Biology subjects

Laub, M. T.

Publications and source records attributed to Laub, M. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Viral SSB-bound ssDNA activates the bacterial anti-phage defense system DARNA

To protect themselves against phage infection, bacteria employ diverse defense systems that are typically activated specifically upon infection. However, the mechanisms of activation and self versus non-self discrimination for most systems remain poorly understood. Here, we show that the bacterial immunity protein DARNA, once activated, cleaves a subset of host tRNAs, thereby inhibiting phage propagation. Although phages escape DARNA-mediated defense through mutations in the gene encoding single-stranded DNA-binding protein (SSB), we find that phage SSBs do not directly stimulate DARNA. Instead, DARNA is activated by single-stranded DNA presented by phage SSB, but not by the host SSB. The recognition of an endogenous nucleic acid signal promoted by a viral protein ensures that DARNA can detect and respond to a broad range of viruses while avoiding auto-immunity.

microbiology↗

bioRxiv2025

A membrane-bound nuclease directly cleaves phage DNA during genome injection

From mammals to bacteria, the direct recognition and cleavage of viral nucleic acids is a potent defense strategy against viral infection, but it requires mechanisms for distinguishing self from non-self1,2. In bacteria, CRISPR-Cas and restriction modification systems achieve this discrimination by recognizing specific DNA sequences or DNA modifications. Alternative mechanisms likely remain to be discovered. Here, we characterize SNIPE, a novel anti-phage defense system that constitutively localizes to the bacterial cell membrane in E. coli to block phage {lambda} infection. Using radiolabeled phage DNA and time-lapse microscopy to track phage genomes we demonstrate that SNIPE directly cleaves phage DNA during genome injection. Based on proximity labeling, we find that SNIPE associates with host proteins essential for {lambda} genome entry and with the {lambda} tape measure protein, which facilitates {lambda} genome injection across the inner membrane. SNIPE also defends against diverse siphoviruses, likely through direct interactions with their tape measure proteins. Our findings establish SNIPE as a widespread bacterial defense system that exploits the spatial organization of phage genome injection to specifically target viral DNA, representing a novel strategy for distinguishing self from non-self in prokaryotic immune systems.

microbiology↗

bioRxiv2025

Unmasking Pathogen Traits for Chronic Colonization in Neurogenic Bladder Patients

Individuals with neurogenic bladder are particularly susceptible to both chronic bacterial colonization of the bladder and urinary tract infections (UTIs). Neurogenic bladder can arise from a variety of diseases such as diabetes, spinal cord injuries, and spina bifida. To study the ecological and evolutionary dynamics of the microbiome in neurogenic bladder, we developed a longitudinal cohort of 77 children and young adults with spina bifida from two medical centers. We used enhanced urine culture, 16S rRNA sequencing, and whole genome sequencing to characterize the microbial composition of urine and fecal samples. In addition to prospective sample collection, we retrieved prior bacterial isolates from enrolled patients from Vanderbilts clinical microbial biobank, MicroVU. This allowed us to compare bacterial isolates from the same patients over a period of five years. Urine samples were characterized by high abundance of urinary pathogens, such as E. coli and Klebsiella. From longitudinal isolates from individual patients, we identified two common patterns of urinary tract colonization. We observed either the rapid cycling of strains and/or species, often following antibiotic treatment, or we observed the persistence of a single strain across timepoints. Neither persistence of a strain nor colonization with a new strain or species was associated with increased antibiotic resistance. Rather, in paired longitudinally collected strains from the same patients, mutations were identified in genes that code for cell envelope components associated with immune or phage evasion. Experimental testing revealed that O-antigen/LPS biosynthesis mutations confer protection from the immune system while altering susceptibility to phage predation, reflecting a fitness trade-off. We argue that this unparalleled cohort offers the opportunity to identify mechanisms of bacterial adaptation to the urinary tract that can be exploited in future therapeutic approaches.

microbiology↗

bioRxiv2025

DefensePredictor: A Machine Learning Model to Discover Novel Prokaryotic Immune Systems

Anti-phage defense systems protect bacteria from viruses. Studying defense systems has begun to reveal the evolutionary roots of eukaryotic innate immunity and produced important biotechnologies such as CRISPR-Cas9. Dozens of new systems have been discovered by looking for systems that co-localize in genomes, but this approach cannot identify systems outside defense islands. Here, we present DefensePredictor, a machine-learning model that leverages embeddings from a protein language model to classify proteins as defensive. We applied DefensePredictor to 69 diverse E. coli strains and validated 45 previously unknown systems, with >750 additional unique proteins receiving high confidence predictions. Our model, provided as open-source software, will help comprehensively map the anti-phage defense landscape of bacteria, further reveal connections between prokaryotic and eukaryotic immunity, and accelerate biotechnology development.

