Abstract
Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.
Highlights
Streptococcus pyogenes (Group A streptococci) is a Gram-positive bacterial pathogen capable of causing a broad spectrum of disease at different sites in the human body
While point mutations in the chromosomal rpsL gene provide a high level of resistance to streptomycin (SmR), the resistance is recessive if a second copy of the gene is expressed in the same cell [56, 57]
Such a system makes it possible to select for rare clones that spontaneously lose the prophage-inserted copy of the rpsLWT allele and to counter-select against genetic elements that harbor a copy of rpsLWT by plating the bacteria on media containing streptomycin [56]
Summary
Streptococcus pyogenes (Group A streptococci) is a Gram-positive bacterial pathogen capable of causing a broad spectrum of disease at different sites in the human body. These range from mild infections like pharyngitis and impetigo to more severe invasive infections such as streptococcal toxic shock syndrome and necrotizing fasciitis (reviewed in Cunningham (1)). When inadequately treated, these infections can lead to the development of post-streptococcal sequelae, such as glomerulonephritis and rheumatic fever [1]. The completed genome revealed that this streptococcal strain is poly-lysogenized, and that the integrated bacteriophages (prophages) encode a number of putative virulence factors, suggesting an intimate relationship between prophage and streptococcal pathogenesis [2]
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