Analysis of 19 Highly Conserved Vibrio cholerae Bacteriophages Isolated from Environmental and Patient Sources Over a Twelve-Year Period

Angermeyer, Angus; Das, Munmee; Singh, D. V.; Seed, Kimberley D.

doi:10.3390/v10060299

Cited by 32 publications

(34 citation statements)

References 29 publications

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“…Once we examined ICP1's effect on V. cholerae transcription, we next sought to document ICP1's transcriptional program. ICP1 has an approximately 126-kb genome and more than 200 predicted open reading frames (46). Less than one-quarter of ICP1's putative coding sequences have activities or functions that can be predicted through bioinformatic analysis.…”

Section: Resultsmentioning

confidence: 99%

A Family of Viral Satellites Manipulates Invading Virus Gene Expression and Can Affect Cholera Toxin Mobilization

et al. 2020

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Many viruses possess temporally unfolding gene expression patterns aimed at subverting host defenses, commandeering host metabolism, and ultimately producing a large number of progeny virions. High-throughput omics tools, such as RNA sequencing (RNA-seq), have dramatically enhanced the resolution of expression patterns during infection. Less studied have been viral satellites, mobile genomes that parasitize viruses. By performing RNA-seq on infection time courses, we have obtained the first time-resolved transcriptomes for bacteriophage satellites during lytic infection. Specifically, we have acquired transcriptomes for the lytic Vibrio cholerae phage ICP1 and all five known variants of ICP1’s parasite, the phage inducible chromosomal island-like elements (PLEs). PLEs rely on ICP1 for both DNA replication and mobilization and abolish production of ICP1 progeny in infected cells. We investigated PLEs’ impact on ICP1 gene expression and found that PLEs did not broadly restrict or reduce ICP1 gene expression. A major exception occurred in ICP1’s capsid morphogenesis operon, which was downregulated by each of the PLE variants. Surprisingly, PLEs were also found to alter the gene expression of CTXΦ, the integrative phage that encodes cholera toxin and is necessary for virulence of toxigenic V. cholerae. One PLE, PLE1, upregulated CTXΦ genes involved in replication and integration and boosted CTXΦ mobility following induction of the SOS response. IMPORTANCE Viral satellites are found in all domains of life and can have profound fitness effects on both the viruses they parasitize and the cells they reside in. In this study, we have acquired the first RNA sequencing (RNA-seq) transcriptomes of viral satellites outside plants, as well as the transcriptome of the phage ICP1, a predominant predator of pandemic Vibrio cholerae. Capsid downregulation, previously observed in an unrelated phage satellite, is conserved among phage inducible chromosomal island-like elements (PLEs), suggesting that viral satellites are under strong selective pressure to reduce the capsid expression of their larger host viruses. Despite conserved manipulation of capsid expression, PLEs exhibit divergent effects on CTXΦ transcription and mobility. Our results demonstrate that PLEs can influence both their hosts’ resistance to phage and the mobility of virulence-encoding elements, suggesting that PLEs can play a substantial role in shaping Vibrio cholerae evolution.

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Section: Resultsmentioning

confidence: 99%

A Family of Viral Satellites Manipulates Invading Virus Gene Expression and Can Affect Cholera Toxin Mobilization

et al. 2020

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“…We isolated eight new ICP1 isolates from cholera patient stool samples collected between 2015 and 2017 and found that all isolates harbour CRISPR-Cas. Thus, it appears that ICP1 isolates lacking CRISPR have not been identified in Bangladesh since 2006 [23]. Analysis of the CRISPR arrays indicates a strong selection for spacers specifically targeting PLE (figure 1 b ; electronic supplementary material, table S5), supporting the function of the ICP1-encoded CRISPR-Cas system as a counter-attack against the anti-phage island PLE [13].…”

Section: Resultsmentioning

confidence: 99%

“…Paired-end sequencing (2 × 150 bp) was performed on an Illumina HiSeq4000 (University of California, Berkeley QB3 Core Facility). Sequencing assembly/mapping and detection of CRISPR was performed as described [23]. The genome of the V. cholerae clinical isolate KS393 was sequenced on Illumina HiSeq4000, PacBio Sequel and Oxford Nanopore MinION sequencers (University of California, Berkeley QB3 Core Facility).…”

