-4 hours, centrifuged at 4500 x g and resuspended 145 in phosphate-buffered saline (PBS). Lysis was performed by a French press (1800 psi). 146 Benzonase (Millipore Sigma, was added and the lysate incubated at room 147 temperature with rotation for 20-30 min. The lysate was centrifuged at 20,000 x g for 20 min and 148 the cleared lysate was passed through a 0.45-µm filter and loaded onto a XK-26/20 column 149 (Cytiva) with 30-35 ml ceramic hydroxyapatite (Bio-Rad, cat no. 1582000). PlyCB WT was eluted 150 from the column with three column volumes of 1 M sodium phosphate buffer (pH 7.2). Protein 151 was subsequently dialyzed in PBS, 10% glycerol and stored at -80°C until use. Protein purification 152 of PlyCB R66E was performed as PlyCB WT [13]. The fluorescent labeling of PlyCB WT and PlyCB R66E 153 was performed using the manufacturer's recommended guidelines (Thermo Fisher, cat. No. 154 A20174). Analytical gel filtration was used to determine the multimeric nature of PlyCB and 155 PlyCB R66E . A total of 100 µl (0.1 mg) of each protein was applied to a Superose 12 10/300 GL 156 column (Cytiva) and run in isocratic conditions in PBS buffer for 1.5 column volumes on an AKTA 157 FPLC system (Cytiva). Gel filtration standards (Bio-Rad) were also run under identical conditions. 158 Purification of GAC 160 GAS cells were grown overnight in THY media at 37˚C. Cultures were centrifuged at 4,500 x g. 161 Pellets were washed and resuspended in 40 ml distilled water per each original liter of media 162 used and combined in an 800 ml beaker. 22.5 ml 4N sodium nitrite (5 ml per liter of culture) was 163 added to the beaker in addition to 22.5 ml glacial acetic acid (5 ml per liter of culture). An orbital 164 shaker was used to gently mix the beaker for 15 min in a hood. The mixture was centrifuged in 165 500 ml bottles at 8000 x g for 15 min. The supernatant was decanted to a new beaker and 166 neutralized with 1M sodium hydroxide. The total solution, about 300 ml, was filtered with a 0.45-167 micron filter assembly. 50-50 ml aliquots were deposited in a 3.5 kDa membrane and dialyzed in 168 a 4-liter beaker overnight with water. The following day, the solution was concentrated using an 169 containing either GAC or L-Rha at known concentrations were added to a 1.5 ml microfuge tube. 179 To this same tube, 320 µL of the anthrone reagent was added. Samples were boiled at 98°C for 180 10 min in a heat block. Samples were cooled to room temperature, transferred to a quartz plate, 181 and the absorbance at 580 nm was recorded using a spectrophotometer. Rha concentration was 182 interpolated using an L-Rha standard curve. 183 184

Precipitation of PlyCB with GAC 185
PlyCB WT and PlyCB R66E samples were defrosted from storage at -80°C. Lyophilized GAC was 186 resuspended in PBS. Both proteins and GAC were added to a 3.5 kDa dialysis membrane and 187 dialyzed overnight in PBS. Protein concentrations were determined using a NanoDrop 188 spectrophotometer (Thermo Fisher ND-2000) at 280 nm and were diluted to 5 mg/ml. The GAC 189 was also assayed and diluted with PBS to 1.6 mg/ml. One-hundred microliters of proteins and 190 100 µl of GAC or PBS were mixed in a 250 µl quartz plate and allowed to incubate without shaking 191 at room temperature. Visible precipitate formed in samples in 5-8 minutes. After recording the 192 precipitate at 340 nm using a Spectramax® M5 (Molecular Devices) spectrophotometer, the total 193 sample volume was transferred to a 1.5 ml microfuge tube. Samples were centrifuged at 14,000 194 x g to pellet the precipitate and supernatants were transferred to new 1.5 ml microfuge tubes. 195 Two-hundred microliters of 8 M urea was added to the pellet. Pellets were resuspended and 5 µl 196 of either pellet or supernatant were added to 40 µl water with 8 µl 6x Laemmli buffer with DTT. 197 Samples were boiled at 98°C for 8 min, and then 12.5 µl were loaded onto a 7.5%  and run for 32 min at 200 V. Proteins were visualized using Coomassie stain. 199

Lysis assay 201
A turbidity reduction assay was used to ascertain strain sensitivity to PlyC. This assay was 202 strains were grown in THY at 37˚C overnight in 5% CO 2 , except for S. mutans, which was grown 210 in THB media. Next day, the bacterial cells were inoculated in 1:100 fresh media and grown until 211 mid-logarithmic phase (OD 600 1.0). The cells were washed in PBS and resuspended to an OD 600 212 of 2.0. In a 96-well plate, to a 100 µl of bacterial cells, 100 µl of PlyC [1 µg/ml] was added and 213 immediately read at an absorbance of OD 600 . The obtained values were standardized by 214 subtracting from the background values. The data is plotted using GraphPad Prism version 9. 215 216