microbiology↗

bioRxiv2024

A versatile bacterial innate immunity protein directly senses two disparate phage proteins

Eukaryotic innate immune systems use pattern recognition receptors (PRRs) to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages1-6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have only been found to sense a single trigger during infection, suggesting a one-to-one relationship between bacterial PRRs and their ligands7-11. Here, we demonstrate that the anti-phage defense protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the remarkable versatility of an immune sensory domain, which may be a common property of anti-phage defense systems and enable them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbor both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phage can only fully evade CapRelSJ46 defense when both triggers are mutated. Our work reveals how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defense systems and complex coevolutionary relationships between them and their phage-encoded triggers.

microbiology↗

bioRxiv2024

Anti-viral defense by an ADP-ribosyltransferase that targets mRNA to block translation

Host-pathogen conflicts are crucibles of molecular innovation. Selection for immunity to pathogens has driven the evolution of sophisticated immunity mechanisms throughout biology, including in bacteria that must evade their viral predators known as bacteriophages. Here, we characterize a widely distributed anti-phage defense system, CmdTAC, that provides robust defense against infection by the T-even family of phages. Our results support a model in which CmdC detects infection by sensing viral capsid proteins, ultimately leading to the activation of a toxic ADP-ribosyltransferase effector protein, CmdT. We show that newly synthesized capsid protein triggers dissociation of the chaperone CmdC from the CmdTAC complex, leading to destabilization and degradation of the antitoxin CmdA, with consequent liberation of the CmdT ADP-ribosyltransferase. Strikingly, CmdT does not target a protein, DNA, or structured RNA, the known targets of other ADP-ribosyltransferases. Instead, CmdT modifies the N6 position of adenine in GA dinucleotides within single-stranded RNAs leading to arrest of mRNA translation and the inhibition of viral replication. Our work reveals a new mechanism of anti-viral defense and a previously unknown but broadly distributed class of ADP-ribosyltransferases that target mRNA.

microbiology↗

bioRxiv2024

Mechanism of phage sensing and abortion by toxin-antitoxin-chaperone systems

Toxin-antitoxins (TAs) are prokaryotic two-gene systems comprised of a toxin neutralised by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilises the antitoxin by recognising its chaperone addiction (ChAD) element. TACs have been shown to mediate antiphage defence, but the mechanisms of viral sensing and restriction are unexplored. We identify and characterise two Escherichia coli antiphage TAC systems containing HigBA and CmdTA TA units, HigBAC and CmdTAC. The HigBAC is triggered through recognition of the gpV major tail protein of phage {lambda}. Both the ChAD and gpV are recognised by the HigC chaperone through analogous aromatic molecular patterns, explaining the mechanism of activation. We show that the CmdT ADP-ribosyltransferase toxin modifies mRNA to shut down protein synthesis. We establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with the HigC chaperone phage sensor. HighlightsE. coli HigBAC and CmdTAC are translation-targeting phage immunity TAC systems HigC chaperone recognises phage {lambda} major tail protein to trigger HigBAC toxicity CmdT ADP-ribosyltransferase toxin abrogates translation through modification of mRNA HigC combined with CmdTA yields hybrid broad-spectrum antiphage defence system

microbiology↗

bioRxiv2023

Toxic anti-phage defense proteins inhibited by intragenic antitoxin proteins

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here we report these proteins are new toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic full-length proteins. The crystal structure of RpnAS revealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms. SignificanceHere we document the function of small genes-within-genes, showing they encode antitoxin proteins that block the functions of the toxic DNA endonuclease proteins encoded by the longer rpn genes. Intriguingly, a sequence present in both long and short protein shows extensive variation in the number of four amino acid repeats. Consistent with a strong selection for the variation, we provide evidence that the Rpn proteins represent a phage defense system.

microbiology↗

bioRxiv2023

Marginal specificity in protein interactions constrains evolution

The evolution of novel functions in biology relies heavily on gene duplication and divergence, creating large paralogous protein families. Selective pressure to avoid detrimental cross-talk often results in paralogs that exhibit exquisite specificity for their interaction partners. But how robust or sensitive is this specificity to mutation? Here, using deep mutational scanning, we demonstrate that a paralogous family of bacterial signaling proteins exhibits marginal specificity, such that many individual substitutions give rise to substantial cross-talk between normally insulated pathways. Our results indicate that sequence space is locally crowded despite overall sparseness, and we provide evidence that this crowding has constrained the evolution of bacterial signaling proteins. These findings underscore how evolution selects for good enough rather than optimized phenotypes, leading to restrictions on the subsequent evolvability of paralogs. Significance StatementLarge paralogous protein families are found throughout biology, the product of extensive gene duplication. To execute different functions inside cells, paralogs typically acquire different specificities, interacting with only desired, cognate partners and avoiding cross-talk with non-cognate proteins. But how robust is this interaction specificity to mutation? Can individual mutations lead to cross-talk or do paralogs diverge enough such that multiple mutations would be required, providing a mutational buffer against cross-talk? To address these questions, we built mutant libraries that produce all possible single substitutions of a bacterial kinase and then screened for cross-talk to non-cognate proteins. Strikingly, we find that many single substitutions can produce cross-talk, meaning that these pathways typically exhibit only marginal specificity, and demonstrate that this restricts their evolvability.

