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Identifying phage proteins that activate the bacterial innate immune system

Aaron Whiteley — Wed, 02 Jul 2025 15:00:00 +0000

Identifying phage proteins that activate the bacterial innate immune system Aaron Whiteley Wed, 07/02/2025 - 09:00

Toni Nagy , Gina Gersabeck , Amy Conte , Aaron Whiteley

BioRxiv (2025). PubMed PMID: 40631175; PubMed Central PMCID: PMC12236752

Abstract

Bacteria have evolved sophisticated antiphage systems that halt phage replication upon detecting specific phage triggers. Identifying phage triggers is crucial to our understanding of immune signaling, however, they are challenging to predict. Here we used an expansive plasmid library that expressed 400 phage protein-coding genes from 6 different phages to identify novel triggers of known and undiscovered antiphage systems. We transformed our library into 72 diverse strains of E. coli . Each strain natively harbors a different suite of antiphage systems whose activation typically inhibits growth. By tracking plasmids that were selectively depleted, we identified over 100 candidate phage trigger- E. coli pairs. Two phage trigger proteins were investigated in detail, revealing a novel antiphage system that detects multiple phage tail fiber proteins and identifying major capsid protein as the activating ligand of the antiphage system Avs8. These experiments provide a unique dataset for continued definition of the molecular details of the bacterial immune system.

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Citation

Nagy TA, Gersabeck GW, Conte AN, Whiteley AT. Identifying phage proteins that activate the bacterial innate immune system. bioRxiv [Preprint]. 2025 Jul 2:2025.07.02.662641. doi: 10.1101/2025.07.02.662641. PMID: 40631175; PMCID: PMC12236752.

Nagy TA, Gersabeck GW, Conte AN, ➤Whiteley AT | BioRxiv 2025

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Functional amyloid proteins confer defence against predatory bacteria

Aaron Whiteley — Wed, 02 Jul 2025 15:00:00 +0000

Functional amyloid proteins confer defence against predatory bacteria Aaron Whiteley Wed, 07/02/2025 - 09:00

Hannah Ledvina , Ryan Sayegh , Ricardo Carale , Burroughs AM , Alexa Macklin , Azadeh AL , Layla Borja , Aravind L , Aaron Whiteley

Nature (2025) PubMed PMID: 40604283

Abstract

Bdellovibrio bacteriovorus is a predatory bacterium that non-selectively preys on Gram-negative bacteria by invading the prey-cell periplasm, leaching host nutrients and ultimately lysing the infected cell to exit and find a new host. The predatory life cycle of B. bacteriovorus is, in many ways, comparable to a bacteriophage. However, unlike phage defence, defence against B. bacteriovorus has not been widely investigated. Here we screened a collection of diverse Escherichia coli strains for resistance to B. bacteriovorus and identified that roughly one-third of strains robustly defended against predation by producing curli fibres. Curli fibres are oligomers of the functional amyloid protein CsgA, which is exceptionally durable3. Using genetics and microscopy, we demonstrate that curli fibres provide a barrier that protects susceptible cells independent of genes required for biofilm formation. This barrier further protected E. coli against attack by the predatory bacterium Myxococcus xanthus and select phages. Bioinformatic analysis of bacterial amyloids showed these systems are diverse and widespread in diderm bacteria (those with both inner and outer membranes). One of these, an evolutionarily distinct amyloid encoded by Pseudomonas aeruginosa, also protected against B. bacteriovorus. This work establishes that functional amyloids defend bacteria against a wide range of threats.

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Citation

Ledvina HE, Sayegh R, Carale RO, Burroughs AM, Macklin AR, Azadeh AL, Borja Najera LD, Aravind L, Whiteley AT. Functional amyloid proteins confer defence against predatory bacteria. Nature. 2025 Jul 2. doi: 10.1038/s41586-025-09204-7. Epub ahead of print. PMID: 40604283.

Ledvina HE, Sayegh R, Carale RO, Burroughs AM, Macklin AR, Azadeh AL, Borja Najera LD, Aravind L, ➤Whiteley AT | Nature 2025

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A minimal CRISPR polymerase produces decoy cyclic nucleotides to detect phage anti-defense proteins

Aaron Whiteley — Sat, 29 Mar 2025 05:00:00 +0000

A minimal CRISPR polymerase produces decoy cyclic nucleotides to detect phage anti-defense proteins Aaron Whiteley Fri, 03/28/2025 - 23:00

Ashley Sullivan , Nabhani A , Kate Schinkel , Dinh DM , Duncan ML , Ednacot EMQ , Charlotte Hoffman , Izrailevsky DS , Emily Kibby , Toni Nagy , Nguyen CM , Uday Tak , Burroughs AM , Aravind L , Aaron Whiteley , Morehouse BR

BioRxiv (2025). PubMed PMID: 40196609; PubMed Central PMCID: PMC11974786.

Abstract

Bacteria use antiphage systems to combat phages, their ubiquitous competitors, and evolve new defenses through repeated reshuffling of basic functional units into novel reformulations. A common theme is generating a nucleotide-derived second messenger in response to phage that activates an effector protein to halt virion production. Phages respond with counter-defenses that deplete these second messengers, leading to an escalating arms race with the host. Here we discover a novel antiphage system we call Panoptes that detects phage infection by surveying the cytosol for phage proteins that antagonize the nucleotide-derived second messenger pool. Panoptes is a two-gene operon, optSE. OptS is predicted to synthesize a second messenger using a minimal CRISPR polymerase (mCpol) domain, a version of the polymerase domain found in Type III CRISPR systems (Cas10) that is distantly related to GGDEF and Thg1 tRNA repair polymerase domains. OptE is predicted to be a transmembrane effector protein that binds cyclic nucleotides. optSE potently restricted phage replication but mutant phages that had loss-of-function mutations in anti-CBASS protein 2 (Acb2) escaped defense. These findings were unexpected because Acb2 is a nucleotide “sponge” that antagonizes second messenger signaling. Using genetic and biochemical assays, we found that Acb2 bound the OptS-synthesized nucleotide, 2′,3′-cyclic adenosine monophosphate (2′,3′-c-di-AMP); however, 2′,3′-c-di-AMP was synthesized constitutively by OptS and inhibited OptE. Nucleotide depletion by Acb2 released OptE toxicity thereby initiating abortive infection to halt phage replication. These data demonstrate a sophisticated immune strategy that hosts use to guard their second messenger pool and turn immune evasion against the virus.

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Citation

Sullivan AE, Nabhani A, Schinkel K, Dinh DM, Duncan ML, Ednacot EMQ, Hoffman CRK, Izrailevsky DS, Kibby EM, Nagy TA, Nguyen CM, Tak U, Burroughs AM, Aravind L, Whiteley AT, Morehouse BR. A minimal CRISPR polymerase produces decoy cyclic nucleotides to detect phage anti-defense proteins. bioRxiv. 2025 Mar 30;. doi: 10.1101/2025.03.28.646047. PubMed PMID: 40196609; PubMed Central PMCID: PMC11974786.

Sullivan AE, Nabhani A, Schinkel K, Dinh DM, Duncan ML, Ednacot EMQ, Hoffman CRK, Izrailevsky DS, Kibby EM, Nagy TA, Nguyen CM, Tak U, Burroughs AM, Aravind L, ➤Whiteley AT†, Morehouse BR† (†co-cor. author) | BioRxiv 2025

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Identifying phage proteins that activate the bacterial innate immune system

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Functional amyloid proteins confer defence against predatory bacteria

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A minimal CRISPR polymerase produces decoy cyclic nucleotides to detect phage anti-defense proteins

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