Identification of novel adhesins from Group B streptococci by use of phage display reveals that C5a peptidase mediates fibronectin binding

Abstract

Group B streptococci (GBS) are a major cause of pneumonia, sepsis, and meningitis in newborns and infants. GBS initiate infection of the lung by colonizing mucosal surfaces of the respiratory tract; adherence of the bacteria to host cells is presumed to be the initial step in and prerequisite for successful colonization (G. S. Tamura, J. M. Kuypers, S. Smith, H. Raff, and C. E. Rubens, Infect. Immun. 62:2450-2458, 1994). We have performed a genome-wide screen to identify novel genes of GBS that mediate adherence to fibronectin. A shotgun phage display library was constructed from chromosomal DNA of a serotype Ia GBS strain and affinity selected on immobilized fibronectin. DNA sequence analysis of different clones identified 19 genes with homology to known bacterial adhesin genes, virulence genes, genes involved in transport or metabolic processes, and genes with yet-unknown function. One of the isolated phagemid clones showed significant homology to the gene (scpB) for the GBS C5a peptidase, a surface-associated serine protease that specifically cleaves the complement component C5a, a chemotaxin for polymorphonuclear leukocytes. In this work we have demonstrated that affinity-purified recombinant ScpB and a peptide ScpB fragment (ScpB-PDF), similar to the peptide identified in the phagemid, bound fibronectin in a concentration-dependent manner. Adherence assays to fibronectin were performed, comparing an isogenic scpB mutant to the wild-type strain. Approximately 50% less binding was observed with the mutant than with the wild-type strain. The mutant phenotype could be fully restored by in trans complementation of the mutant with the cloned wild-type scpB gene, providing further evidence for the role of ScpB in fibronectin adherence. Our results suggest that C5a peptidase is a bifunctional protein, which enzymatically cleaves C5a and mediates adherence to fibronectin. Since binding of fibronectin has been implicated in attachment and invasion of eukaryotic cells by streptococci, our results may imply a second important role for this surface protein in the pathogenesis of GBS infections.

FIG. 1.

Enrichment of the phage display library as a consequence of subsequent cycles of Fn binding (panning). Enrichment is indicated by the amplification of the number of bound phage with each panning, which also indicates specific binding. Peptide-carrying phages have been enriched by a factor of 10 to 100 in each cycle.

FIG. 2.

Schematic presentation of ScpB. Shaded area, Fn binding fragment (ScpB-PDF, aa 116 to 227); black area, cell wall anchor motif LPTTND at the C terminus; S, signal sequence; Asp-130, His-193, and Ser-512, amino acids in the serine protease active site. The insertion site of the Ω-kan2 cassette for construction of the mutant TOH97 is indicated as a large arrowhead above the protein.

FIG. 3.

Binding of purified rScpB (▪) and rScpB-PDF (⧫) fusion proteins and the control rGST protein (•) to immobilized Fn. Fn-coated microtiter plates were blocked with 5% BSA in PBS, and various concentrations of recombinant proteins were allowed to bind for 2 h. Detection of bound proteins was performed with an anti-GST antibody and a secondary HRP-conjugated anti-goat IgG antibody (see Materials and Methods). The plates were developed with ortho-phenyldiamine, and color development was analyzed by optical density (OD) at 490 nm. Points represent means of triplicates, and standard deviations are indicated. Recombinant proteins bound in a concentration-dependent manner.

FIG. 4.

Adherence of radiolabeled COH1 and TOH97 to immobilized Fn. Microtiter plates were coated with various concentrations of Fn, and [³H]leucine-labeled COH1 (▪) and TOH97 (⧫) were added. Shown is the percentage of adherent bacteria relative to the input. Points represent means of triplicates, and bars represent the standard deviation. TOH97 adhered to Fn approximately 50% less than wild-type COH1.

FIG. 5.

Fn binding of radiolabeled COH1 (▪), TOH97 (⧫), and mutant BEC971 containing the complementation plasmid pBEC101 (▴). Microtiter plates were coated with four different concentrations of Fn, and adherence of radiolabeled bacteria was performed as described in Materials and Methods. Points represent means of triplicates, and standard deviations are indicated. pBEC101 expressed ScpB in the complementing strain BEC971 and restored the binding to immobilized Fn to at least wild-type levels.