Biology subjects

Westley, J.

Publications and source records attributed to Westley, J..

6 recordsLinked to original sources

bioRxiv2026

Maintenance of restriction-modification system diversity creates pan-immunity

Bacterial antiviral defense systems are highly diverse and unevenly distributed across populations, such that different strains carry distinct immune repertoires that together form a pan-immune system. Restriction-modification (RM) systems are the most abundant bacterial defense systems and exemplify this architecture, exhibiting remarkable diversity in structure, function, specificity, and efficacy. How and why such diversity is maintained, why weaker systems are not outcompeted, and whether diversity provides benefits beyond protection of individual cells remain unclear. Here, we experimentally tracked bacteria and phages across populations differing in RM diversity. We find no consistent evidence of a trade-off between defense strength and host fitness. Instead, we identify a trade-off between defense strength and horizontal transferability: stronger RM systems are less able to spread between strains, likely because factors that increase defense strength also increase toxicity risk upon transfer to unmethylated hosts. These weaker yet more mobile systems may therefore act as more accessible pan-immune components that bacterial populations can acquire in response to shifting parasite pressures. Furthermore, we show RM systems experience negative frequency-dependent selection, such that common specificities are more readily overcome by phages while rare ones remain protective, causing the benefits of potent systems to erode as they spread. Together, these dynamics maintain RM diversity and explain why weaker systems persist alongside stronger ones. Finally, we show that population-level RM diversity generates collective protection by constraining the emergence of RM-escaping phages and limiting the invasion of bacterial surface mutants that avoid phage adsorption, providing experimental support for the pan-immune model.

microbiology↗

bioRxiv2026

Dual ATPase-activated DNA hemimethylation and cleavage by the MANTIS defence system

Bacterial defence systems are diverse and form major barriers to horizontal gene transfer and bacteriophage (phage) infection. Though restriction-modification systems were the first such defences discovered and remain the most widespread, the basis of self/non-self discrimination in many newly identified methylation-based systems remains unclear. Here, we define the mechanism of a widely distributed BREX-related defence family, previously termed Gao_RL, which we rename MANTIS (Methyltransferase, ATPases and Nuclease Targeting Inverted-repeat Sequences). Using phage infection and plasmid transformation assays in Pseudomonas aeruginosa, biochemical reconstitution, and structural biology, we show that MANTIS establishes self-identity through adenine hemimethylation of short asymmetric DNA sequences. Unexpectedly, a AAA+ ATPase (MtsA) and a co-regulator (MtsB) are both required to recruit and activate the methyltransferase MtsC, revealing a previously unknown ATP-dependent mode of epigenetic modification. DNA recognition by the MtsABC complex then triggers restriction by a dimeric helicase-nuclease effector (MtsD), with activity dependent on target-site architecture: inverted pairs of unmodified sites support robust restriction, whereas direct repeats or single sites are restricted less efficiently. Consistent with this model, virulent phages show strand-biased depletion of MANTIS target sites, indicating evolutionary pressure to evade restriction. Together, these findings reveal how dual ATPases couple self-modification to non-self restriction and provide a framework for understanding the expanding landscape of methylation-based defence systems.

molecular biology↗

bioRxiv2025

Escape from antimicrobial CRISPR-Cas9 in E. coli ST131 depends on the genetic context of the target gene.