SDS-PAGE and blotting analysis 217
PlyCB WT binding to recombinant E. coli expressing pRha was conducted using blot analysis. 218 Briefly, the lysate from the overnight cultures was analyzed in 20% tricine gels. SDS-PAGE and 219 protein transfers were performed according to manufactures instructions, Atto Ae-6050 Mini Gel 220 chamber and Novex protein separation from Thermo Fisher, respectively. The PVDF membranes 221 were blocked with 5% non-fat dry milk with Tris-Buffered Saline, 0.1% Tween® 20 detergent prior 222 to incubation with PlyCB WT labelled with Alexa Fluor® 647 (1:1000) for one hour at room 223 temperature. Goat anti-rabbit GAC antibodies conjugated with IRDye® 800CW were used as a 224 positive control (abcam ab216773). The resulting blots were imaged using the Licor Odyssey FC 225 Imaging System. All the blots were processed in parallel under the same conditions. 226 cluster in parts with the GAC gene cluster [33][34][35]. We therefore expanded the previously reported 264 PlyC streptococci cell lysis assay used by Nelson et al. [2] to investigate those new isolates named 265 SDSE_gac. We also tested if PlyC was able to lyse a selection of GAS serotypes including a 266 newly emerged isolate M1 UK [36], and included negative controls GGS isolates and S. mutans 267 serotype C ( 277 Importantly, all strains tested in this study that are susceptible to PlyC lysis produce a cell wall 278 polysaccharide that contains the pRha backbone and a -linked sugar substituent on the 1,2-279 linked Rha (Fig 1A). We therefore suggest that the pRha backbone with and without a side chain 280 are both vital ligands to assist PlyC activity and the new SDSE_gac isolates will also be 281 susceptible to PlyC treatment due to production of the GAC. 282 283 Purified GAC precipitates PlyCB -but not PlyCB R66E 284 The lysis assay of GAS cells, and in particular of the SDSE_gac variants, suggests that either the 285 ubiquitous pRha or GAC in GAS cells is the ligand for PlyCB. We propose that the PlyCB 286 octameric CBD binds GAC and/or the GAC pRha backbone. We tested this hypothesis by 287 investigating the binding of PlyC to partially purified GAC. We hypothesized if the GAC was able 288 to precipitate PlyCB, an interaction of the two systems must have occurred [38]. As a negative 289 control, we employed the previously published inactive mutant PlyCB R66E , which lost the ability to 290 bind to GAS cells [13]. Consistent with prior analytical gel filtration and dynamic light scattering 291 results [12], as well as the crystal structures [13,39,40], both PlyCB and its R66E mutant self-292 assemble into stable octameric structures (Fig 1C, SF 1). The purified proteins were incubated 293 with the extracted GAC, and precipitation was monitored at 340 nm, a standard wavelength for 294 measuring protein aggregation [41, 42] ( Fig. 2A, B). Whilst keeping the PlyCB concentration 295 constant, we varied the concentration of GAC. Within five minutes at room temperature the 296 solution became turbid, suggesting aggregation ( Fig. 2A). When the PlyCB concentration was 297 kept constant and the GAC concentration was varied, the turbidity correlated with PlyCB 298 concentration in a dose dependent manner, suggesting that PlyCB requires GAC to aggregate. 299 Importantly, PlyCB did not self-aggregate when no GAC was added in the assay. Furthermore, 300 no aggregation was detected when PlyCB R66E was incubated with purified GAC (Fig. 2B). To 301 demonstrate the presence of PlyCB in the precipitates, we analyzed the soluble and pellet 302 fractions (Fig. 2C, D). A higher yield of aggregated PlyCB was found in the pelleted samples when 303 compared to the soluble fraction (Fig. 2D). A similar precipitation effect was observed when we 304 varied the PlyCB concentration and kept the GAC concentration constant (Fig. 2E, F), 305 demonstrating that both species are necessary for an interaction. 