Biology subjects

LeRoux, M.

Publications and source records attributed to LeRoux, M..

6 recordsLinked to original sources

bioRxiv2026

Helicase family proteins promote antiviral ADP-ribosylation of DNA purines

DarTG defense systems studied to date contain a DarT1 or DarT2 toxin that blocks phage replication by ADP-ribosylating DNA and a NADAR or macrodomain antitoxin, DarG, that reverses this modification at homeostasis. Here, we identify additional DarTG subfamilies, one of which comprises a DarT3 toxin, an ADP-ribosylhydrolase (ARH)-domain antitoxin, and a helicase family protein, DarH. The Escherichia coli DarHTG3 system confers broad phage defense, requires DarH and, unlike previously characterized DarTG systems, modifies adenine. Characterization of additional DarH-containing DarTG systems reveals that a subset also target guanine. Our data support a model in which ADP-bound DarH functions as a processivity factor by recruiting DarT to its single-stranded DNA substrate and allowing for ADP-ribosylation of multiple target sites. These findings reveal how a helicase family protein regulates an antiviral effector by promoting DNA engagement rather than unwinding and thereby expanding the functional repertoire of helicase family proteins in bacterial immunity.

microbiology↗

bioRxiv2025

Acinetobacter phages use distinct strategies to breach the capsule barrier

Acinetobacter baumannii is an opportunistic pathogen that is a growing threat in hospital settings due to its alarmingly high rates of antibiotic resistance. Alternative therapies are urgently needed to manage the growing burden of untreatable A. baumannii infections. Phage therapy is a promising avenue that has already seen some success in isolated compassionate-use cases, including the famous "Patterson case". A. baumannii capsule is highly diverse both in structure and composition, and provides the first immunity barrier against phages. Here, we perform a detailed molecular characterization of three recently isolated, distinct A. baumannii phages that breach the capsule via different mechanisms. Like many previously described A. baumannii phages, a specific capsule type is necessary and sufficient for StAb1 infection. We found that StAb2 and its relatives adsorb to either a specific capsule type or the conserved outer membrane protein CarO, a porin normally occluded by the capsule. Thus, this phage has a narrow host range amongst capsulated strains, but can broadly infect A. baumannii strains lacking capsule. We also show that an unclassified siphophage, StAb3, requires a conserved and uncharacterized glycan, likely containing ManNAc, that enables StAb3 to infect a broad range of A. baumannii strains without depolymerizing the capsule. We demonstrate how rationally combining phages with distinct capsule interactions reduces the rapid emergence of phage escape mutants, with potential applications for more effective phage therapy.

microbiology↗

bioRxiv2024

Phages carry orphan antitoxin-like enzymes to neutralize the DarTG1 toxin-antitoxin defense system

The astounding number of anti-phage defenses encoded by bacteria is countered by an elaborate set of phage counter-defenses, though their evolutionary origins are often unknown. Here, we discover an orphan antitoxin counter-defense element in T4-like phages that can overcome the bacterial toxin-antitoxin phage defense system, DarTG1. The DarT1 toxin, an ADP-ribosyltransferase, modifies phage DNA to prevent replication while its cognate antitoxin, DarG1, is an ADP-ribosylglycohydrolase that reverses these modifications in uninfected bacteria. The orphan phage DarG1-like protein, which we term anti-DarT factor NADAR (AdfN), removes ADP-ribose modifications from phage DNA during infection thereby enabling replication in DarTG1-containing bacteria. AdfN, like DarG1, is in the NADAR superfamily of ADP-ribosylglycohydrolases found across domains of life. We find divergent NADAR proteins in unrelated phages that likewise exhibit anti-DarTG1 activity, underscoring the importance of ADP-ribosylation in bacterial-phage interactions, and revealing the function of a substantial subset of the NADAR superfamily.

microbiology↗

bioRxiv2022

Direct activation of an innate immune system in bacteria by a viral capsid protein

Bacteria have evolved sophisticated and diverse immunity mechanisms to protect themselves against a nearly constant onslaught of bacteriophages1-3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns (PAMPs)4, many bacterial immune systems that respond to bacteriophage infection require a phage-specific trigger to be activated. However, the identities of such triggers and the mechanistic basis of sensing remain almost completely unknown. Here, we discover and investigate the anti-phage function of a fused toxin-antitoxin (TA) system called CapRelSJ46 that protects E. coli against diverse phages. Through genetic, biochemical, and structural analysis, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both an antitoxin element and a phage-infection sensor. Following infection by certain phages, the newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to then pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for TA-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a Red Queen conflict5, revealing a new front in the intense coevolutionary battle being waged by phage and bacteria. With capsid proteins of some eukaryotic viruses known to stimulate innate immune signaling in mammalian hosts6-10, our results now reveal an ancient, deeply conserved facet of immunity.

microbiology↗

bioRxiv2021

The DarTG toxin-antitoxin system provides phage defense by ADP-ribosylating viral DNA

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, the bacteriophage. To identify TA systems that protect E. coli MG1655 against phage, we searched for those frequently encoded near known phage defense genes in bacterial genomes. Two of the systems tested provide strong protection against phage infection and are homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. We demonstrate that phage infection triggers the release of DarT toxin, a DNA ADP-ribosyltransferase, to modify viral DNA and prevent replication, thereby blocking the production of mature virions. Phages can evolve to overcome DarTG defense either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defense may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

microbiology↗

bioRxiv2020

Stress induces the transcription of toxin-antitoxin systems but does not activate toxin

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacterial genomes, but their functions are controversial. Although they are frequently postulated to regulate cell growth following stress, few null phenotypes for TA systems have been reported. Here, we show that TA transcript levels can increase substantially in response to stress, but toxin is not liberated. We find that the growth of an Escherichia coli strain lacking 10 TA systems encoding endoribonuclease toxins is not affected following exposure to six stresses that each trigger TA transcription. Additionally, using RNA-sequencing, we find no evidence of mRNA cleavage following stress. Stress-induced transcription arises from antitoxin degradation and relief of transcriptional autoregulation. Importantly, although free antitoxin is readily degraded in vivo, antitoxin bound to toxin is protected from proteolysis, preventing release of active toxin. Thus, transcription is not a reliable marker of TA activity, and TA systems likely do not strongly promote survival following stress.

microbiology↗