Escherichia coli (E. coli) is a common bacterium in the human gut and an important cause of intestinal and extraintestinal infections. Some E. coli sequence types (ST) are associated with high pathogenicity. The Extraintestinal Pathogenic E. coli (ExPEC) ST131 is a globally distributed multidrug-resistant human pathogen associated with urinary tract and bloodstream infections. Antibiotic-resistant infections often lead to antibiotic treatment failure, underscoring the need of developing alternative treatments. The highly selective antimicrobial potential of CRISPR-Cas9 has been demonstrated in a range of model organisms. However, the effectiveness of CRISPR-Cas9 in combating ST131-associated infections and the consequences of CRISPR-Cas9 treatment, such as the emergence of escapers, remains unclear. Here, we investigated the antimicrobial activity of CRISPR-Cas9 against ST131 and assessed the frequency and genetic basis of escape. We conjugatively delivered CRISPR-Cas9 to ST131 isolates which carried cefotaxime-resistance-encoding target gene blaCTX-M-15 in the chromosome and characterized escape subpopulations. Two main types of escapers emerged: blaCTX-M-15-positive escapers carried dysfunctional CRISPR-Cas9 systems and arose at a [~]10-5 frequency. Instead, blaCTX-M-15-negative escapers presented chromosomal deletions involving blaCTX-M-15 loss. The frequency of blaCTX-M-15 loss depended on the blaCTX-M-15 genetic context. Specifically, blaCTX-M-15-negative escapers emerged at low frequency ([~]10-5) in isolates where blaCTX-M-15 was located downstream of insertion sequence (IS) ISEcp1, while escapers emerged with high frequency ([~]10-3) in isolates where blaCTX-M-15 was flanked by IS26. This work emphasizes how the genetic context of target genes can drive the outcome of CRISPR-Cas9 tools, where the presence of IS26 may drive increased frequencies of escape. IMPORTANCEIn the past decade CRISPR-Cas9 has emerged as an efficient antimicrobial tool capable of selective elimination of targeted bacteria. Even though it has been well described that bacteria can evolve to escape targeting by CRISPR-Cas9, the mechanisms of bacterial escape and their consequences remain largely elusive. In this study, we demonstrate the antimicrobial efficacy of CRISPR-Cas9 against natural isolates of Escherichia coli ST131, a clinically relevant pathogen, and elucidate the mechanism of escape from antimicrobial activity. We identify two distinct mechanisms of escape, which involve either dysfunctional CRISPR-Cas9 activity, or loss of the target gene (blaCTX-M-15), with the latter occurring at frequencies that depend on the genetic context of the target gene. These findings provide important insights into the frequency and mechanisms of bacterial escape from CRISPR-Cas9-based antimicrobials and offer a foundation for the development of more effective treatments.

microbiology↗

bioRxiv2025

Eco-evolutionary feedbacks drive the co-occurrence of restriction-modification systems and antimicrobial resistance genes

Bacterial pathogens commonly become drug resistant via horizontal acquisition of antimicrobial resistance genes (ARGs), which are often encoded on mobile genetic elements (MGEs). Although bacterial defence systems are typically considered barriers to horizontal gene transfer (HGT), previous studies revealed that bacteria with more restriction-modification (RM) systems (the most abundant bacterial defences) frequently carry more MGEs. It was suggested that this counterintuitive relationship might result from stronger selection for RM systems when exposure to costly MGEs increases. Here, we test this hypothesis using a combination of modelling and bioinformatics analysis of >40,000 bacterial genomes to better understand how eco-evolutionary feedbacks between selection for RM and acquisition of MGEs shape bacterial genome evolution. Our model predicts negative associations between HGT and RM, but only if RM diversity is high. By contrast, at low RM diversity, eco-evolutionary feedbacks drive the emergence of positive associations between HGT and RM. Consistent with these predictions, we identified negative relationships between acquired ARG counts and RM counts across species but positive relationships within individual species. Collectively, our work helps to understand how RM systems shape patterns of HGT of ARGs, which may offer opportunities for targeted surveillance of strains at higher risk of horizontally acquiring novel drug resistance alleles. Significance statementPrevious research shows positive associations between bacterial restriction-modification (RM) systems and mobile genes, despite RM being well-evidenced as a barrier to mobile gene acquisition. Eco-evolutionary feedbacks have been hypothesised to drive this; higher HGT exposure will select for greater RM investment, while still leading to increased mobile gene acquisition. By formalising this hypothesis into a mathematical model and analysing bacterial genomes, we reveal novel insights into how the relationship between RM and mobile antimicrobial resistance genes (ARGs) depends on ecological variables. This study illustrates that to better comprehend how defence systems regulate gene transfer in natural communities, it is essential to consider both their mechanisms and the complex eco-evolutionary dynamics at play.

