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Phage-mediated horizontal transfer of a Staphylococcus aureus virulence-associated genomic island.

Moon BY, Park JY, Hwang SY, Robinson DA, Thomas JC, Fitzgerald JR, Park YH, Seo KS - Sci Rep (2015)

Bottom Line: The genomic islands νSaα and νSaβ are found in almost all S. aureus strains and are characterized by extensive variation in virulence gene content.The transfer of the νSaβ appears to have been accomplished by multiple conversions of transducing phage particles carrying overlapping segments of the νSaβ.Our findings solve a long-standing mystery regarding the diversification and spread of the genomic island νSaβ, highlighting the central role of bacteriophages in the pathogenic evolution of S. aureus.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi state, MS 39762, United States [2] Department of Microbiology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, Seoul 151-742, South Korea.

ABSTRACT
Staphylococcus aureus is a major pathogen of humans and animals. The capacity of S. aureus to adapt to different host species and tissue types is strongly influenced by the acquisition of mobile genetic elements encoding determinants involved in niche adaptation. The genomic islands νSaα and νSaβ are found in almost all S. aureus strains and are characterized by extensive variation in virulence gene content. However the basis for the diversity and the mechanism underlying mobilization of the genomic islands between strains are unexplained. Here, we demonstrated that the genomic island, νSaβ, encoding an array of virulence factors including staphylococcal superantigens, proteases, and leukotoxins, in addition to bacteriocins, was transferrable in vitro to human and animal strains of multiple S. aureus clones via a resident prophage. The transfer of the νSaβ appears to have been accomplished by multiple conversions of transducing phage particles carrying overlapping segments of the νSaβ. Our findings solve a long-standing mystery regarding the diversification and spread of the genomic island νSaβ, highlighting the central role of bacteriophages in the pathogenic evolution of S. aureus.

No MeSH data available.


Related in: MedlinePlus

Identification of a tranducing phage particle, φSaBovLUK, harboring linear phage DNA.(A) A schematic map of linear phage DNA, based on PCR results (see below). Coloring of genes is as in Fig. 1. (B) Based on genome sequencing results of MNKN and CTH96 transductants, various sets of primer (see above map) were designed and tested to locate a linear form of phage DNA containing a bacteriocin gene cluster and LukD/E genes. PCR was positive with primer pairs p1654/p1655 and p1691/p1694 but not with p1651/p1655 and p1691/pseg, indicating a linear form of phage DNA with left flanking near SAB1654, and right flanking near SAB1694. (C) Southern blot analysis of RF122 chromosomal DNA (C) and phage DNA (P) digested with EcoRI restriction enzyme using a probe specific to the lukE gene (the membrane used in this figure is the same as in Fig. 1).
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f2: Identification of a tranducing phage particle, φSaBovLUK, harboring linear phage DNA.(A) A schematic map of linear phage DNA, based on PCR results (see below). Coloring of genes is as in Fig. 1. (B) Based on genome sequencing results of MNKN and CTH96 transductants, various sets of primer (see above map) were designed and tested to locate a linear form of phage DNA containing a bacteriocin gene cluster and LukD/E genes. PCR was positive with primer pairs p1654/p1655 and p1691/p1694 but not with p1651/p1655 and p1691/pseg, indicating a linear form of phage DNA with left flanking near SAB1654, and right flanking near SAB1694. (C) Southern blot analysis of RF122 chromosomal DNA (C) and phage DNA (P) digested with EcoRI restriction enzyme using a probe specific to the lukE gene (the membrane used in this figure is the same as in Fig. 1).

