Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique

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.

Strains of bacterial species induce a greatly varied acute adaptive immune response: The contribution of the accessory genome

“…Using the same approach we compared the adaptive immune response to wild type S . pyogenes M1 (SF370) vs its complete phage KO (CEM1ΔΦ) [ 19 ]. Similar to S .…”

Bacteriophages Contribute to Shaping Clostridioides (Clostridium) difficile Species

“…Therefore, one way to investigate the impact of a prophage on its host is to introduce a given temperate phage into a susceptible bacterial host to create a new lysogen and to study various phenotypes in comparison with the parental strain lacking that prophage. However, bacterial genomes often carry multiple prophages and phenotypes can sometimes result from the cumulative effects of more than one prophages, like reported for the DNAses secreted by Streptococcus pyogenes SF370 ( Euler et al, 2016 ). In addition, natural lysogens have been carrying prophages for extensive periods of time and as such, the prophages’ regulatory circuits are often seamlessly integrated into the host network ( Ehrbar and Hardt, 2005 ).…”

“…Reports of successful curing using extensive screening for spontaneous prophage-cured mutants, or using allelic exchange with counter selection methods have been published in Gram-negative (e.g., E. coli ) and Gram-positive bacteria (e.g., S. pyogenes, S. aureus ). These studies have shed light on the role of individual prophages as well as their combined contribution to virulence of their host ( Bae et al, 2006 ; Wang et al, 2010 ; Euler et al, 2016 ). Of note, curing of one of the two prophages from C. difficile strain 630 has been recently reported, and involved the use of the CRISPR technology ( Hong et al, 2018 ).…”

Specific Integration of Temperate Phage Decreases the Pathogenicity of Host Bacteria

“…However, it is well-known that prophages can switch to the lytic life cycle, in which the prophage DNA is excised from the host genome. Although such process often accompanied by lysis of the host cells, curing of prophages from host bacteria was reported in some bacteria (Euler et al, 2016;Aucouturier et al, 2018). Therefore, it is necessary to ensure there is no spontaneous release of prophage before using a lysogenized bacterium as a live attenuated vaccine.…”