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Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

Blower TR, Evans TJ, Przybilski R, Fineran PC, Salmond GP - PLoS Genet. (2012)

Bottom Line: The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination.This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system.Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

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ΦTE-F expresses ToxI RNA during infection.(A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46]. The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. DH5α 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45]. Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.
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pgen-1003023-g007: ΦTE-F expresses ToxI RNA during infection.(A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46]. The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. DH5α 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45]. Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.

Mentions: Having ascertained that expression of pseudo-ToxI during infection could alter the Abi outcome, we studied the levels of antitoxic RNA and ToxN protein during ΦTE infections of Pba containing FLAG-tagged ToxIN plasmid pMJ4 [12]. When infecting Pba with ΦTE wt, there were no detectable differences in ToxI or ToxN levels between the infected and uninfected controls, as measured by S1 nuclease assays and Western blotting of the ToxN-FLAG protein, respectively (Figure 7A). To see if an ‘escape’ ΦTE phage caused an alteration in the ToxI:ToxN ratio, the same experiment was performed with phage ΦTE-F. No detectable differences were observed following infection relative to the uninfected controls (Figure 7B).


Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

Blower TR, Evans TJ, Przybilski R, Fineran PC, Salmond GP - PLoS Genet. (2012)

ΦTE-F expresses ToxI RNA during infection.(A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46]. The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. DH5α 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45]. Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.
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pgen-1003023-g007: ΦTE-F expresses ToxI RNA during infection.(A) Upper panel; an S1-nuclease protection assay was used to detect ToxI levels from a ToxIN plasmid during ΦTE wt infection, using an antisense probe against the full 5.5 repeat ToxI sequence [46]. The antisense ToxI-probe was first hybridised to 10 µg of total RNA prepared from Pba ToxIN (pMJ4) at different times after ΦTE infection, and then followed by S1-nuclease treatment. Numbers (+) indicate the time (min) after infection or (−) without the addition of phage. Pba ToxIN-FS (pTA47) and Pba serve as positive and negative controls, respectively. A non-hybridized S1-digested probe (+S1) serves as a further negative control. DH5α 1.5 repeats (pTA96), a non-S1 digested probe (−S1) and an in vitro transcribed Hammerhead ribozyme (HHRz), which cleaves itself during transcription, serve as size markers. HHRz was prepared as described previously [45]. Lower panel; Western blot targeting C-terminal FLAG tagged ToxN contained within total protein harvested from Pba ToxIN (pMJ4) at different time points, with (+, left) and without (−, right) phage infection. Time 0 indicates a sample taken immediately after infection. Total protein from Pba ToxIN (pMJ4) (−) serves as positive control. (B) Infection with escape phage ΦTE-F. Levels of ToxI were determined by S1-assay (upper) as described in (A) with and without infection. ToxN levels were estimated by Western blotting (lower) as described in (A). (C) Expression of the ΦTE-F ToxI locus. An S1-nuclease assay targeting ToxI was performed on total RNA of Pba (pBR322) at different times during ΦTE-F infection. Pba ToxIN (pMJ4) and DH5α serve as positive and negative controls, respectively.
Mentions: Having ascertained that expression of pseudo-ToxI during infection could alter the Abi outcome, we studied the levels of antitoxic RNA and ToxN protein during ΦTE infections of Pba containing FLAG-tagged ToxIN plasmid pMJ4 [12]. When infecting Pba with ΦTE wt, there were no detectable differences in ToxI or ToxN levels between the infected and uninfected controls, as measured by S1 nuclease assays and Western blotting of the ToxN-FLAG protein, respectively (Figure 7A). To see if an ‘escape’ ΦTE phage caused an alteration in the ToxI:ToxN ratio, the same experiment was performed with phage ΦTE-F. No detectable differences were observed following infection relative to the uninfected controls (Figure 7B).

Bottom Line: The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination.This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system.Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

Show MeSH
Related in: MedlinePlus