Section: Methodsmentioning

confidence: 99%

“…Previous analysis revealed that ICP1-encoded CRISPR-Cas can adapt and acquire new spacers against PLE under laboratory conditions [13], however the rules governing spacer acquisition and targeting efficacy for this system are not known. Further, recent comparative genomics of 18 ICP1 isolates collected from Bangladesh between 2001 and 2012 found that 50% carry CRISPR-Cas [23], however the contemporary state of circulating ICP1 and V. cholerae PLE in the region are not known.…”

Section: Introductionmentioning

confidence: 99%

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Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race

McKitterick

LeGault

Angermeyer

et al. 2019

Phil. Trans. R. Soc. B

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CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae . Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

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“…Once we examined ICP1's effect on V. cholerae transcription, we next sought to document ICP1's transcriptional program. ICP1 has an approximately 126kb genome and more than 200 predicted open reading frames [42]. Less than a quarter of ICP1's putative coding sequences have activities or functions that can be predicted through bioinformatic analysis.…”

Section: Establishing the Icp1 Transcriptional Programmentioning

confidence: 99%

A family of viral satellites manipulates invading virus gene expression and affects cholera toxin mobilization

Barth

Netter

Angermeyer

et al. 2020

Preprint

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Many viruses possess temporally unfolding gene expression patterns aimed at subverting host defenses, commandeering host metabolism, and ultimately producing a large number of progeny virions. High throughput -omics tools, such as RNA-seq, have dramatically enhanced resolution of expression patterns during infection. Less studied have been viral satellites, mobile genomes that parasitize viruses and have far reaching effects on host-cell fitness. By performing RNA-seq on infection time courses, we have obtained the first time-resolved transcriptomes for bacteriophage satellites during lytic infection. Specifically, we have acquired transcriptomes for the lytic Vibrio cholerae phage ICP1 and all five known variants of ICP1's parasite, the Phage Inducible Chromosomal Island-Like Elements (PLEs). PLEs rely on ICP1 for both DNA replication and mobilization, and abolish production of ICP1 progeny in infected cells. We investigated PLEs impact on ICP1 gene expression and found that PLEs did not broadly restrict or reduce ICP1 gene expression. A major exception occurred in ICP1's capsid morphogenesis operon, which was downregulated by each of the PLE variants. This transcriptional manipulation, conserved among PLEs, has also evolved independently in at least one other phage satellite, suggesting that viral satellites may be under strong selective pressure to reduce the capsid expression of their larger host viruses. Surprisingly, PLEs were also found to alter the gene expression of CTXφ, the integrative phage that encodes cholera toxin and is necessary for virulence of toxigenic V. cholerae. One PLE, PLE1, upregulated CTXφ genes involved in replication and integration, and boosted CTXφ mobility following induction of the SOS response. Our data show that PLEs exhibit conserved manipulation of their host-phage's gene expression, but divergent effects on CTXφ, revealing that PLEs can influence both their hosts' resistance to phage and the mobility of virulence encoding elements.So far, few insights have been gained into the gene expression programs of ICP1 and PLEs. PLE1 expresses its integrase in uninfected cells [22], expression of PLE1's replication initiator, RepA, is induced following infection of ICP1 [23], and the PLE's lysis modulator, LidI, is detectable by Western blot late during ICP1 infection [25]. The PLE integrase's recombination directionality factor (necessary for directing integrase excision activity) is PexA, an ICP1 protein whose native function is unknown, but whose expression can be detected by 5 minutes post infection [22]. While these limited observations have provided insight into key PLE and ICP1 genes, the gross expression patterns of ICP1 and PLEs remain unknown. Until now, we have not known the degree to which ICP1 alters cellular expression patterns, and whether PLEs alter ICP1's gene expression or reproduce and restrict ICP1 without such alterations. To address these questions, we performed RNA-seq on V. cholerae infected by ICP1 over the course of the infection cycle. We sequenced the transc...

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Analysis of 19 Highly Conserved Vibrio cholerae Bacteriophages Isolated from Environmental and Patient Sources Over a Twelve-Year Period

Cited by 32 publications

References 29 publications

A Family of Viral Satellites Manipulates Invading Virus Gene Expression and Can Affect Cholera Toxin Mobilization

A Family of Viral Satellites Manipulates Invading Virus Gene Expression and Can Affect Cholera Toxin Mobilization

Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race

A family of viral satellites manipulates invading virus gene expression and affects cholera toxin mobilization

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