306 307 308 PlyCB binds to recombinantly produced pRha backbone 309 The purified GAC from bacteria contains a mixture of carbohydrates, including the fully decorated 310 GAC with GroP[21] and a small proportion of the polysaccharide lacking the side chains [43]. PlyC 311 is able to lyse a number of GAS mutants including GAVS and dgacI_M1 [2,11,25,44], that 312 decorate the cell wall with the unmodified GAC lacking the side chains (Fig. 1A, B), suggesting 313 that the pRha backbone of the GAC is the minimal carbohydrate structure required for PlyCB 314 binding. To test this hypothesis, we recombinantly produced the pRha backbone in E. coli cells. 315 We and others have previously reported that the S. mutans sccABCDEFG gene cluster, when 316 transformed into E. coli cells, functionally produces the pRha backbone attached to the lipid A 317 [23,28]. Additionally, to understand if PlyCB recognizes a specific pRha backbone, we 318 engineered E. coli cells expressing the GAC gene cluster gacABCDEFG required for the GAC 319 pRha production. E. coli cells carrying an empty plasmid were used as a negative control. 320 321 Next, we investigated the binding of PlyCB conjugated with Alexa Fluor® 647 (PlyCB AF647 ) to an 322 E. coli total cell lysate expressing the pRha backbone of the SCC or GAC, respectively (Fig. 3A). 323 The blotted membranes were incubated with PlyCB AF647 , and a positive interaction between 324 PlyCB AF647 and the E. coli produced pRha is visualized as a prominent band around 40 kDa. The 325 size of the band agrees with the band detected by anti-GAC antibodies that were previously 326 reported to recognize pRha [23] (Fig. 3B). Importantly, PlyCB AF647 and GAC antibodies do not 327 interact with the cell lysate of E. coli expressing an empty plasmid (Fig. 3 A, B). Contrary, the 328 R66E-mutant protein was not able to detect the pRha of the PAGE separated sample (SF 2). We 329 further confirmed the ability of PlyCB to bind to E. coli cells decorated with the pRha by fluorescent 330 microscopy (SF 3). Only cells that produce the pRha are detected by the PlyCB AF647 , in agreement 331 with the results of the blot analysis. 332

333
To gather additional evidence that PlyCB interacts with the pRha backbone, we established a flow 334 cytometry assay to analyse the binding of PlyCB AF647 to pRha-producing E. coli. Unstained E. coli 335 cells that express pRha or carry an empty plasmid sort in the identical range (Fig. 4A). The GAC 336 antibodies label exclusively the cells producing pRha (Fig. 4A). A similar pattern of the GAC 337 antibodies binding is observed when the cells were incubated with PlyCB AF647 (Fig. 4B). Contrary, 338 the PlyCB R66E AF647 mutant protein does not bind to E. coli cells, and PlyCB AF647 does not interact 339 with the cells expressing an empty vector (Fig. 4B). Taken together, these data provide the first 340 definitive evidence that the pRha backbone of GAC and SCC is a binding receptor of the PlyCB 341 octameric subunit. 342 343 PlyC lyses engineered S. mutans producing the GAC 344 Despite the fact that the SCC pRha backbone is identical to the GAC, S. mutans is resistant to 345 PlyC lysis (Fig. 1A). To get a better understanding why S. mutans is resistant to PlyC, we 346 compared PlyC-induced lysis of the sacculi purified from S. mutans WT and a number of mutant 347 strains producing different SCC variants (Fig. 5). First, we examined the sccH mutant producing 348 the GroP-deficient SCC [21]. Similar to S. mutans WT, sccH was resistant to PlyC-mediated 349 lysis (Fig. 5). Second, we tested the sccN deletion mutant, sccN, that is deficient in the enzyme 350 required for generation of the Glc side chains [45]. A time dependent lysis is observed for sccN. 351 Expression of the WT copy of sccN in sccN (the sccN:psccN strain) fully restored the resistance 352 of the bacteria to PlyC (Fig. 5). These observations clearly suggest that PlyC is able to bind to the 353 S. mutans cells producing the unmodified pRha backbone, and the Glc side chains in SCC hinders 354 PlyC binding. We then investigated whether the addition of the GAC GlcNAc side chains to the 355 pRha backbone affects sensitivity of the engineered S. mutans sacculi to PlyC-induced lysis. We 356 expressed the GAS genes gacHIJKL required for the formation and addition of the GlcNAc side 357 chains and GroP to GAC [21], in the sccN background strain in two versions, creating the 358 sccN:pgacHI*JKL and sccN:pgacHIJKL strains [45]. The plasmid pgacHI*JKL contains an 359 inserted stop codon in the gacI gene required for generation of the GlcNAc side chain, and, 360 therefore, the sccN:pgacHI*JKL strain produces the unmodified SCC lacking any side chains 361 (Fig. 5). As expected, the sacculi isolated from this strain remains susceptible to PlyC lysis. We 362 previously showed that in sccN:pgacHIJKL, the Glc side chains are replaced with the GlcNAc 363 side chains [45]. Interestingly, expression of gacHIJKL in sccN did not restore the resistance of 364 the bacteria to PlyC (Fig. 5), indicating that the GlcNAc side chains do not obstruct PlyC binding. 