Mentions: Mitomycin C treatment of strain RF122 can induce heterogeneous transducing phages harboring the egc, and these induced phages have a broad host specificity range, suggesting the egc could be transferred to other S. aureus by this phage. To test this possibility, the tetM gene, conferring tetracycline resistance, was introduced into the sem gene of the egc, resulting in RF122 sem::tetM. The phage induced from this strain was successfully transduced to various recipients. Similar to phage spot results, the transduction frequency to bovine (ST151) and USA400 (ST1-SCCmecIV) strains was much higher than those to USA300 and USA200 strains (Table 1). To further confirm the transfer of the egc, a draft genome sequence of the recipients MNKN (ST1-SCCmecIV) and CTH96 (CC151), and phage transduced strains (transductant) was determined. Strikingly, it was shown that both transductants have an identical sequence with the donor strain RF122 from the 141 bp downstream of the start codon of the SAB1676 gene (bsaG) to the attNR sequence at the tRNA-Ser, even preserving SNPs at direct repeats, totaling to 65,756 bp. This result indicates that not only the integrase gene (from φSaBovN) and the egc (from φSaBovEGC), but also the region upstream of the egc containing a bacteriocin gene cluster and leukotoxin D/E genes, were transferred (Supplementary Figure S3). Southern blot analysis using a probe specific to the lukE gene demonstrated the presence of the transducing phage particle harboring the region upstream of the egc containing a bacteriocin gene cluster and leukotoxin D/E genes (Fig. 2C). To test whether this type of transducing phage particle also carries a circular form of phage DNA, outward PCR using various sets of primers was attempted from freshly prepared phage DNA templates and repeated more than 10 times but failed (data not shown). We then investigated the possibility of the existence of a linear form of phage DNA. Indeed, PCR was positive with primer pairs p1654/p1655 and p1691/p1694 but not with p1651/p1655 and p1691/pseg (Fig. 2B), suggesting a linear form of phage DNA with left flanking near SAB1654 and right flanking near SAB1694 (Fig. 2A). However, one cannot rule out the possibility that several intermediates might be detectable as a result of imperfect excision of φSaBovN or φSaBovEGC or a stochastic event (e.g. nucleases digested at the ends of the linear DNA that was possibly fragmented by the phage). This type of transducing phage particle harboring a bacteriocin gene cluster and leukotoxin D/E genes was designated as φSaBovLUK. To confirm the transduction activity of φSaBovLUK, the tetM gene was introduced at the lukE gene (RF122 lukE::tetM). The phage induced from this strain was also successfully transduced the lukE gene to various recipients with a much lower transduction frequency (Table 1).


Phage-mediated horizontal transfer of a Staphylococcus aureus virulence-associated genomic island.

Moon BY, Park JY, Hwang SY, Robinson DA, Thomas JC, Fitzgerald JR, Park YH, Seo KS - Sci Rep (2015)

Identification of a tranducing phage particle, φSaBovLUK, harboring linear phage DNA.(A) A schematic map of linear phage DNA, based on PCR results (see below). Coloring of genes is as in Fig. 1. (B) Based on genome sequencing results of MNKN and CTH96 transductants, various sets of primer (see above map) were designed and tested to locate a linear form of phage DNA containing a bacteriocin gene cluster and LukD/E genes. PCR was positive with primer pairs p1654/p1655 and p1691/p1694 but not with p1651/p1655 and p1691/pseg, indicating a linear form of phage DNA with left flanking near SAB1654, and right flanking near SAB1694. (C) Southern blot analysis of RF122 chromosomal DNA (C) and phage DNA (P) digested with EcoRI restriction enzyme using a probe specific to the lukE gene (the membrane used in this figure is the same as in Fig. 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402969&req=5

