The putative glycosyltransferase-encoding gene cylJ and the group B Streptococcus (GBS)-specific gene cylK modulate hemolysin production and virulence of GBS

Abstract

Group B streptococcus (GBS) expresses a hemolysin/cytolysin that plays an important role in pathogenesis. Using the Himar1 transposon mutagenesis system, a hypohemolytic mutant carrying an interrupted cylJ gene was characterized. cylJ, encoding a putative glycosyltransferase, and cylK, whose product is unknown, are both required for the full hemolytic/cytolytic activity, pigment formation, and virulence of GBS.

FIG. 1.

Structure of the cyl operon in the serotype III GBS strain NEM316 (3). The 12 genes belonging to this cluster are shown, and similarities of the deduced proteins to gene products with known functions in the databases are indicated by shading. The CovR binding site in the promoter region is depicted.

FIG. 2.

Hemolytic/cytolytic activities and pigment formation in GBS strains. The in-frame deletion mutants NEM2456 (ΔcylE), NEM2457 (ΔcylJ), and NEM2458 (ΔcylK) derived from the hyperhemolytic and hyperpigmented strain CCH206 (Pcyl+). (A) Hemolytic phenotype of the mutants on horse blood agar plates (BioMérieux). (B) Pigment formation on Granada medium plates. (C) The cytolytic activity of GBS on A549 human alveolar epithelial cells (multiplicity of infection, 100 bacteria per cell) was assessed using the cytotoxicity detection kit (LDH) (Roche) at different time points (30 min, 1 h, and 2 h). The values were standardized such that the positive control phosphate-buffered saline-Triton 0.1% gave 100% of LDH release. (D) Absorbance profile of pigment extracts from the parental strain Pcyl+ and its ΔcylE, ΔcylJ, and ΔcylK isogenic mutants. The results shown are representative of three independent experiments.

FIG. 3.

Mortality curves in neonatal rats infected with the GBS parental strain Pcyl+ and its ΔcylE, ΔcylJ, and ΔcylK isogenic mutants. Two-day-old Sprague-Dawley rat pups (12 per strain) were inoculated i.p. with 5 × 10⁶ bacteria.