Expression and functional properties of the Streptococcus intermedius surface protein antigen I/II

Abstract

Streptococcus intermedius is associated with deep-seated purulent infections. In this study, we investigated expression and functional activities of antigen I/II in S. intermedius. The S. intermedius antigen I/II appeared to be cell surface associated, with a molecular mass of approximately 160 kDa. Northern blotting indicated that the S. intermedius NCTC 11324 antigen I/II gene was transcribed as a monocistronic message. Maximum expression was seen during the early exponential phase. Insertional inactivation of the antigen I/II gene resulted in reduced hydrophobicity during early exponential phase, whereas no effect was detected during mid- and late exponential phases. Binding to human fibronectin and laminin was reduced in the isogenic mutant, whereas binding to human collagen types I and IV and to rat collagen type I was not significant for either the wild type or the mutant. Compared to the wild type, the capacity of the isogenic mutant to induce interleukin 8 (IL-8) release by THP-1 monocytic cells was significantly reduced. The results indicate that the S. intermedius antigen I/II is involved in adhesion to human receptors and in IL-8 induction.

FIG. 1

Strategy used for insertional inactivation of the S. intermedius antigen I/II gene. The antigen I/II gene sequence amplified by PCR was cut with Tsp509I, and the resulting fragments were ligated to pSF151 digested with EcoRI. The ligation mixture was used to transform S. intermedius NCTC 11324. Km^r mutants were selected.

FIG. 2

Immunoblot detection of antigen I/II in S. intermedius with antibodies raised to S. mutans OMZ 175 antigen I/II (anti-I/II SR). Lanes: 1, S. intermedius NCTC 11324; 2, S. intermedius CCUG 28191; 3, S. intermedius CCUG 43515. Std, Standard Protein Molecular Weight Marker (Bio-Rad). Molecular masses are given in kilodaltons.

FIG. 3

Immunoblot analysis of antigen I/II expression in the cell extract (lanes 1 to 4) and supernatant (lanes 5 to 8) of S. intermedius NCTC 11324 (1, 5), the S. intermedius isogenic mutants IB08981 (2, 6) and IB08982 (3, 7), and S. mutans OMZ 175 (4, 8). Std, Standard Protein Molecular Weight Marker (Bio-Rad). Molecular masses are given in kilodaltons.

FIG. 4

Northern blot analysis of total RNA from S. intermedius NCTC 11324 (lane 11324) hybridized with the antigen I/II digoxigenin-labeled probe. Std, Standard RNA Molecular Weight Marker II (Roche Diagnostics). kb, kilobases.

FIG. 5

S. intermedius NCTC 11324 growth-related antigen I/II transcription and expression. (a) Northern blot analysis of total RNA hybridized with the antigen I/II probe. (b) Immunoblot detection of antigen I/II in the cell extract with anti-I/II SR. EP, early exponential phase; MP, mid-exponential phase; LP, late exponential phase.

FIG. 6

CSLM image of the S. intermedius NCTC 11324. (I) Cells present in a selected field of view. (II) Fluorescence detection with antibodies raised to S. mutans OMZ 175 antigen I/II.

FIG. 7

Southern blot analysis of XbaI- and PstI-digested chromosomal DNA from the wild-type S. intermedius NCTC 11324 (W) and the isogenic mutant IB08981 (M), probed with pSF151 (a) or antigen I/II (b) digoxigenin-labeled probes. Std, Standard DNA Molecular Weight Marker III (Roche Diagnostics). kb, kilobases.

FIG. 8

Hydrophobicity of S. intermedius NCTC 11324 (●) and the isogenic mutant IB08981 (○) during growth (top graph). Hydrophobicity was calculated as percent adsorption to hexadecane. Growth was monitored by measuring OD₆₅₀ (bottom graph). The x axis for both graphs indicates time in hours (h). Error bars indicate standard deviations of triplicate samples.