The group B streptococcal C5a peptidase is both a specific protease and an invasin

Abstract

The group B streptococcus (GBS) is a major cause of pneumonia, sepsis, and meningitis in neonates and a serious cause of mortality or morbidity in immunocompromised adults. Although these streptococci adhere efficiently and invade a variety of tissue-specific epithelial and endothelial cells, adhesins and invasins are still unknown. All serotypes of GBS studied to date express C5a peptidase (SCPB) on their surface. This investigation addresses the possibility that this relatively large surface protein has additional activities. Rabbit anti-SCPB serum inhibited invasion of lung epithelial A549 cells by the serotype Ia strain O90R, suggesting that SCPB is an invasin. This was confirmed by inserting an in-frame 25-amino-acid deletion into the scpB gene. Invasion of HEp2 and A549 human cell lines was significantly reduced by the mutation. Enzyme-linked immunosorbent assays were used to demonstrate that purified SCPB protein binds directly to HEp2 and A549 cells and also binds the extracellular matrix protein fibronectin. Binding was dose dependent and saturable. These results suggested that SCPB is one of several potential invasins essential for GBS colonization of damaged epithelium.

FIG. 1.

Comparison of adherence (A) and invasion (B) of SCPB⁺ O90R (wild type [WT]) and an SCPB⁻ deletion mutant. Assays were carried out in the absence of serum. Percent adherence is normalized for growth in wells that contain epithelial cells and equals CFU associated with the monolayer divided by total CFU in the well at 2 h postinfection. Invasion is intracellular CFU divided by initial inoculum of CFU. The data are means ± standard errors of the means from three independent experiments. The percentage is indicated above the bars.

FIG. 2.

SCP binding to epithelial cells and Fn. Purified SCP protein was incubated with HEp2 cells (A), A549 cells (B), and Fn (C) for 2 h at 37°C. Bound SCP was detected by rabbit anti-SCPB followed by HRP-goat anti-rabbit IgG. Standard curves for each protein were developed to compensate for the fact that they have somewhat different affinities for the primary antibody. SCPB(del) protein has residues Ala⁴⁹² to Val⁵¹⁷ deleted. Standard errors were less than 8% for all experiments.

FIG. 3.

Binding of Fn to SCPA⁺ O90R and SCPA⁻ O90R(del) streptococci. Increasing amounts of human Fn were added to microtiter plates coated with 10⁶ CFU of fixed wild-type O90R (WT) or SCPB-O90R(del). Bound Fn was detected by sheep anti-human Fn followed by HRP-donkey anti-sheep IgG. Data are means ± standard errors of the means from a single experiment performed in triplicate but represent three independent experiments. Control wells were without bacteria. OD, optical density.

FIG. 4.

Effect of soluble Fn on invasion of epithelial cells. (A) Invasion of A549 cells; (B) invasion of HEp2 cells. Assays were carried out with or without 10 μg of Fn per ml in the well. Bacteria grew at comparable rates in wells with or without Fn. Percent invasion is intracellular CFU divided by the initial inoculum. Data are from three independent experiments and are means ± standard errors of the means.