evolutionary biology↗

bioRxiv2023

Selection of a de novo gene that can promote survival of E. coli by modulating protein homeostasis pathways

Cells sometime adapt to challenging environments by turning non-functional loci into functional genes in a process termed de novo gene birth. But how proteins with random amino acid sequences integrate into existing cellular pathways to provide a benefit remains poorly understood. Here, we screened [~]108 random genes for their ability to rescue growth arrest of E. coli cells producing the ribonuclease toxin MazF. Approximately 2,000 random genes could promote growth by reducing transcription from the promoter driving mazF expression. Additionally, one gene, named random antitoxin of MazF (ramF), whose protein product was well-tolerated in cells and neutralized MazF by interacting with chaperones, leading to MazF proteolysis. We show that the specificity of RamF for MazF relative to other toxins relies on the degron-like function of MazFs first 10 amino acids. Finally, we demonstrate that random proteins can improve during evolution by identifying beneficial mutations that turned RamF into a more efficient inhibitor. Our work provides a mechanistic basis for how de novo gene birth can produce new, functional proteins that are integrated into complex cellular systems and provide a benefit to cells.

evolutionary biology↗

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↗

bioRxiv2022

Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome

The ancient, ongoing coevolutionary battle between bacteria and their viral predators, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Dozens of additional anti-phage systems have been identified based on their co-location within so-called defense islands, but whether these computational screens are exhaustive is unclear. Here, we developed an experimental selection scheme agnostic to genomic context to identify defense systems encoded in 71 diverse E. coli strains. Our results unveil 21 new and conserved defense systems, none of which were previously detected as enriched in defense islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defense systems in E. coli, with defense systems typically carried in specific locations, or hotspots. These hotspots in homologous prophages and mobile genetic elements encode dozens of additional, as-yet uncharacterized defense system candidates. Collectively, our findings reveal an extended landscape of antiviral immunity in E. coli and provide a generalizable approach for mapping defense systems in other species.

microbiology↗

bioRxiv2022

The evolution of a counter-defense mechanism in a virus constrains its host range

Bacteria use diverse immunity mechanisms to defend themselves against their viral predators, bacteriophages. In turn, phages can acquire counter-defense systems, but it remains unclear how such mechanisms arise and what factors constrain viral evolution. Here, we experimentally evolved T4 phage to overcome a phage-defensive toxin-antitoxin system, toxIN, in E. coli. Through recombination, T4 rapidly acquires segmental amplifications of a previously uncharacterized gene, now named tifA, encoding an inhibitor of the toxin, ToxN. These amplifications subsequently drive large deletions elsewhere in T4s genome to maintain a genome size compatible with capsid packaging. The deleted regions include accessory genes that help T4 overcome defense systems in alternative hosts. Thus, our results reveal a trade-off in viral evolution; the emergence of one counter-defense mechanism can lead to loss of other such mechanisms, thereby constraining host range. We propose that the accessory genomes of viruses reflect the integrated evolutionary history of the hosts they infected. HighlightsO_LIExperimentally evolved T4 to overcome E. coli toxIN, a phage-defensive TA system C_LIO_LIDiscovered TifA, a phage-encoded protein inhibitor of ToxN C_LIO_LIAmplification of the tifA locus drives large deletions elsewhere in the T4 genome C_LIO_LIDeleted genes in evolved T4 clones include those necessary to infect alternative hosts C_LI

microbiology↗

bioRxiv2022

Ancestral reconstruction of duplicated signaling proteins reveals the evolution of signaling specificity

Gene duplication is crucial to generating novel signaling pathways during evolution. However, it remains unclear how the redundant proteins produced by gene duplication ultimately acquire new interaction specificities to establish insulated paralogous signaling pathways. Here, we used ancestral sequence reconstruction to resurrect and characterize a bacterial two-component signaling system that duplicated in -proteobacteria. We determined the interaction specificities of the signaling proteins that existed before and immediately after this duplication event and then identified key mutations responsible for establishing specificity in the two systems. Just three mutations, in only two of the four interacting proteins, were sufficient to establish specificity of the extant systems. Some of these mutations weakened interactions between paralogous systems to limit crosstalk. However, others strengthened interactions within a system, indicating that the ancestral interaction, although functional, had the potential to be strengthened. Our work suggests that protein-protein interactions with such latent potential may be highly amenable to duplication and divergence.

