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

Abstract

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.

Keywords: Streptococcus pyogenes; bacteriophage; cell wall; endolysin; polysaccharide; rhamnose.

Fig 1)

A) Symbolic drawings of the carbohydrate structures of GAS (GAC), polyrhamnose backbone (pRha) in GAVS and M1_dgacI, GCS (GCC), GGS (GGC) and S. mutans (SCC). For simplicity, the reported glycerol phosphate of occasionally present on the GAC and SCC side chains have been omitted. Repeat units are marked with brackets. The pRha backbone with alternating (α1->2) and (α1->3) linkages, whilst the α1->2 Rha being decorated with a β1->3 side chain is a commonality among all PlyC susceptible strains. B) PlyC lysis of streptococcal pathogens. Group A and Group C Streptococci serotypes are susceptible to PlyC-mediated lysis. Group G Streptococci show limited susceptibility and S. mutans is resistant to PlyC lysis. Three SDSE isolates that produce the GAC instead of GGC (SDSE_gac) are susceptible to PlyC treatment. C) Bottom and top view onto WT PlyCB octameric structure with the GlyH and EAD domains omitted (4f78.pdb).

Fig 2)

Precipitation studies of purified 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 at 8 kDa.

Fig 3)

Representation of immunoblot analysis of the cell lysate of E. coli expressing the SCC and GAC pRha and carrying an empty control plasmid (-ve). A) Blot was incubated with PlyCB-Alexa Fluor® 647 (PlyCB_WT^AF647). B) Probing the same samples with the GAC antibodies confirms the presence of GAC in the bands. Molecular mass markers are given in kDa.

Fig 4)

PlyCB binding to E. coli cells were investigated by flow cytometry after labelling with PlyCB_WT^AF647 and PlyCB_R66E^AF647 mutant proteins. Blue: -ve control cells without pRha. Red: pRha producing E. coli cells. Representative histograms are shown. A) Left panel: unstained cells. Right panel: The anti-GAC antibodies (GAC-FITC) were used as a positive control to label the E. coli cells producing pRha. The antibodies do not bind to the E. coli cells carrying an empty plasmid (-ve). B) Left panel: PlyCB_WT^AF647 binds to the E. coli cells producing pRha, but not to the E. coli cells carrying an empty plasmid (-ve). Right panel: PlyCB_R66E^AF647 does not binds to the E. coli cells producing pRha.

Fig 5)

The PlyC-mediated lysis of sacculi purified from S. mutans strains. The lysis was monitored after 10, 20, 30, 40, 50 and 60 min as a decrease in OD₆₀₀. Results are presented as a fold change in OD₆₀₀ of the sacculi incubated with PlyC vs. the sacculi incubated without PlyC. Data points and error bars represent mean values of four biological replicates and standard deviation, respectively. P-values were determined by two-way ANOVA with Dunnett’s multiple comparisons test.