Streptococcal erythrogenic toxin B abrogates fibronectin-dependent internalization of Streptococcus pyogenes by cultured mammalian cells

Abstract

Streptococcus pyogenes secretes several proteins that influence host-pathogen interactions. A tissue-culture model was used to study the influence of the secreted cysteine protease streptococcal erythrogenic toxin B (SPE B) on the interaction between S. pyogenes strain NZ131 (serotype M49) and mammalian cells. Inactivation of the speB gene enhanced fibronectin-dependent uptake of the pathogen by Chinese hamster ovary (CHO-K1) cells compared to that in the isogenic wild-type strain. Preincubation of the NZ131 speB mutant with purified SPE B protease significantly inhibited fibronectin-dependent uptake by both CHO-K1 and CHO-pgs745 cells. The effect was attributed to an abrogation of fibronectin binding to the surface of the bacteria that did not involve either the M49 protein or the streptococcal fibronectin-binding protein SfbI. In contrast, pretreatment of the NZ131 speB mutant with SPE B did not influence sulfated polysaccharide-mediated uptake by CHO-pgs745 cells. The results indicate that the SPE B protease specifically alters bacterial cell surface proteins and thereby influences pathogen uptake.

FIG. 1

Inactivation of the speB gene in NZ131 enhanced internalization by CHO-K1 cells. S. pyogenes NZ131 was grown to the mid-exponential (exp) and stationary (stat) phases in THY broth. Aliquots were centrifuged, and the bacteria were resuspended in DMEM prior to the addition of 10⁷ CFU to preconfluent CHO-K1 cell monolayers with (+) or without (−) 5% FCS. The infection proceeded for 2 h prior to the assessment of internalization by differential immunogold-silver staining. (A) Internalization of M49 strain NZ131 (open bars) and NZ131 speB (solid bars). (B) Internalization of M12 strain 86-858. The data represent the mean number of streptococci per cell ± standard error from at least three separate experiments.

FIG. 2

SPE B protease decreased Fn-mediated internalization. NZ131 speB was suspended in sterile spent medium prepared from the speB mutant. The zymogen preparation (hatched bars) or activated SPE B protease (solid bars) was added or not (open bars), and the bacteria were incubated for 1 h at 37°C. Following preincubation, the bacteria were collected by centrifugation and resuspended in DMEM. Approximately 10⁷ CFU were added to each well that contained CHO-pgs745 cells, and internalization was assessed. (A) Internalization of NZ131 speB in the presence of 5% FCS. Preincubation with the SPE B zymogen preparation (hatched bar in panel B) was not done. (B) Internalization of NZ131 speB in the presence of 2 μg of human Fn per ml. The data represent the mean number of streptococci per cell ± standard error from at least three separate experiments.

FIG. 3

SPE B protease abrogated Fn binding to NZ131. NZ131 speB was grown to mid-exponential phase, collected by centrifugation, and incubated with spent medium alone (lane 1) or spent medium containing the SPE B protease (lane 2), as described for the internalization experiments. After being washed, the bacteria were incubated with purified human Fn. Fn associated with the bacteria was detected by immunoblotting with an Fn-specific antibody. The experiment was repeated twice, and a representative result is shown. The migration and size of the molecular mass standards are indicated. The lower band in lane 1 probably represents the 70-kDa N-terminal fragment of Fn that is frequently observed in commercial Fn preparations.

FIG. 4

M49 protein was not required for Fn-mediated internalization of NZ131. The internalization of NZ131 speB and NZ131 speB emm49 by CHO-K1 and CHO-pgs745 cells was determined following preincubation with the SPE B protease (solid bars), SPE B zymogen preparation (hatched bars), or spent medium alone (open bars). Data represent the mean number of streptococci per cell ± standard error from at least three separate experiments.