Induction of Lysogenic Bacteriophage and Phage-Associated Toxin from Group A Streptococci during Coculture with Human Pharyngeal Cells

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 ϳ10 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.Streptococcus pyogenes (group A streptococcus) is responsible for a large number of serious diseases worldwide, the most common of which is pharyngitis. If gone untreated, this infection could result in rheumatic heart disease in about 3% of cases. While a great deal of data have been accumulated regarding this organism, including its structures and genetic composition, our knowledge of its interaction with host cells during the early stages of infection remains at a rudimentary level. In deciphering the interactions of the streptococcus with its host, both surface and extracellular proteins are likely involved. While the majority of surface proteins act as adhesins, soluble proteins tend to exhibit a variety of activities, some of which are enzymatic.In an effort to conserve energy, bacterially secreted proteins involved in pathogenesis may not necessarily be constitutively expressed, but instead are induced when required for infection. Upon culture of bacteria together with their host cells, upregulation of specific bacterial genes (some of which are virulence factors) has been observed with both intracellular (1, 2, 9) and nonintracellular organisms (12,14). The streptococcal class of toxins includes some of the most potent molecules secreted by S. pyogenes, which often results in the destruction of host cells. Thus, analysis of the secretion of toxins by S. pyogenes in response to its host cell environment may help illuminate the regulation of these molecules in one of the earliest stages of streptococcal pathogenesis. MATERIALS AND METHODSGrowth conditions for pharyngeal cells and S. pyogenes. The human pharyngeal cell line Detroit 562 (ATCC CCL 138) was grown in minimal essential medium (MEM; Gibco-BRL, Gaithersburg, Md.) containing 10% fetal bovine serum. Cells were grown in Falcon six-well plates (35 mm in diameter) at 37°C under 5% CO 2 . The strains of S. pyogenes used included D471 (from the Rockefeller University collection), lysogenized strain CS112, and its indicator strain, CS24 (kindly supplied by Patrick Schlievert). All bacteria were cultured at 37°C in Todd-Hewitt broth containing 1% yeast extract (THY; Difco Laboratories, Detroit, Mich.).S. pyogenes-human pharyngeal cell coculture system. Pathogenic S. pyogenes (strain D471) cells were grown overnight and suspended in phosphate-buffered saline (PBS). After adjustment of the optical density at 650 nm (OD 650 ) to 1.0 (ϳ5 ϫ 10 8 CFU/ml), the bacteria were ...

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