Identification and characterization of a novel heme-associated cell surface protein made by Streptococcus pyogenes

Abstract

Analysis of the genome sequence of a serotype M1 group A Streptococcus (GAS) strain identified a gene encoding a previously undescribed putative cell surface protein. The gene was cloned from a serotype M1 strain, and the recombinant protein was overexpressed in Escherichia coli and purified to homogeneity. The purified protein was associated with heme in a 1:1 stoichiometry. This streptococcal heme-associated protein, designated Shp, was produced in vitro by GAS, located on the bacterial cell surface, and accessible to specific antibody raised against the purified recombinant protein. Mice inoculated subcutaneously with GAS and humans with invasive infections and pharyngitis caused by GAS seroconverted to Shp, indicating that Shp was produced in vivo. The blood of mice actively immunized with Shp had significantly higher bactericidal activity than the blood of unimmunized mice. The shp gene was cotranscribed with eight contiguous genes, including homologues of an ABC transporter involved in iron uptake in gram-negative bacteria. Our results indicate that Shp is a novel cell surface heme-associated protein.

FIG. 1.

Reverse transcription-PCR analysis demonstrating a polycistronic transcript containing shp mRNA. RNA from strain MGAS5005 was reverse transcribed into cDNA, and PCR was done with internal ORF-specific primers. Templates used for PCR were RNA (lane R), DNA (lanes D), and cDNA (lanes C). Forward (F) and reverse (R) primers used for PCR amplification are designated according to the corresponding locus in the sequenced GAS strain SF370. Dashes indicate DNA sizes in the 100-bp ladder (lane M, left) and λ HindIII marker (lane M, right). The schematic shows the arrangement of the genes in strain MGAS5005. A putative promoter (P_shp) and rho-independent terminator (stem-loop structure) were present in the regions upstream and downstream of the gene cluster, respectively.

FIG. 2.

TaqMan analysis of shp transcription. TaqMan assays were performed with a probe and primers specific for shp. Cultures of GAS were harvested at OD₆₀₀s of 0.05, 0.1, 0.2, 0.4, and 0.8. The level of shp gene transcript was normalized to the level of transcript derived from gyrA, a gene expressed constitutively throughout the GAS growth cycle (2). The mean level ± standard deviation of triplicate measurements is presented.

FIG. 3.

SDS-PAGE showing purified recombinant Shp. The gel was stained with Coomassie brilliant blue. Lane 1, molecular mass markers; lane 2, E. coli with empty (control) vector; lane 3, E. coli lysate containing Shp; lane 4, purified recombinant Shp.

FIG. 4.

Shp binds heme. (A) UV-visible absorption spectrum of 17 μM Shp in 50 mM Tris-HCl, pH 8.0. (B) Reduction spectra of pyridine hemochrome derived from Shp (solid curve) and purified recombinant M. tuberculosis KatG (open circles), as described in Materials and Methods. The spectra were normalized for comparison.

FIG. 5.

Detection of iron associated with purified Shp by ICP-MS. (A) Mass spectrum of 1.7 μM Shp displaying the iron peak at m/z 56. (B) Iron concentrations in blank, 50 μM recombinant streptococcal dipeptide-binding protein (Dpp) (negative control protein), and 50 μM Shp.

FIG. 6.

Flow cytometric analysis of in vitro production and cell surface location of Shp. (A to E) MGAS5005 (serotype M1) cells harvested at OD₆₀₀s of 0.2 (early exponential phase [EE]) (A), 0.4 (mid-exponential phase [ME]) (B), and 0.8 (early stationary phase [ES]) (C) were treated with Shp-specific polyclonal rabbit antibody (solid lines) or control rabbit antibody (dashed lines), stained with phycoerythrin-conjugated donkey anti-rabbit IgG, and analyzed by flow cytometry. The results at EE (red), ME (blue), and ES (green) were overlaid, showing a higher fluorescence intensity at ES for bacteria treated with Shp-specific antibody (D) but no fluorescence shift in the control antibody treatment (E). (F) MGAS5005 grown in THY (dashed line) or DTHY (solid line) was harvested at an OD₆₀₀ of 0.3 and analyzed as described above.

FIG. 7.

Anti-Shp antibody titers and bactericidal activity of immune mouse blood. (A) The titers of the sera from mice before immunization (open bars) and after the third immunizations (closed bars) were determined by ELISA as described in the text. (B) Direct bactericidal activity. MGAS 5005 bacteria were inoculated in 0.15 ml of heparinized blood from a control mouse (sham immunized) or a mouse actively immunized with Shp. The samples were rotated end-to-end at 37°C for 3 h, and the number of viable bacteria was determined by plating on blood agar plates. The mean colony number ± standard deviation for five mice in each group is presented.