Elimination of Chromosomal Island SpyCIM1 from Streptococcus pyogenes Strain SF370 Reverses the Mutator Phenotype and Alters Global Transcription

Hendrickson, Christina G.; Euler, Chad W.; Nguyen, Scott V.; Rahman, Maliha; McCullor, Kimberly A.; King, C.; Fischetti, Vincent A.; McShan, W. Michael

doi:10.1371/journal.pone.0145884

Cited by 18 publications

(11 citation statements)

References 62 publications

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“…However, all resulting mutants (> 500 CFU) were Sm R / Kan R , suggesting a rate of recombination, gene conversion or mutation in the rpsL WT gene of the Janus cassette was significantly higher than the rate of loss of SpyCIM1 [ 46 ]. This confirmed previous results that SpyCIM1 was difficult to cure from SF370 [ 5 , 7 , 58 ].…”

Section: Resultssupporting

confidence: 92%

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

et al. 2016

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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.

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Section: Resultssupporting

confidence: 92%

“…pyogenes (or potentially other bacteria) that differ solely in phage content. Such KO strains will be useful tools to study the dynamics of phage lysogeny, the role phage play in both bacterial virulence and the regulation of chromosomal genes [ 58 ], and can provide insight into the evolution of streptococcal pathogenicity.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2016

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“…A mutator phenotype was found in a serotype M1 GAS strain, SF370, where a defective phage, SpyClM1, may exist as lysogens or episomes depending on the growth status (36,43). The switching status of the phage altered the global transcription profile, probably by controlling the regulation of the DNA mismatch repair (MMR) operon, thus facilitating alternative adaptation for survival of the bacteria in host cells (44). In this regard, it is possible that the phages in the strain studied here also have evolved to influence virulence at the gene regulation level in addition to the genetic level.…”

Section: Discussionmentioning

confidence: 99%

Genomic Characterization of a Pattern D Streptococcus pyogenes emm53 Isolate Reveals a Genetic Rationale for Invasive Skin Tropicity

et al. 2016

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The genome of an invasive skin-tropic strain (AP53) of serotype M53 group A Streptococcus pyogenes (GAS) is composed of a circular chromosome of 1,860,554 bp and carries genetic markers for infection at skin locales, viz., emm gene family pattern D and FCT type 3. Through genome-scale comparisons of AP53 with other GAS genomes, we identified 596 candidate single-nucleotide polymorphisms (SNPs) that reveal a potential genetic basis for skin tropism. The genome of AP53 differed by ϳ30 point mutations from a noninvasive pattern D serotype M53 strain (Alab49), 4 of which are located in virulence genes. One pseudogene, yielding an inactive sensor kinase (CovS ؊ ) of the two-component transcriptional regulator CovRS, a major determinant for invasiveness, severely attenuated the expression of the secreted cysteine protease SpeB and enhanced the expression of the hyaluronic acid capsule compared to the isogenic noninvasive AP53/CovS ؉ strain. The collagen-binding protein transcript sclB differed in the number of 5=-pentanucleotide repeats in the signal peptides of AP53 and Alab49 (9 versus 15), translating into different lengths of their signal peptides, which nonetheless maintained a full-length translatable coding frame. Furthermore, GAS strain AP53 acquired two phages that are absent in Alab49. One such phage (⌽AP53.2) contains the known virulence factor superantigen exotoxin gene tandem speK-slaA. Overall, we conclude that this bacterium has evolved in multiple ways, including mutational variations of regulatory genes, short-tandem-repeat polymorphisms, large-scale genomic alterations, and acquisition of phages, all of which may be involved in shaping the adaptation of GAS in specific infectious environments and contribute to its enhanced virulence. IMPORTANCEInfectious strains of S. pyogenes (GAS) are classified by their serotypes, relating to the surface M protein, the emm-like subfamily pattern, and their tropicity toward the nasopharynx and/or skin. It is generally agreed that M proteins from pattern D strains, which also directly bind human host plasminogen, are skin tropic. We have sequenced and characterized the genome of an invasive pattern D GAS strain (AP53) in comparison to a very similar strain (Alab49) that is noninvasive and developed a genomic rationale as to possible reasons for the skin tropicity of these two strains and the greater invasiveness of AP53. G roup A Streptococcus pyogenes (GAS) is a beta-hemolytic, Gram-positive, human-pathogenic bacterium that infects multiple epithelial surfaces and, in some cases, is able to invade deeper soft tissue and cause severe disease (1). An estimated 700 million cases of GAS infection occur worldwide per year (2), ranging from benign treatable clinical conditions, e.g., pharyngitis and impetigo, to more invasive and potentially lethal infections, including necrotizing fasciitis (NF), streptococcal toxic shock syndrome (STSS), acute rheumatic fever (ARF), and acute glomerulonephritis (AGN) (3, 4). The wide range of infectious niches and broad varianc...