evolutionary biology↗

bioRxiv2021

Coevolution of interacting proteins through non-contacting and non-specific mutations

Proteins often accumulate neutral mutations that do not affect current functions1 but can profoundly influence future mutational possibilities and functions2-4. Understanding such hidden potential has major implications for protein design and evolutionary forecasting5-7, but has been limited by a lack of systematic efforts to identify potentiating mutations8,9. Here, through the comprehensive analysis of a bacterial toxin-antitoxin system, we identified all possible single substitutions in the toxin that enable it to tolerate otherwise interface-disrupting mutations in its antitoxin. Strikingly, the majority of enabling mutations in the toxin do not contact, and promote tolerance non-specifically to, many different antitoxin mutations, despite covariation in homologs occurring primarily between specific pairs of contacting residues across the interface. In addition, the enabling mutations we identified expand future mutational paths that both maintain old toxin-antitoxin interactions and form new ones. These non-specific mutations are missed by widely used covariation and machine learning methods10,11. Identifying such enabling mutations will be critical for ensuring continued binding of therapeutically relevant proteins, such as antibodies, aimed at evolving targets12-14.

evolutionary biology↗

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↗

bioRxiv2021

Global analysis of the specificities and targets of endoribonucleases from E. coli toxin-antitoxin systems

Toxin-antitoxin systems are widely distributed genetic modules typically featuring toxins that can inhibit bacterial growth and antitoxins that can reverse inhibition. Although Escherichia coli encodes 11 toxins with known or putative endoribonuclease activity, the target of most of these toxins remain poorly characterized. Using a new RNA-seq pipeline that enables the mapping and quantification of RNA cleavage with single-nucleotide resolution, we characterize the targets and specificities of 9 endoribonuclease toxins from E. coli. We find that these toxins use low-information cleavage motifs to cut a significant proportion of mRNAs in E. coli, but not tRNAs or the rRNAs from mature ribosomes. However, all the toxins, including those that are ribosome-dependent and cleave only translated RNA, inhibit ribosome biogenesis. This inhibition likely results from the cleavage of ribosomal protein transcripts, which disrupts the stoichiometry and biogenesis of new ribosomes and causes the accumulation of aberrant ribosome precursors. Collectively, our results provide a comprehensive, global analysis of endoribonuclease-based toxin-antitoxin systems in E. coli and support the conclusion that, despite their diversity, each disrupts translation and ribosome biogenesis. ImportanceToxin-antitoxin systems are widespread genetic modules found in almost all bacteria that can regulate their growth and may play prominent roles in phage defense. Escherichia coli encodes 11 TA systems in which the toxin is a known or predicted endoribonuclease. The targets and cleavage specificities of these endoribonucleases have remained largely uncharacerized, precluding an understanding of how each impacts cell growth and an assessment of whether they have distinct or overlapping targets. Using a new and broadly applicable RNA-seq pipeline, we present a global analysis of 9 endoribonulease toxins from E. coli. We find that each uses a relatively low-information cleavage motif to cut a large proportion of mRNAs in E. coli, but not tRNAs or mature rRNAs. Notably, although the precise set of targets varies, each toxin efficiently disrupts ribosome biogenesis, primarily by cleaving the mRNAs of ribosomal proteins. In sum, the analyses presented provide new, comprehensive insights into the cleavage specificities and targets of almost all endoribonuclease toxins in E. coli. Despite different specificities, our work reveals a striking commonality in function as each toxin disrupts ribosome biogenesis and translation.

microbiology↗

bioRxiv2021

Activation of a signaling pathway by the physical translocation of a chromosome

In every organism, the cell cycle requires the execution of multiple cellular processes in a strictly defined order. However, the mechanisms used to ensure such order remain poorly understood, particularly in bacteria. Here, we show that the activation of the essential CtrA signaling pathway that triggers cell division in Caulobacter crescentus is intrinsically coupled to the successful initiation of DNA replication via the physical translocation of a newly-replicated chromosome, powered by the ParABS system. We demonstrate that ParA accumulation at the new cell pole during chromosome segregation recruits ChpT, an intermediate component of the CtrA signaling pathway. ChpT is normally restricted from accessing the selective PopZ polar microdomain until the new chromosome and ParA arrive. Consequently, any disruption to DNA replication initiation prevents the recruitment of ChpT and, in turn, cell division. Collectively, our findings reveal how major cell-cycle events are coordinated in Caulobacter and, importantly, how the physical translocation of a chromosome triggers an essential signaling pathway.

microbiology↗