f2: Identification of a tranducing phage particle, φSaBovLUK, harboring linear phage DNA.(A) A schematic map of linear phage DNA, based on PCR results (see below). Coloring of genes is as in Fig. 1. (B) Based on genome sequencing results of MNKN and CTH96 transductants, various sets of primer (see above map) were designed and tested to locate a linear form of phage DNA containing a bacteriocin gene cluster and LukD/E genes. PCR was positive with primer pairs p1654/p1655 and p1691/p1694 but not with p1651/p1655 and p1691/pseg, indicating a linear form of phage DNA with left flanking near SAB1654, and right flanking near SAB1694. (C) Southern blot analysis of RF122 chromosomal DNA (C) and phage DNA (P) digested with EcoRI restriction enzyme using a probe specific to the lukE gene (the membrane used in this figure is the same as in Fig. 1).
Mentions: Mitomycin C treatment of strain RF122 can induce heterogeneous transducing phages harboring the egc, and these induced phages have a broad host specificity range, suggesting the egc could be transferred to other S. aureus by this phage. To test this possibility, the tetM gene, conferring tetracycline resistance, was introduced into the sem gene of the egc, resulting in RF122 sem::tetM. The phage induced from this strain was successfully transduced to various recipients. Similar to phage spot results, the transduction frequency to bovine (ST151) and USA400 (ST1-SCCmecIV) strains was much higher than those to USA300 and USA200 strains (Table 1). To further confirm the transfer of the egc, a draft genome sequence of the recipients MNKN (ST1-SCCmecIV) and CTH96 (CC151), and phage transduced strains (transductant) was determined. Strikingly, it was shown that both transductants have an identical sequence with the donor strain RF122 from the 141 bp downstream of the start codon of the SAB1676 gene (bsaG) to the attNR sequence at the tRNA-Ser, even preserving SNPs at direct repeats, totaling to 65,756 bp. This result indicates that not only the integrase gene (from φSaBovN) and the egc (from φSaBovEGC), but also the region upstream of the egc containing a bacteriocin gene cluster and leukotoxin D/E genes, were transferred (Supplementary Figure S3). Southern blot analysis using a probe specific to the lukE gene demonstrated the presence of the transducing phage particle harboring the region upstream of the egc containing a bacteriocin gene cluster and leukotoxin D/E genes (Fig. 2C). To test whether this type of transducing phage particle also carries a circular form of phage DNA, outward PCR using various sets of primers was attempted from freshly prepared phage DNA templates and repeated more than 10 times but failed (data not shown). We then investigated the possibility of the existence of a linear form of phage DNA. Indeed, PCR was positive with primer pairs p1654/p1655 and p1691/p1694 but not with p1651/p1655 and p1691/pseg (Fig. 2B), suggesting a linear form of phage DNA with left flanking near SAB1654 and right flanking near SAB1694 (Fig. 2A). However, one cannot rule out the possibility that several intermediates might be detectable as a result of imperfect excision of φSaBovN or φSaBovEGC or a stochastic event (e.g. nucleases digested at the ends of the linear DNA that was possibly fragmented by the phage). This type of transducing phage particle harboring a bacteriocin gene cluster and leukotoxin D/E genes was designated as φSaBovLUK. To confirm the transduction activity of φSaBovLUK, the tetM gene was introduced at the lukE gene (RF122 lukE::tetM). The phage induced from this strain was also successfully transduced the lukE gene to various recipients with a much lower transduction frequency (Table 1).

Bottom Line: The genomic islands νSaα and νSaβ are found in almost all S. aureus strains and are characterized by extensive variation in virulence gene content.The transfer of the νSaβ appears to have been accomplished by multiple conversions of transducing phage particles carrying overlapping segments of the νSaβ.Our findings solve a long-standing mystery regarding the diversification and spread of the genomic island νSaβ, highlighting the central role of bacteriophages in the pathogenic evolution of S. aureus.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi state, MS 39762, United States [2] Department of Microbiology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, Seoul 151-742, South Korea.

ABSTRACT
Staphylococcus aureus is a major pathogen of humans and animals. The capacity of S. aureus to adapt to different host species and tissue types is strongly influenced by the acquisition of mobile genetic elements encoding determinants involved in niche adaptation. The genomic islands νSaα and νSaβ are found in almost all S. aureus strains and are characterized by extensive variation in virulence gene content. However the basis for the diversity and the mechanism underlying mobilization of the genomic islands between strains are unexplained. Here, we demonstrated that the genomic island, νSaβ, encoding an array of virulence factors including staphylococcal superantigens, proteases, and leukotoxins, in addition to bacteriocins, was transferrable in vitro to human and animal strains of multiple S. aureus clones via a resident prophage. The transfer of the νSaβ appears to have been accomplished by multiple conversions of transducing phage particles carrying overlapping segments of the νSaβ. Our findings solve a long-standing mystery regarding the diversification and spread of the genomic island νSaβ, highlighting the central role of bacteriophages in the pathogenic evolution of S. aureus.

No MeSH data available.


Related in: MedlinePlus