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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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Related in: MedlinePlus

DNA of pp1 and EFCIV583 are packaged in separated capsids.Presentation of two working hypotheses for pp1 and EFCIV583 DNA packaging in a dilysogen strain and experimental results corroborating one of them. On the left, DNAs are packaged inside the same capsid. The resulting virions are predicted to deliver both DNA during infection and to form plaques containing both P1 and EFCIV583 virions since pp1 is required for formation of EFCIV583 virions on indicator strains pp− and pp1−pp7−, both deleted for pp1 and EFCIV583. On the right, pp1 and EFCIV583 DNAs are packaged separately in two different capsids. The resulting virions would deliver either pp1 or EFCIV583 DNA during infection of strains pp− and pp1−pp7−, and would form only P1 plaques since pp1 is required for formation of EFCIV583 virions and co-infection by two particles is highly improbable. However, EFCIV583 virions would be detected on the indicator strain pp7−, which harbors pp1. Lysates of strain pp1+ pp7+ were tested on indicator strains pp−, pp1−pp7− and pp7− and the resulting plaques were identified by pp1- and EFCIV583-specific PCRs. Our results strongly support that P1 and EfCIV583 genomes are packaged in two different capsids since plaques formed by pp1+ pp7+ lysates on indicator strains pp− and pp1− pp7− were identified as P1 plaques only, while EFCIV583 virions were detected on indicator strain pp7−.
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pgen-1003539-g005: DNA of pp1 and EFCIV583 are packaged in separated capsids.Presentation of two working hypotheses for pp1 and EFCIV583 DNA packaging in a dilysogen strain and experimental results corroborating one of them. On the left, DNAs are packaged inside the same capsid. The resulting virions are predicted to deliver both DNA during infection and to form plaques containing both P1 and EFCIV583 virions since pp1 is required for formation of EFCIV583 virions on indicator strains pp− and pp1−pp7−, both deleted for pp1 and EFCIV583. On the right, pp1 and EFCIV583 DNAs are packaged separately in two different capsids. The resulting virions would deliver either pp1 or EFCIV583 DNA during infection of strains pp− and pp1−pp7−, and would form only P1 plaques since pp1 is required for formation of EFCIV583 virions and co-infection by two particles is highly improbable. However, EFCIV583 virions would be detected on the indicator strain pp7−, which harbors pp1. Lysates of strain pp1+ pp7+ were tested on indicator strains pp−, pp1−pp7− and pp7− and the resulting plaques were identified by pp1- and EFCIV583-specific PCRs. Our results strongly support that P1 and EfCIV583 genomes are packaged in two different capsids since plaques formed by pp1+ pp7+ lysates on indicator strains pp− and pp1− pp7− were identified as P1 plaques only, while EFCIV583 virions were detected on indicator strain pp7−.

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)

DNA of pp1 and EFCIV583 are packaged in separated capsids.Presentation of two working hypotheses for pp1 and EFCIV583 DNA packaging in a dilysogen strain and experimental results corroborating one of them. On the left, DNAs are packaged inside the same capsid. The resulting virions are predicted to deliver both DNA during infection and to form plaques containing both P1 and EFCIV583 virions since pp1 is required for formation of EFCIV583 virions on indicator strains pp− and pp1−pp7−, both deleted for pp1 and EFCIV583. On the right, pp1 and EFCIV583 DNAs are packaged separately in two different capsids. The resulting virions would deliver either pp1 or EFCIV583 DNA during infection of strains pp− and pp1−pp7−, and would form only P1 plaques since pp1 is required for formation of EFCIV583 virions and co-infection by two particles is highly improbable. However, EFCIV583 virions would be detected on the indicator strain pp7−, which harbors pp1. Lysates of strain pp1+ pp7+ were tested on indicator strains pp−, pp1−pp7− and pp7− and the resulting plaques were identified by pp1- and EFCIV583-specific PCRs. Our results strongly support that P1 and EfCIV583 genomes are packaged in two different capsids since plaques formed by pp1+ pp7+ lysates on indicator strains pp− and pp1− pp7− were identified as P1 plaques only, while EFCIV583 virions were detected on indicator strain pp7−.
© Copyright Policy
Related In: Results  -  Collection

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pgen-1003539-g005: DNA of pp1 and EFCIV583 are packaged in separated capsids.Presentation of two working hypotheses for pp1 and EFCIV583 DNA packaging in a dilysogen strain and experimental results corroborating one of them. On the left, DNAs are packaged inside the same capsid. The resulting virions are predicted to deliver both DNA during infection and to form plaques containing both P1 and EFCIV583 virions since pp1 is required for formation of EFCIV583 virions on indicator strains pp− and pp1−pp7−, both deleted for pp1 and EFCIV583. On the right, pp1 and EFCIV583 DNAs are packaged separately in two different capsids. The resulting virions would deliver either pp1 or EFCIV583 DNA during infection of strains pp− and pp1−pp7−, and would form only P1 plaques since pp1 is required for formation of EFCIV583 virions and co-infection by two particles is highly improbable. However, EFCIV583 virions would be detected on the indicator strain pp7−, which harbors pp1. Lysates of strain pp1+ pp7+ were tested on indicator strains pp−, pp1−pp7− and pp7− and the resulting plaques were identified by pp1- and EFCIV583-specific PCRs. Our results strongly support that P1 and EfCIV583 genomes are packaged in two different capsids since plaques formed by pp1+ pp7+ lysates on indicator strains pp− and pp1− pp7− were identified as P1 plaques only, while EFCIV583 virions were detected on indicator strain pp7−.
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