Induction of lysogenic bacteriophage and phage-associated toxin from group a streptococci during coculture with human pharyngeal cells - PubMed (original) (raw)

Induction of lysogenic bacteriophage and phage-associated toxin from group a streptococci during coculture with human pharyngeal cells

T B Broudy et al. Infect Immun. 2001 Mar.

Free PMC article

Abstract

We found that when group A streptococci are cocultured with human pharyngeal cells, they upregulate and secrete a 25-kDa toxin, determined to be the bacteriophage-encoded streptococcal pyrogenic exotoxin C (SpeC). This prompted us to determine if the bacteriophage themselves are induced during coculture conditions. We found that bacteriophage induction does occur, resulting in the release of approximately 10(5) phage particles during the 3-h coculture. Furthermore, we show that the bacteriophage induction event is mediated by a pharyngeal cell soluble factor for which we provide an initial characterization.

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Figures

FIG. 1

FIG. 1

Detection of secreted toxin during coculture. Streptococcal strains were grown alone or in coculture with Detroit human pharyngeal cells for 3 h. Proteins in the culture medium supernatants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotted, and probed with anti-toxin-specific antibody 442.

FIG. 2

FIG. 2

Identification of bacteriophage during coculture. Streptococcal strains were grown alone or in coculture with Detroit human pharyngeal cells for 3 h. At timed intervals, the culture medium was examined for bacterial CFU of the CS112 control (--×--) and Detroit-CS112 coculture (--●--) versus PFU of the CS112 control (—∗—) and Detroit-CS112 coculture (—●—). The assay detection limit is 2 × 102 PFU/ml. The values plotted are mean concentrations of duplicate wells ± standard deviations. At the 3-h time point, the PFU concentrations found in the coculture media were significantly higher than those found in the control, even when assuming the highest undetectable concentration of 2 × 102 PFU/ml (P = 0.01). (Inset) Electron micrograph (with direct negative stain) of the bacteriophage found in the Detroit-CS112 coculture medium.

FIG. 3

FIG. 3

SpeC and bacteriophage induction during coculture or SPIF exposure. (A) Streptococcal strain CS112 was incubated in coculture with Detroit 562 pharyngeal cells (Det/CS112), in the presence of SPIF (CS112+SPIF), or alone (CS112 Only). Proteins in the culture medium supernatants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotted, and probed with anti-toxin-specific antibody 442. (B) The culture medium supernatants were also analyzed by plaque assay for CS112 bacteriophage induction.

FIG. 4

FIG. 4

Characteristics of SPIF. Detroit pharyngeal cell culture medium supernatants were analyzed by plaque assay in order to detect CS112 bacteriophage induction. To characterize SPIF, spent medium from Detroit 562 pharyngeal cells was filtered through a 10-kDa membrane, filtered through a 1-kDa membrane, boiled, or pronase digested prior to CS112 bacterial incubation in the medium. CS112 bacteria were also incubated in serum-free MEM and serum-containing MEM as controls. CS112 bacteriophage induction during Detroit-CS112 coculture was examined in parallel and is included as a reference mark. The assay detection limit is 2 × 102 PFU/ml. The values plotted are mean concentrations from duplicate trials ± standard deviations. Phage induction was found to be statistically significant with either spent medium from Detroit 562 pharyngeal cells (Whole Det. Media;P = 0.02), <10-kDa Detroit cell medium (<10kDa Det. Media; P = 0.007), boiled Detroit cell medium (Boiled Det. Media; P = 0.0009), pronase-digested Detroit cell medium (Pronase+ Det. Media;P = 0.001) or Detroit-CS112 coculture (Det/CS112;P = 0.001).

FIG. 5

FIG. 5

Bacterial growth is insufficient for phage induction.S. pyogenes CS112 was incubated in MEM (without serum) for 3 h. At 3 h, either MEM containing 30% fetal calf serum (▪), spent Detroit medium containing SPIF (░⃞), or additional MEM containing 0% serum (□) was added to the incubating culture. The phage titer is represented by the bar graph, and the OD is represented by the line curve. The values plotted are averages from duplicate trials ± standard deviations. Addition of either SPIF or serum to the CS112 culture resulted in statistically insignificant differences in bacterial growth (P = 0.56); however, SPIF addition resulted in a marked bacteriophage induction (P = 0.002).

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