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Enterococcus faecalis prophage dynamics and contributions to pathogenic traits.

Matos RC, Lapaque N, Rigottier-Gois L, Debarbieux L, Meylheuc T, Gonzalez-Zorn B, Repoila F, Lopes Mde F, Serror P - PLoS Genet. (2013)

Bottom Line: The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci.Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis.Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1319 Micalis, Jouy-en-Josas, France.

ABSTRACT
Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.

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Interaction between E. faecalis pp1 and pp7 (EfCIV583).(A) Semi-quantitative PCR detection of EfCIV583 circular forms (attP) and excision sites (attB) in wild-type (WT) and strains pp1−, pp1+ pp7+ and pp7+. Excision and circularization products probed by semi-quantitative PCR on 100, 10 and 1 pg of total bacterial DNA prepared from cultures of WT and strains pp1−, pp1+ pp7+ and pp7+ induced for 2 h with 2 µg/ml of ciprofloxacin at 37°C. Twenty cycles were used to amplify products of pp1 and EfCIV583. These results are representative of two independent experiments. (B) Prophage DNA extracted from precipitated phage particles obtained from lysates of WT and strains pp1−, pp1+pp7+ and pp7+ was separated by FIGE and analyzed by Southern-blot and hybridized sequentially using specific probes for pp1 and EfCIV583 genomes. The approximately 38.2 kb and 12 kb band corresponds to P1 and EfCIV583 genome, respectively. As ascertained by pp1-specific hybridization, migration of P1 DNA was delayed in lane pp1+ and pp7− compared to lanes WT and pp1+pp7+. Lambda DNA mono-cut mix (NEB) was run next to the samples to validate band sizes. (C) Scanning electron microscopy images of bacterial cells from strains pp− and pp1+pp7+ after ciprofloxacin treatment. (D) Transmission electron microscopy images of phages produced by strain pp1+pp7+ after ciprofloxacin treatment. White and black arrows indicate big and small sized particles attributed to P1 and EfCIV583, respectively. Enlarged images of EfCIV583 and P1 (renamed vB_EfaS_V583-P1) are shown on the right.
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pgen-1003539-g004: Interaction between E. faecalis pp1 and pp7 (EfCIV583).(A) Semi-quantitative PCR detection of EfCIV583 circular forms (attP) and excision sites (attB) in wild-type (WT) and strains pp1−, pp1+ pp7+ and pp7+. Excision and circularization products probed by semi-quantitative PCR on 100, 10 and 1 pg of total bacterial DNA prepared from cultures of WT and strains pp1−, pp1+ pp7+ and pp7+ induced for 2 h with 2 µg/ml of ciprofloxacin at 37°C. Twenty cycles were used to amplify products of pp1 and EfCIV583. These results are representative of two independent experiments. (B) Prophage DNA extracted from precipitated phage particles obtained from lysates of WT and strains pp1−, pp1+pp7+ and pp7+ was separated by FIGE and analyzed by Southern-blot and hybridized sequentially using specific probes for pp1 and EfCIV583 genomes. The approximately 38.2 kb and 12 kb band corresponds to P1 and EfCIV583 genome, respectively. As ascertained by pp1-specific hybridization, migration of P1 DNA was delayed in lane pp1+ and pp7− compared to lanes WT and pp1+pp7+. Lambda DNA mono-cut mix (NEB) was run next to the samples to validate band sizes. (C) Scanning electron microscopy images of bacterial cells from strains pp− and pp1+pp7+ after ciprofloxacin treatment. (D) Transmission electron microscopy images of phages produced by strain pp1+pp7+ after ciprofloxacin treatment. White and black arrows indicate big and small sized particles attributed to P1 and EfCIV583, respectively. Enlarged images of EfCIV583 and P1 (renamed vB_EfaS_V583-P1) are shown on the right.

