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. 2007 Nov;75(11):5405-14.
doi: 10.1128/IAI.00717-07. Epub 2007 Aug 20.

The surface protein Srr-1 of Streptococcus agalactiae binds human keratin 4 and promotes adherence to epithelial HEp-2 cells

Ulrike Samen  1 , Bernhard J Eikmanns, Dieter J Reinscheid, Frédéric Borges
Affiliations

The surface protein Srr-1 of Streptococcus agalactiae binds human keratin 4 and promotes adherence to epithelial HEp-2 cells

Ulrike Samen et al. Infect Immun. 2007 Nov.

Abstract

Streptococcus agalactiae is frequently the cause of bacterial sepsis and meningitis in neonates. In addition, it is a commensal bacterium that colonizes the mammalian gastrointestinal tract. During its commensal and pathogenic lifestyles, S. agalactiae colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present study, the serine-rich repeat protein Srr-1 from S. agalactiae was functionally investigated. Immunofluorescence microscopy showed that Srr-1 was localized on the surface of streptococcal cells. The Srr-1 protein was shown to interact with a 62-kDa protein in human saliva, which was identified by matrix-assisted laser desorption ionization-time-of-flight analysis as human keratin 4 (K4). Immunoblot and enzyme-linked immunosorbent assay experiments allowed us to narrow down the K4 binding domain in Srr-1 to a region of 157 amino acids (aa). Furthermore, the Srr-1 binding domain of K4 was identified in the C-terminal 255 aa of human K4. Deletion of the srr-1 gene in the genome of S. agalactiae revealed that this gene plays a role in bacterial binding to human K4 and that it is involved in adherence to epithelial HEp-2 cells. Binding to immobilized K4 and adherence to HEp-2 cells were restored by introducing the srr-1 gene on a shuttle plasmid into the srr-1 mutant. Furthermore, incubation of HEp-2 cells with the K4 binding domain of Srr-1 blocked S. agalactiae adherence to epithelial cells in a dose-dependent fashion. This is the first report describing the interaction of a bacterial protein with human K4.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of the Srr-1 protein of S. agalactiae (A) and ability of truncated Srr-1 derivatives to bind human K4 (B and C). In the full-length Srr-1 protein, the K4 binding domain (K4-BD), the serine-rich repeat regions (vertical hatching), the membrane-spanning region (diagonal hatching), and the LPXTG cell wall-anchoring motif are indicated. The Srr-1 protein fragments produced in E. coli as hexahistidyl-tagged proteins are schematically represented in panel B. The results of immunoblot analysis presented in panel C are summarized in panel B; effective binding and undetected binding to human K4 are represented by + and −, respectively. Localization of the K4 binding domain in Srr-1 is shown in panel C. After SDS-PAGE, K4 was blotted on nitrocellulose. The membranes were incubated with anti-K4 antibodies (control) or with the protein Srr-1-N (a), Srr-1-N1 (b), Srr-1-N2N3 (c), Srr-1-N2 (d), or Srr-1-N3 (e), followed by incubation with anti-His tag antibodies.
FIG. 2.
FIG. 2.
Intraspecies size variability of srr-1 in different S. agalactiae isolates. Chromosomal DNAs from 98 human S. agalactiae isolates belonging to serotypes Ia, Ib, II, III, IV, and V were used as templates for amplification by PCR of the 5′ (A) and 3′ (B) regions of srr-1. The 5′ and 3′ regions of srr-1 are bp 1 to 1928 and bp 1929 to 3930, respectively, in strain NEM316. A representative sample of the 98 tested strains is presented.
FIG. 3.
FIG. 3.
Localization of Srr-1 on the surface of S. agalactiae. S. agalactiae strain 6313 and the Δsrr-1 mutant were immobilized on slides and incubated either with anti-FbsA monoclonal antibodies (A), with anti-Srr-1 serum (B), or, as a negative control, without primary antibodies (C). Subsequently, bound antibodies were detected by measuring the fluorescence of TRITC-labeled anti-mouse IgG Fab fragments, and pictures were taken after phase-contrast and fluorescence microscopy visualization.
FIG. 4.
FIG. 4.
Binding of Srr-1-N to human K4. (A) Binding of Srr-1-N to proteins of bovine, murine, or human origin was investigated by immunoblotting. Crude extracts from different organs (lane 1, bovine brain endothelium; lane 2, bovine lung; lane 3, bovine joint liquid; lane 4, bovine brain; lane 6, murine lung) and human saliva (lane 5) were size separated by SDS-PAGE and blotted onto nitrocellulose. The membrane was incubated with Srr-1-N fusion protein and subsequently with anti-Srr-1 antibodies. Bound primary antibodies were detected with peroxidase-labeled anti-mouse IgG Fab fragments with subsequent visualization by chemiluminescence. (B) Binding of Srr-1 to human K4 and K6 was tested by immunoblotting. Purified human K6 (lanes 1 and 3) and K4 (lanes 2 and 4) were size separated by SDS-PAGE (lanes 1 and 2), blotted onto nitrocellulose, and tested for binding to Srr-1-N (lanes 3 and 4) as described for panel A. (C) Identification of Srr-1 binding site in human K4. Full-length human K4 and the truncated proteins K4N and K4C, comprising the N-terminal and C-terminal regions of K4, respectively, were size separated by SDS-PAGE, blotted onto nitrocellulose, and tested for binding to Srr-1-N as described for panel A.
FIG. 5.
FIG. 5.
Binding of Srr-1-derived proteins to immobilized human K4 in a capture ELISA (A) and attachment of S. agalactiae to immobilized human K4 (B). (A) In the ELISA, microtiter wells were coated with a fixed amount of K4, followed by the addition of increasing concentrations of the fusion proteins Srr-1-N (diamonds), Srr-1-N2N3 (squares), Srr-1-N3 (triangles), and Srr-1-N1 (circles). Bound Srr-1 fusion proteins were detected by anti-His tag antibodies, followed by peroxidase-labeled anti-mouse IgG Fab fragments. Color development was initiated by the addition of tetramethyl-benzidine substrate and stopped with H2SO4. The absorbance of the microtiter wells was measured at 450 nm. Values represent the means and standard deviations from three independent experiments, each performed in triplicate. (B) Bacterial attachment to immobilized K4 was quantified with FITC-labeled bacteria. The ordinate represents the percentage of bacteria bound to immobilized K4 in relation to the number of input bacteria. The asterisk indicates a significant (P < 0.01) difference in the binding abilities of the indicated strains. Each assay was performed at least four times in triplicate.
FIG. 6.
FIG. 6.
Role of Srr-1 in bacterial adherence to and invasion of HEp-2 cells. The epithelial cell line HEp-2 was infected with equal amounts of bacteria of each strain, and the numbers of cell-adherent (A) and internalized (B) bacteria were related to the number of input bacteria. The asterisk indicates a significant difference in the adherence abilities of the indicated strains (P < 0.05). For competition experiments with soluble purified proteins (C), HEp-2 cells were incubated, prior to infection, with increasing concentrations of proteins. The adherence assay was subsequently performed with the wild-type strain S. agalactiae 6313 as for panel A. Error bars indicate standard deviations.
FIG. 7.
FIG. 7.
Accessibility of K4 on the surface of epithelial HEp-2 cells. Cells were incubated with either monoclonal anti-K4 antibodies (white) or monoclonal anti-rabbit IgG antibodies as a negative control (black). After washing, cells were probed with FITC-labeled Fab fragments raised against mouse IgG, and fluorescent HEp-2 cells were counted with a flow cytometer.
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