Biology subjects

Huiting, E.

Publications and source records attributed to Huiting, E..

6 recordsLinked to original sources

bioRxiv2026

Intracellular barriers and receptor masking limit success of a Pseudomonas aeruginosa clinical phage family

Bacteriophage therapy is needed to treat antibiotic resistant infections; however, when a clinical isolate resists a given phage, it is often unclear why. It is therefore currently unknown how to rationally fortify phage therapies to circumvent a priori resistance. Using a family of broad host range therapeutic Pseudomonas aeruginosa phages (Pbunaviruses), we show that cell surface receptor masking and intracellular defenses are both common barriers in distinct clinical isolates. In some cases these barriers can be bypassed by intrafamily phage engineering. Using unbiased genome-wide CRISPRi screens, we reveal that the broadly conserved L-Rhamnose in the core polysaccharide is the receptor for Pbunavirus family. This molecule is often masked by diverse O-antigen structures. In other isolates with the L-Rha receptor accessible, internal defense mechanisms commonly prevent Pbunavirus DNA replication. A single anti-defense locus often encoding 8-11 different genes within the Pbunavirus family is required for optimal host range, providing anti-defense genes that enable replication of both Pbunavirus phages and phages of other families. Our work demonstrates the importance of both internal and surface defense mechanisms in clinical isolates causally antagonizing a commonly used phage therapeutic and presents phage engineering strategies to circumvent a priori resistance.

microbiology↗

bioRxiv2026

CBASS limits bacteriophage production while maintaining cell viability in Pseudomonas aeruginosa

CBASS is an immune pathway that recognizes phage infection and generates cyclic nucleotide signals, which directly activate effectors that stop phage replication. Membrane-acting effectors are proposed to induce cell death to prevent phage replication; however, this mechanism has not been assessed with endogenous expression levels of the effector. We therefore sought to determine the cell viability outcomes of the CBASS phospholipase effector (CapV) upon activation with 3,3-cGAMP signals in Pseudomonas aeruginosa. Here, we surprisingly observe that constitutive 3,3-cGAMP signaling from the synthase (CdnA) enables robust cell growth and viability while effectively abolishing phage production in a CapV-dependent manner. Exogenous 3,3-cGAMP also enhances CBASS anti-phage activity and cell growth. Moreover, constitutive activation of the CapV effector induces no cell fitness cost, and blocks replication of many, but not all, phages. This demonstrates that a cyclic nucleotide-activated CBASS effector possess a degree of phage specificity that has been previously overlooked. When CBASS is active, phage transcription and initiation of DNA replication proceed normally, but phages do not reach maximum DNA levels and fewer mature virions are produced. Based on these findings, we propose that CapV interferes with the early stages of phage capsid assembly at the cell membrane and resultantly disrupts DNA packaging. Collectively, we demonstrate that a successful CBASS response antagonizes a late-stage of the phage replication cycle while maintaining cell viability.

microbiology↗

bioRxiv2024

Exploring the diversity of anti-defense systems across prokaryotes, phages, and mobile genetic elements

The co-evolution of prokaryotes, phages, and mobile genetic elements (MGEs) over the past billions of years has driven the emergence and diversification of defense and anti-defense systems alike. Anti-defense proteins have diverse functional domains, sequences, and are typically small, creating a challenge to detect anti-defense homologs across the prokaryotic genomes. To date, no tools comprehensively annotate anti-defense proteins within a desired genome or MGE. Here, we developed "AntiDefenseFinder" - a free open-source tool and web service that detects 156 anti-defense systems (of one or more proteins) in any genomic sequence. Using this dataset, we identified 47,981 anti-defense systems distributed across prokaryotes, phage, and MGEs. We found that some genes co-localize in "anti-defense islands", including E. coli T4 and Lambda phages, although many are standalone. Out of the 112 systems detected in bacteria, 100 systems localize only or preferentially in prophages, plasmids, phage satellites, integrons, and integrative and conjugative elements. However, over 80% of anti-Pycsar protein 1 (Apyc1) resides in non-mobile regions of bacteria. Evolutionary and functional analyses revealed that Apyc1 likely originated in bacteria to regulate cNMP signaling, but was co-opted multiple times by phages to overcome cNMP-utilizing defenses. With the AntiDefenseFinder tool, we hope to facilitate the identification of the full repertoire of anti-defense systems in MGEs, the discovery of new protein functions, and a deeper understanding of host-pathogen arms race.