Mentions: To identify the step at which pp1 was required for production of EfCIV583 virions, we analyzed both the excision and replication of EfCIV583 and the packaging of EfCIV583 DNA in WT and the isogenic strains pp1−, pp1+pp7+, pp7+ and pp1+ by semi-quantitive PCR. Excision (attB region) and replication (attP region) products of EfCIV583 were detected in pp1− and pp7+ strains at the same level as strains wild type and pp1+pp7+ (Figure 4A), showing that pp1 is not required for EfCIV583 excision and replication. Next, DNA from phage particles produced by ciprofloxacin-treated WT and the isogenic strains pp1−, pp1+pp7+, pp7+ and pp1+ was recovered and analyzed as described above. Particles containing EfCIV583 DNA were recovered from WT and pp1+pp7+ strains, while EfCIV583 DNA was no longer packaged in the absence of pp1 (strain pp1−) or when present as a single element (strain pp7+) (Figure 4B), indicating that pp1 is required for packaging of EfCIV583 DNA. Independent hybridizations revealed that EfCIV583 DNA is encapsidated as monomers only since no signal was detected at high molecular weight (data not shown). Noticeably, while the amount of the EfCIV583 DNA was similar between strains, the amount of pp1 DNA increased significantly when EfCIV583 was deleted (strain pp1+), suggesting that EfCIV583 DNA hijacks P1 proteins at the expense of P1 particles production. The above molecular evidences for EfCIV583 pirating P1 proteins correlate with respective phage titers (Table 3). First, EfCIV583 titer was 10-fold higher than the titer of P1 in lysates from strain pp1+pp7+, supporting that when present, EfCIV583 outnumbers P1 particles. Secondly, P1 titer of lysates from strain pp1+ was 100-fold higher than in lysates from strain pp1+pp7+, indicating that EfCIV583 impairs the production of P1 particles. Interestingly, P1 particles are infectious on strains pp1−pp7− and pp−, but not on strains pp1− nor pp7+ (Figure 5 and Table 3), further supporting that EfCIV583 interferes with P1 growth.


Enterococcus faecalis prophage dynamics and contributions to pathogenic traits.

Matos RC, Lapaque N, Rigottier-Gois L, Debarbieux L, Meylheuc T, Gonzalez-Zorn B, Repoila F, Lopes Mde F, Serror P - PLoS Genet. (2013)

Interaction between E. faecalis pp1 and pp7 (EfCIV583).(A) Semi-quantitative PCR detection of EfCIV583 circular forms (attP) and excision sites (attB) in wild-type (WT) and strains pp1−, pp1+ pp7+ and pp7+. Excision and circularization products probed by semi-quantitative PCR on 100, 10 and 1 pg of total bacterial DNA prepared from cultures of WT and strains pp1−, pp1+ pp7+ and pp7+ induced for 2 h with 2 µg/ml of ciprofloxacin at 37°C. Twenty cycles were used to amplify products of pp1 and EfCIV583. These results are representative of two independent experiments. (B) Prophage DNA extracted from precipitated phage particles obtained from lysates of WT and strains pp1−, pp1+pp7+ and pp7+ was separated by FIGE and analyzed by Southern-blot and hybridized sequentially using specific probes for pp1 and EfCIV583 genomes. The approximately 38.2 kb and 12 kb band corresponds to P1 and EfCIV583 genome, respectively. As ascertained by pp1-specific hybridization, migration of P1 DNA was delayed in lane pp1+ and pp7− compared to lanes WT and pp1+pp7+. Lambda DNA mono-cut mix (NEB) was run next to the samples to validate band sizes. (C) Scanning electron microscopy images of bacterial cells from strains pp− and pp1+pp7+ after ciprofloxacin treatment. (D) Transmission electron microscopy images of phages produced by strain pp1+pp7+ after ciprofloxacin treatment. White and black arrows indicate big and small sized particles attributed to P1 and EfCIV583, respectively. Enlarged images of EfCIV583 and P1 (renamed vB_EfaS_V583-P1) are shown on the right.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3675006&req=5