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“…These isogenic pairs were M1 strains SF370 and SF370ΔSpyCIM1 and M6 strains MGAS10395 and MGAS10395ΔSpyCIM6. In SF370, our laboratory has demonstrated that SpyCIM1 is involved in altering global transcription (50). Genes upregulated in the presence of SpyCIM1 include emm and capsule gene hasB.…”

Section: Discussionmentioning

confidence: 99%

Genomic Sequencing of High-Efficiency Transducing Streptococcal Bacteriophage A25: Consequences of Escape from Lysogeny

et al. 2018

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Lytic bacteriophage A25, which infects and several related species, has been used to better understand phage-microbe interactions due to its ability to mediate high-efficiency transduction. Most of these studies, however, are decades old and were conducted prior to the advent of next-generation sequencing and bioinformatics. The aim of our study was to gain a better understanding of the mechanism of high-efficiency transduction through analysis of the A25 genome. We show here that phage A25 is related to a family of genome prophages and became a lytic phage following escape from lysogeny. A lambdoid-like residual lysogeny module consisting of an operator site with two promoters and a-like antirepressor gene was identified, but the genes for the cI-like repressor and integrase are missing. Additionally, the genetic organization of the A25 genome was found to be modular in nature and similar to that of many prophages of as well as from other streptococcal species. A study of A25 homology to all annotated prophages within revealed near identity within the remnant lysogeny module of the A25 phage genome to the corresponding regions in resident prophages of genome strains MGAS10270 (M2), MGAS315 (M3), MGAS10570 (M4), and STAB902 (M4). Host range studies of MGAS10270, MGAS315, and MGAS10750 demonstrated that these strains were resistant to A25 infection. The resistance mechanism of superinfection immunity was confirmed experimentally through complementation of the operator region and -like repressor from prophage MGAS10270.2 into susceptible strains SF370, CEM1Δ4 (SF370ΔSpyCIM1), and ATCC 12204, which rendered all three strains resistant to A25 infection. prediction of packaging through homology analysis of the terminase large subunit from bacteriophages within the known packaging mechanism of Gram-positive bacteria as well as the evidence of terminally redundant and/or circularly permuted sequences suggested that A25 grouped with phages employing the less stringent -type packaging mechanisms, which likely explains the characteristic A25 high-efficiency transduction capabilities. Only a few examples of lytic phages appearing following loss of part or all of the lysogeny module have been reported previously, and the genetic mosaicism of A25 suggests that this event may not have been a recent one. However, the discovery that this lytic bacteriophage shares some of the genetic pool of prophages emphasizes the importance of genetic and biological characterization of bacteriophages when selecting phages for therapeutics or disinfectants, as phage-phage and phage-microbe interactions can be complex, requiring more than just assessment of host range and carriage of toxoid or virulence genes. Bacteriophages (bacterial viruses) play an important role in the shaping of bacterial populations as well as the dissemination of bacterial genetic material to new strains, resulting in the spread of virulence factors and antibiotic resistance genes. This study identified the genetic origins of phage A25 and uncovered the molecular mechan...

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Elimination of Chromosomal Island SpyCIM1 from Streptococcus pyogenes Strain SF370 Reverses the Mutator Phenotype and Alters Global Transcription

Cited by 18 publications

References 62 publications

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

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

Genomic Characterization of a Pattern D Streptococcus pyogenes emm53 Isolate Reveals a Genetic Rationale for Invasive Skin Tropicity

Genomic Sequencing of High-Efficiency Transducing Streptococcal Bacteriophage A25: Consequences of Escape from Lysogeny

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