microbiology↗

bioRxiv2023

Single phage proteins sequester TIR- and cGAS-generated signaling molecules

Prokaryotic anti-phage immune systems use TIR (toll/interleukin-1 receptor) and cGAS (cyclic GMP-AMP synthase) enzymes to produce 1-3/1-2 glycocyclic ADPR (gcADPR) and cyclid di-/tri-nucleotides (CDNs and CTNs) signaling molecules that limit phage replication, respectively 1-3. However, how phages neutralize these common systems is largely unknown. Here, we show that Thoeris anti-defense proteins Tad1 4 and Tad2 5 both have anti-CBASS activity by simultaneously sequestering CBASS cyclic oligonucleotides. Strikingly, apart from binding Thoeris signals 1-3 and 1-2 gcADPR, Tad1 also binds numerous CBASS CDNs/CTNs with high affinity, inhibiting CBASS systems using these molecules in vivo and in vitro. The hexameric Tad1 has six binding sites for CDNs or gcADPR, which are independent from two high affinity binding sites for CTNs. Tad2 also sequesters various CDNs in addition to gcADPR molecules, inhibiting CBASS systems using these CDNs. However, the binding pockets for CDNs and gcADPR are different in Tad2, whereby a tetramer can bind two CDNs and two gcADPR molecules simultaneously. Taken together, Tad1 and Tad2 are both two-pronged inhibitors that, alongside anti-CBASS protein 2, establish a paradigm of phage proteins that flexibly sequester a remarkable breadth of cyclic nucleotides involved in TIR- and cGAS-based anti-phage immunity.

microbiology↗

bioRxiv2023

Phage anti-CBASS protein simultaneously sequesters cyclic trinucleotides and dinucleotides

CBASS is a common anti-phage immune system that uses cyclic oligonucleotide signals to activate effectors and limit phage replication. In turn, phages encode anti-CBASS (Acb) proteins. We recently uncovered a widespread phage anti-CBASS protein Acb2 that acts as a "sponge" by forming a hexamer complex with three cGAMP molecules. Here, we identified that Acb2 binds and sequesters many CBASS and cGAS-produced cyclic dinucleotides in vitro and inhibits cGAMP-mediated STING activity in human cells. Surprisingly, Acb2 also binds CBASS cyclic trinucleotides 333-cyclic AMP-AMP-AMP (cA3) and 333-cAAG with high affinity. Structural characterization identified a distinct binding pocket within the Acb2 hexamer that binds two cyclic trinucleotide molecules and another binding pocket that binds to cyclic dinucleotides. Binding in one pocket does not allosterically alter the other, such that one Acb2 hexamer can simultaneously bind two cyclic trinucleotides and three cyclic dinucleotides. Phage-encoded Acb2 provides protection from Type III-C CBASS that uses cA3 signaling molecules in vivo and blocks cA3-mediated activation of the endonuclease effector in vitro. Altogether, Acb2 sequesters nearly all known CBASS signaling molecules through two distinct binding pockets and therefore serves as a broad-spectrum inhibitor of cGAS-based immunity.

microbiology↗

bioRxiv2022

Bacteriophages antagonize cGAS-like immunity in bacteria

The recently discovered cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is related to eukaryotic cGAS-STING anti-viral immunity and is present in diverse prokaryotes. However, our understanding of how CBASS detects, inhibits, and co-evolves with phages is limited because CBASS function has only been studied in reconstituted heterologous systems. Here, we identify a phage-encoded CBASS antagonist (acbIIA1, anti-cbass type II-A gene 1) necessary for phage replication in the presence of endogenous CBASS immunity in Pseudomonas aeruginosa. acbIIA1 homologs are encoded by numerous lytic and temperate phages infecting Gram-negative bacteria. Deletion of acbIIA1 renders multiple phages susceptible to CBASS, but phages can then escape immune function via mutations in the major capsid gene. These mutants suggest that CBASS is activated by, or targets, the late-expressed phage capsid. Together, we establish a native model system to study CBASS and identify a common phage-encoded CBASS antagonist, demonstrating that CBASS is a bona fide anti-phage immune system in nature. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/486325v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a4bb64org.highwire.dtl.DTLVardef@10ade0forg.highwire.dtl.DTLVardef@78e1b0org.highwire.dtl.DTLVardef@28f556_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

microbiology↗