microbiology↗

bioRxiv2024

Heterogeneous efflux pump expression underpins phenotypic resistance to antimicrobial peptides

Antimicrobial resistance threatens the viability of modern medical interventions. There is a dire need of developing novel approaches to counter resistance mechanisms employed by starved or slow-growing pathogens that are refractory to conventional antimicrobial therapies. Antimicrobial peptides have been advocated as potential therapeutic solutions due to low levels of genetic resistance observed in bacteria against these compounds. However, here we show that subpopulations of stationary phase Escherichia coli and Pseudomonas aeruginosa survive tachyplesin treatment without genetic mutations. These phenotypic variants display enhanced efflux activity to limit intracellular peptide accumulation. Differential regulation of genes involved in outer membrane vesicle section, membrane modification, and protease activity were also found between phenotypically resistant and susceptible cells. We discovered that formation of these phenotypic variants could be prevented by administering tachyplesin in combination with sertraline, a clinically used antidepressant, suggesting a novel approach for combatting antimicrobial-refractory stationary phase bacteria.

microbiology↗

bioRxiv2019

Human antiviral protein MxA forms novel metastable membrane-less cytoplasmic condensates exhibiting rapid reversible \"crowding\"-driven phase transitions

Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles in the mammalian cell cytoplasm and nucleus. We report that the interferon (IFN)-inducible human \"myxovirus resistance protein A\" (MxA) forms membrane-less metastable condensates in the cytoplasm. Light and electron microscopy studies revealed that transient expression of HA- or GFP-tagged MxA in Huh7, HEK293T or Cos7 cells, or exposure of Huh7 cells to IFN-2a led to the appearance of MxA in the cytoplasm in membrane-less variably-sized spherical or irregular bodies, in filaments and even a reticulum. 1,6-Hexanediol treatment led to rapid disassembly of these condensates; however, FRAP revealed a relative rigidity with a mobile fraction of only 0.24{+/-}0.02 within condensates. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with GFP-MxA condensates. Remarkably, the cytoplasmic GFP-MxA condensates disassembled within 1-3 min of exposure of cells to hypotonic medium (40-50 milliosmolar) and reassembled within 0.5-2 min of re-exposure of cells to isotonic medium (310-325 milliosmolar) through multiple cycles. Mechanistically, the extent of cytoplasmic \"crowding\" regulated this phase-separation process. GFP-MxA condensates also included the DNA sensor protein cyclic GMP-AMP synthase (cGAS), another protein known to be associated with liquid-like condensates. Functionally, GFP-MxA expression inhibited DNA/cGAS-responsive ISG54-luciferase activity but enhanced relative inducibility of ISG54-luc by IFN-, revealing a physical separation between condensate- and cytosol-based signaling pathways in the cytoplasm. Taken together, the data reveal a new aspect of the cell biology of MxA in the cell cytoplasm.\n\nImportanceThe human interferon-inducible \"myxovirus resistance protein A\" (MxA), which displays antiviral activity against several RNA and DNA viruses, exists in the cytoplasm in phase-separated membrane-less metastable condensates of variably-sized spherical or irregular bodies, in filaments and even in a reticulum. MxA condensate formation appeared necessary but not sufficient for antiviral activity. Remarkably, MxA condensates showed the unique property of rapid (within 1-3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions Mechanistically, these phase transitions were regulated by the extent of cytoplasmic \"crowding.\" Moreover, GFP-MxA condensates included the DNA sensor protein cyclic GMP-AMP synthase (cGAS). Functionally, GFP-MxA expression inhibited DNA/cGAS-responsive ISG54-luciferase activity but enhanced inducibility of ISG54-luc by IFN-, revealing a biological distinction between condensate- and cytosol-based signaling pathways. Since intracellular edema and ionic changes are hallmarks of cytopathic viral effects, the rapid hypotonicity-driven disassembly of MxA condensates may modulate MxA.function during virus infection.

cell biology↗