365 Lastly, we analyzed PlyC-mediated lysis of sacculi purified from the rgpG mutant, which is 366 deficient in SCC expression [45]. The RgpG protein catalyzes the first step in SCC biosynthesis 367 [46]. In comparison to sccN, PlyC-induced lysis of rgpG was less pronounced (Fig. 5), 368 indicating the importance of the pRha backbone of SCC in PlyC activity and supporting the 369 findings that the pRha backbone is a ligand contributing to PlyC binding. These studies reveal 370 that if the SCC is 'unmasked' i.e., stripped of the Glc and Glc-GroP side chains, it becomes a 371 ligand for PlyCB and that S. mutans is PlyC susceptible if SCC is replaced with GAC. 372 373 374

Concluding remarks 375 376
A structural feature of the PlyCB protein remains to be discovered that explains why only certain 377 streptococci are susceptible to PlyC's lytic activity. The pRha decorated with an linked side 378 chain sugar appears not compatible with the PlyCB ligand binding site and therefore only those 379 streptococci expressing pRha decorated with -linked substituents, such as GlcNAc and GlcNAc-380 GroP are susceptible. This could potentially be exploited for diagnostic purposes or in the case of 381 SCC and Group G Streptococci, opens up the potential for novel therapeutic approaches. If SCC 382 was treated by PlyC in combination with an additional enzyme that removes the linked side 383 chains in these streptococcal carbohydrates, this would expose the pRha backbone and 384 subsequently make these strains susceptible. A recently accepted manuscript by Boendum et al. 385 [40] reported 19 potential binding states of tetrarhamnose to PlyCB. Once the pRha ligand site 386 has been identified, this could also be further exploited by directed evolution approaches to 387 generate PlyCB protein variants that are capable to bind the carbohydrates from, for example, 388 SCC and Group G Streptococci. 389 390 Whilst much has been learned about the structure and function of PlyC in the past 20 years, many 391 questions remain, specifically with respect to its interaction with the PG. Considering the average 392 length of the cellular pRha is 7-10 kDa [25] and that the 1,2-1,3-pRha with or without -393 configured GlcNAc/GalNAc-side chains bind to PlyCB, it is inviting to speculate that an element 394 of avidity is responsible for tight binding of the PlyCB octamer to the streptococcal surface, in 395 agreement with the recent accepted manuscript [40]. Further proof is needed to substantiate this 396 hypothesis. Another question lies in the actions of the EADs relative to the PlyCB octamer. PlyC 397 clearly has a high turnover as demonstrated in multiple biochemical assays. However, it is 398 unknown if PlyCB "docks" to the surface and the flexibility of the holoenzyme allows the PlyC 399 EADs to cleave multiple bonds in a localized area weakening the overall superstructure of the 400 PG. Alternatively, the enzymatic turnover could be dictated by a balance of on and off rates of the 401 EADs and CBD monomers leading to widespread hydrolysis of the PG. Lastly, it is unknown 402 whether PlyC binds, cleaves, and releases the PG at random points on the streptococcal surface 403 or works its way down a single strand of PG in a processive manner. It is noteworthy that cellulase  Walker, M. J., Rush, J. S., Korotkov,K. V.,Widmalm,G.,van Sorge,N. M. and Korotkova,N. 548 ( Precipitation studies of puri ed PlyCB and GAC reveal direct interaction of PlyCB with GAC. A) The PlyCB concentration is kept constant whilst the GAC concentration is varied. Visible precipitate forms at the higher concentrations. B) The precipitate level is measured spectrophotometrically at 340 nm and compared to the mutant PlyCB_R66E, which does not bind the GAC. C) Coomassie stained and D) densitometry analysis of PlyCB protein from the supernatant fraction and aggregates (pellets). E, F) The same dose dependency is observed when the PlyCB concentration is varied. Arrowhead depicts PlyCB protein (8 kDa