Biology subjects

Carion, H.

Publications and source records attributed to Carion, H..

5 recordsLinked to original sources

bioRxiv2023

Parallelized screening of virus accessory genes reveals diverse defense and counter-defense mechanisms

Viruses have been evolving host-modifying factors for billions of years. Genomes of bacterial and archaeal viruses are replete with fast-evolving, uncharacterized accessory genes (AGs), most of which likely antagonize host defenses or other viruses1, 2. Systematic investigation of AGs could uncover a multitude of biological mechanisms involved in virus-host competition, but AG identification in genomic databases remains a challenge. We developed an integrated computational and high-throughput discovery platform to identify AGs in virus genomes and assay their functions in complementary phage infection-dependent and -independent contexts. Our approach showcases how phages interact with the principal layers of antiviral immunity, including cell surface modifications, restriction systems, and abortive infection (Abi) mechanisms, which operate simultaneously in the same host. We discovered multiple Enterobacteriophage AGs associated with counter-defense functions that activate rather than inhibit antiviral immunity in cells, including the surprising finding that anti-restriction AGs elicit programmed cell death (PCD) activity of some restriction-modification (R-M) systems. We propose that counter-defense AGs that trigger PCD create a conundrum for phages whereby keeping the AGs causes PCD but losing them exposes the phage to restriction by bacteria. Strategies employed by viruses to avoid this double jeopardy could be an important factor in virus evolution that remains to be explored.

microbiology↗

bioRxiv2022

Translation-dependent downregulation of Cas12a mRNA by an anti-CRISPR protein

Bacteria have evolved multiple defense systems, including CRISPR-Cas, to cleave the DNA of phage and mobile genetic elements (MGE). In turn, phage have evolved anti-CRISPR (Acr) proteins that use novel and co-opted mechanisms to block DNA binding or cleavage. Here, we report that an anti-CRISPR (AcrVA2) unexpectedly inhibits Cas12a biogenesis by triggering translation-dependent destruction of its mRNA. AcrVA2 specifically clears the mRNA of Cas12a by recognizing and binding its N-terminal polypeptide. Mutating conserved N-terminal amino acids in Cas12a prevents binding and inhibition by AcrVA2 but also decreases Cas12a anti-phage activity. This mechanism therefore enables AcrVA2 to specifically inhibit divergent Cas12a orthologs while constraining its ability to escape inhibition. AcrVA2 homologs are found on diverse MGEs across numerous bacterial classes, typically in the absence of Cas12a, suggesting that this protein family may induce similar molecular outcomes against other targets. These findings reveal a new gene regulatory strategy in bacteria and create opportunities for polypeptide-specific gene regulation in prokaryotes and beyond.

microbiology↗

bioRxiv2022

Functional screens of barcoded expression libraries uncover new gene functions in carbon utilization among gut Bactereroidales

A mechanistic understanding of host-microbe interactions in the gut microbiome is hindered by poorly annotated bacterial genomes. While functional genomics can generate large gene-to- phenotype datasets to accelerate functional discovery, their applications to study gut anaerobes have been limited. For instance, most gain-of-function screens of gut-derived genes have been performed in Escherichia coli and assayed in a small number of conditions. To address these challenges, we developed Barcoded Overexpression BActerial shotgun library sequencing (Boba-seq). We demonstrate the power of this approach by assaying genes from diverse gut Bacteroidales overexpressed in Bacteroides thetaiotaomicron. From hundreds of experiments, we identified new functions and novel phenotypes for 29 genes involved in carbohydrate metabolism or tolerance to antibiotics or bile salts. Highlights include the discovery of a D- glucosamine kinase, a raffinose transporter, and several routes that increase tolerance to bile salts through lipid biosynthesis. This approach can be readily applied to develop screens in other strains and additional phenotypic assay types.

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↗

bioRxiv2022

High-throughput suppressor screens unveil functional convergence of single-gene lysis proteins

In contrast to dsDNA phages where multiple proteins are involved in programmed host lysis, lysis in ssRNA Fiersviridae and ssDNA Microviridae phages requires only a single gene (sgl for single gene lysis) to meet the size constraints of some of the smallest genomes in the biosphere. To achieve lysis, Sgl proteins exploit evolutionary "weak spots" in bacterial cell wall biogenesis. In several cases, this is done by inhibiting specific steps in Lipid II synthesis. Recently metatranscriptomics has revealed thousands of novel ssRNA phage genomes, each of which must carry at least one sgl gene. Determining the targets of these Sgl proteins could reveal novel vulnerabilities in bacterial envelope biogenesis and may lead to new antibiotics. Here, we employ a high-throughput genetic screen to uncover genome-wide host suppressors of Sgl activity and apply it to a set of diverse Sgls with unknown molecular targets. In addition to validating known molecular mechanisms, we determined that the Sgl of PP7, an ssRNA phage of P. aeruginosa, targets MurJ, the flippase responsible for Lipid II export which was previously shown to be the target of the Sgl of coliphage M. These two Sgls, which are unrelated and predicted to have opposite membrane topology, thus represent a case of convergent evolution. Another set of Sgls which are thought to cause lysis without inhibiting cell wall synthesis elicit a common set of multicopy suppressors, suggesting these Sgls act by the same or similar mechanism.

microbiology↗