Structural and functional analysis of RopB: a major virulence regulator in Streptococcus pyogenes

Abstract

Group A Streptococcus (GAS) is an exclusive human pathogen that causes significant disease burden. Global regulator RopB of GAS controls the expression of several major virulence factors including secreted protease SpeB during high cell density. However, the molecular mechanism for RopB-dependent speB expression remains unclear. To understand the mechanism of transcription activation by RopB, we determined the crystal structure of the C-terminal domain of RopB. RopB-CTD has the TPR motif, a signature motif involved in protein-peptide interactions and shares significant structural homology with the quorum sensing RRNPP family regulators. Characterization of the high cell density-specific cell-free growth medium demonstrated the presence of a low molecular weight proteinaceous secreted factor that upregulates RopB-dependent speB expression. Together, these results suggest that RopB and its cognate peptide signals constitute an intercellular signalling machinery that controls the virulence gene expression in concert with population density. Structure-guided mutational analyses of RopB dimer interface demonstrated that single alanine substitutions at this critical interface significantly altered RopB-dependent speB expression and attenuated GAS virulence. Results presented here suggested that a properly aligned RopB dimer interface is important for GAS pathogenesis and highlighted the dimerization interactions as a plausible therapeutic target for the development of novel antimicrobials.

Figure 1

Crystal structure of RopB‐CTD. A. Ribbon representation of the individual subunit of RopB‐CTD highlighting the positions of the 5 TPR motifs. Each TPR motif and the capping helix are colour coded and labelled. The amino‐ and carboxy‐termini of the molecule are labelled as N and C, respectively. B. Ribbon diagram of the crystallographic RopB‐CTD dimer. Individual subunits of a dimer molecule are colour‐coded. The N‐ and C‐termini are labelled and the ′ indicates the structural elements from the second subunit of a dimer. The putative ligand‐binding pocket predicted by the program ‘COACH’ is depicted with spheres. C. Amino acid sequence of RopB‐CTD with the corresponding structure elements are marked and colour‐coded as in panel A.

Figure 2

Expression of speB is activated by a secreted product present in the high cell density‐specific culture supernatant. speB transcript level analysis of wild type and isogenic ΔropB mutant strains in the indicated cell‐free culture supernatants. Secretome preparation and secretome swap assay were performed as described in the Methods section. The data were graphed as the mean ± standard deviation. ME, mid exponential phase of growth; ME SEC, total secretome prepared from mid‐exponential growth phase; STAT SEC, total secretome prepared from stationary growth phase; <3 kDa SEC, filtrate prepared by subjecting the STAT‐SEC to <3 kDa cut‐off filtration; and Prot‐K, STAT SEC digested by proteinase‐K.

Figure 3

Characterization of isoallelic ropB mutant strains at the dimer interface. A. Ribbon representation of the crystal structure of RopB‐CTD dimer. The individual subunits of RopB‐CTD dimer are colour‐coded. The positions of C_α atoms of amino acids included in the dimer interface mutational analysis are indicated as spheres and are labelled. B. A close up view of the dimer interfaces I and II, boxed in panel A, of RopB‐CTD. The dimer interfaces are boxed and labelled, and the side chains of amino acids involved in dimerization interactions are shown as spheres. The participating amino acids of dimer interface I is coloured in green, whereas the dimer interface II is shaded in red. Analogous residues from the same interface of opposing subunits are coloured in dark and light shades. C. qRT‐PCR analysis of ropB gene transcript levels in ropB isoallelic mutant strains. The data were graphed as the mean ± standard deviation. D. Immunoblotting detection of recombinant WT RopB‐CTD and its mutant derivatives by anti‐RopB polyclonal rabbit antibody and chemiluminescence. E. qRT‐PCR analysis of speB transcript levels in WT and ropB isoallelic mutant strains as determined in B. F. Western immunoblot analysis of secreted SpeB in filtered growth media from indicated strains. Growth media collected were probed using anti‐SpeB polyclonal rabbit antibody and chemiluminescence. The masses of molecular weight markers (M) in kilodaltons (kDa) are marked. G. Milk plate assay to assess SpeB protease activity. Protease activity was determined by the presence of clear zone around the bacterial growth.

Figure 4

Effect of single alanine substitutions at RopB dimer interface on secondary structure and oligomerization. A. Far‐UV CD spectra of recombinant wild type (WT) and mutant RopB‐CTD in 20 mM Tris pH 7.5 were recorded in the range 200–260 nm at room temperature. A protein concentration of 0.2 mg/ml was used and the spectra were reported in units of mean residue molar ellipticity and plotted against wavelength. B. Purified recombinant WT and mutant RopB‐CTD proteins were run on a superdex 200 size exclusion column. The minor peak at elution volume 8.5 ml observed for defective recombinant mutant proteins is indicated with arrows. The elution volumes corresponding to molecular weight markers (in kDa) are shown on the top. C and D. The presence of RopB‐CTD in the fractions of peaks 1 (C) and 2 (D), as assessed by Western immunoblotting.

Figure 5

Effect of naturally occurring amino acid substitutions at the RopB dimer interface on the secondary structure and oligomerization. A. Far‐UV CD spectra of recombinant wild type (WT) and mutant RopB‐CTD were recorded and analysed as described in Fig. 4. B. Analyses of purified recombinant WT and mutant RopB‐CTD proteins on a size exclusion column. The minor peak at elution volume 8.5 ml observed for defective recombinant mutant proteins is indicated with arrows. C and D. The presence of RopB‐CTD in the fractions of peaks 1 (C) and 2 (D), as assessed by Western immunoblotting.

Figure 6

Characterization of isoallelic ropB mutant strains at the putative ligand‐binding pocket and interdomain interface. A. Ribbon representation of RopB‐CTD dimer. The individual subunits of RopB‐CTD dimer are colour‐coded and the N‐ and C‐termini of one subunit is marked. The location of N152 and N192 within the binding pocket of one subunit are marked. The side chain of Y182 from the C‐terminal domain and E59 of helix α1 from a symmetry‐related molecule are depicted as ball and stick models. B. Characterization of isoallelic ropB mutant strains for ropB gene transcript levels by qRT‐PCR. C. Immunoblotting to detect recombinant WT RopB‐CTD and its mutant derivatives. Characterization of isoallelic ropB mutant strains for speB transcript levels (D), secreted SpeB levels (E), and SpeB protease activity (F).

Figure 7

Single alanine substitutions at key residues identified in RopB‐CTD structure decreases GAS virulence in mouse models of infection. A. Ten outbred CD‐1 mice per strain were injected intramuscularly with 1 × 10⁷ CFU of each strain. Kaplan‐Meier survival curve with P values derived by log rank test. B. Histopathologic analysis of hindlimb lesions from mice infected with each indicated strain. SpeB producing strains caused large lesions with extensive muscle damage (boxed), whereas the SpeB‐deficient strains caused small confined lesions with markedly less tissue damage (arrows).