pgen-1003539-g004: Interaction between E. faecalis pp1 and pp7 (EfCIV583).(A) Semi-quantitative PCR detection of EfCIV583 circular forms (attP) and excision sites (attB) in wild-type (WT) and strains pp1−, pp1+ pp7+ and pp7+. Excision and circularization products probed by semi-quantitative PCR on 100, 10 and 1 pg of total bacterial DNA prepared from cultures of WT and strains pp1−, pp1+ pp7+ and pp7+ induced for 2 h with 2 µg/ml of ciprofloxacin at 37°C. Twenty cycles were used to amplify products of pp1 and EfCIV583. These results are representative of two independent experiments. (B) Prophage DNA extracted from precipitated phage particles obtained from lysates of WT and strains pp1−, pp1+pp7+ and pp7+ was separated by FIGE and analyzed by Southern-blot and hybridized sequentially using specific probes for pp1 and EfCIV583 genomes. The approximately 38.2 kb and 12 kb band corresponds to P1 and EfCIV583 genome, respectively. As ascertained by pp1-specific hybridization, migration of P1 DNA was delayed in lane pp1+ and pp7− compared to lanes WT and pp1+pp7+. Lambda DNA mono-cut mix (NEB) was run next to the samples to validate band sizes. (C) Scanning electron microscopy images of bacterial cells from strains pp− and pp1+pp7+ after ciprofloxacin treatment. (D) Transmission electron microscopy images of phages produced by strain pp1+pp7+ after ciprofloxacin treatment. White and black arrows indicate big and small sized particles attributed to P1 and EfCIV583, respectively. Enlarged images of EfCIV583 and P1 (renamed vB_EfaS_V583-P1) are shown on the right.
Mentions: To identify the step at which pp1 was required for production of EfCIV583 virions, we analyzed both the excision and replication of EfCIV583 and the packaging of EfCIV583 DNA in WT and the isogenic strains pp1−, pp1+pp7+, pp7+ and pp1+ by semi-quantitive PCR. Excision (attB region) and replication (attP region) products of EfCIV583 were detected in pp1− and pp7+ strains at the same level as strains wild type and pp1+pp7+ (Figure 4A), showing that pp1 is not required for EfCIV583 excision and replication. Next, DNA from phage particles produced by ciprofloxacin-treated WT and the isogenic strains pp1−, pp1+pp7+, pp7+ and pp1+ was recovered and analyzed as described above. Particles containing EfCIV583 DNA were recovered from WT and pp1+pp7+ strains, while EfCIV583 DNA was no longer packaged in the absence of pp1 (strain pp1−) or when present as a single element (strain pp7+) (Figure 4B), indicating that pp1 is required for packaging of EfCIV583 DNA. Independent hybridizations revealed that EfCIV583 DNA is encapsidated as monomers only since no signal was detected at high molecular weight (data not shown). Noticeably, while the amount of the EfCIV583 DNA was similar between strains, the amount of pp1 DNA increased significantly when EfCIV583 was deleted (strain pp1+), suggesting that EfCIV583 DNA hijacks P1 proteins at the expense of P1 particles production. The above molecular evidences for EfCIV583 pirating P1 proteins correlate with respective phage titers (Table 3). First, EfCIV583 titer was 10-fold higher than the titer of P1 in lysates from strain pp1+pp7+, supporting that when present, EfCIV583 outnumbers P1 particles. Secondly, P1 titer of lysates from strain pp1+ was 100-fold higher than in lysates from strain pp1+pp7+, indicating that EfCIV583 impairs the production of P1 particles. Interestingly, P1 particles are infectious on strains pp1−pp7− and pp−, but not on strains pp1− nor pp7+ (Figure 5 and Table 3), further supporting that EfCIV583 interferes with P1 growth.

Bottom Line: The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci.Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis.Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1319 Micalis, Jouy-en-Josas, France.

ABSTRACT
Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.

Show MeSH
Related in: MedlinePlus