Group A Streptococcus produce pilus-like structures containing protective antigens and Lancefield T antigens

Abstract

Although pili have long been recognized in Gram-negative pathogens as important virulence factors involved in adhesion and invasion, very little is known about extended surface organelles in Gram-positive pathogens. Here we report that Group A Streptococcus (GAS), a Gram-positive human-specific pathogen that causes pharyngitis, impetigo, invasive disease, necrotizing fasciitis, and autoimmune sequelae has long, surface-exposed, pilus-like structures composed of members of a family of extracellular matrix-binding proteins. We describe four variant pili and show that each is recognized by a specific serum of the Lancefield T-typing system, which has been used for over five decades to characterize GAS isolates. Furthermore, we show that immunization of mice with a combination of recombinant pilus proteins confers protection against mucosal challenge with virulent GAS bacteria. The data indicate that induction of a protective immune response against these structures may be a useful strategy for development of a vaccine against disease caused by GAS infection.

Fig. 1.

Schematic representation of the FCT region from seven GAS strains. Genes coding for LPXTG-containing proteins are represented with orange arrows, whereas transcriptional regulators (rofA, nra, and msmRL) are in green and conserved flanking genes (Spy0123 and Spy0136) are in gray. At least one sortase is present in every FCT region (black arrows), and a signal peptidase, lepA, is present in three of four regions (white arrows). In the FCT region of the M1 strains, two transposable elements are also present (blue arrows).

Fig. 2.

FCT proteins form high-molecular-mass structures. (A–D) Immunoblots of cell-wall fractions of GAS strains with antisera specific for LPXTG proteins of strain M6_ISS3650 (A), M1_SF370 (B), M5_ISS4883 (C), and M12_20010296 (D). (E–H) Immunoblots of cell-wall fractions of deletion mutants M1_SF370Δ128 (E), M1_SF370Δ130 (F), M1_SF370ΔSrtC1 (G), and the M1_128 deletion strain complemented with plasmid pAM::128, which contains the M1_128 gene (H) with antisera specific for the pilin components of M1_SF370. The antisera used are given above the lanes, and the strains used are indicated below the lanes.

Fig. 3.

Pilus-like structures observed by electron microscopy. Immunogold labeling and transmission electron microscopy of four GAS strains with antisera raised against pilus proteins. (A) Anti-T6 antisera staining of strain M6_ISS3650. (B) Anti-M1_128 staining of strain M1_SF370. (C) Anti-M5_orf80 staining of strain M5_ISS4883. (D) Anti-M12_EftLSL.A staining of strain M12_20010296. (E) Anti-M6_Cpa staining of strain M6_ISS3650. (F) Anti-M1_Cpa staining of strain M1_SF370. (G) Anti-M1_130 staining of strain M1_SF370. (H) Anti-M1_128 staining of strain M1_SF370Δ128, which lacks the M1_128 gene. (Scale bars, 200 nm.)

Fig. 4.

Pili are trypsin-resistant structures. Flow cytometry of GAS bacteria treated or not with trypsin and stained with sera specific for the major pilus component or a trypsin-sensitive control protein of the respective strains. The histograms obtained by using immune sera on trypsin-treated bacteria are shown in blue, and those obtained by staining untreated bacteria are shown in green. Preimmune staining is shown in black. Shown are strain M6_ISS3650 stained with sera specific for M6 protein (A) or anti-M6_T6 (B), strain M1_SF370 stained with anti-M1 protein (C) or anti-M1_128 (D), strain M5_ISS4883 stained with anti-PrtF (E) or anti-M5_orf80 (F), and strain M12_20010296 with anti-M12 protein (G) or anti-EftLSL.A (H)

Fig. 5.

Pilin proteins are Lancefield T antigens. (A–C) Immunoblots of recombinant pilin components with polyvalent Lancefield T-typing sera. The recombinant proteins are shown above the blot, and the sera pool used is shown below the blot. (D–G) Immunoblots of pilin proteins with monovalent T-typing sera. The recombinant proteins are shown below the blot, and the sera used are shown above the blot. (H and I) Flow cytometry analysis of strain M1_SF370 (H) and the deletion strain M1_SF370Δ128 (I) with T-typing antisera pool T.