Biology subjects

Davidson, A. R.

Publications and source records attributed to Davidson, A. R..

16 recordsLinked to original sources

bioRxiv2026

A dramatic protein fold switch powers a bactericidal nanomachine

Fold switching, where a protein region interconverts between entirely distinct three-dimensional structures, is emerging as vital for certain protein functions. Here, we report a remarkable example in the F7 pyocin, a phage tail-like bactericidal nanomachine. Cryogenic electron microscopy and tomography reveal that a 163-residue segment of the central tail fiber undergoes a dramatic transition--from a trimeric -helical coiled-coil to a triangular {beta}-prism--upon binding to the bacterial cell surface. This massive fold switch remodels the tail tip, ejects the internal tape measure protein, and drives membrane puncture. Site-directed mutations that selectively destabilize the {beta}-prism conformation completely abolish bactericidal activity without impairing particle assembly, implying that the energy released during this transition powers penetration. AlphaFold-based analyses further predict similar large-scale coiled-coil to {beta}-prism switches in diverse non-contractile phage tails. This discovery reveals a sophisticated, ATP-independent strategy for microbial warfare and opens exciting possibilities for engineering next-generation bacteriocins to combat multidrug-resistant pathogens.

microbiology↗

bioRxiv2025

GNAT: An Interactive Web Tool for Gene Neighbourhood Analysis

Given the sequence of a protein, the Gene Neighbourhood Analysis Tool (GNAT) identifies homologues within microbial (bacterial, archaeal, or fungal) or viral databases, aligns and clusters their genomic neighbourhoods (GNs) by similarity, reports the taxonomic distribution of matched genomes, and generates interactive, publication-quality visualizations with customizable annotations, colours, and display options, along with categorical and quantitative summary analytics. GNATs intuitive interface enables a broad community of researchers to generate biological insights from GNs without requiring computational expertise and serves as a comprehensive and versatile platform for easily comparing GNs across the domains of life. GNAT is accessible at https://gnat.usask.ca.

bioinformatics↗

bioRxiv2025

A novel method for detection of Cas9 gRNAs using a fluorophore-labeled DNA oligo

Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) has become one of the most important gene editing tools. CRISPR guide RNA (gRNA) serves as an important component in guiding Cas proteins to a target site in gene editing processes. For effective gene editing or site specific gene integration, levels of gRNA present in a target cell are critical. Especially, for prime editing, even the ratio of different gRNAs present in target cells has a major effect on gene editing efficiency. Therefore, having a convenient, highly sensitive method for direct detection of gRNAs can improve editing design and optimize targeting efficiency. Here, we have developed a convenient, highly sensitive method for direct detection of gRNAs, which may be used to improve editing design and optimize targeting efficiency.

molecular biology↗

bioRxiv2024

Prophages express a type IV pilus component to provide anti-phage defence

Phage genomes integrated within bacterial genomes, known as prophages, frequently encode proteins that provide defence against further phage infection. These proteins often function at the cell surface and prevent phages from attaching to their host receptor. Here, we describe the discovery of prophage-encoded proteins that resemble FimU, a component of the Pseudomonas aeruginosa type IV pilus. These phage FimU proteins are incorporated into the pilus without altering its function, yet they mediate robust protection against infection by phages that bind to the tip of the pilus, where FimU is likely located. The phage FimU proteins and the phage tail proteins that interact with FimU are highly diverse, suggesting that evolution in this system is driven by phage versus phage competition. To our knowledge, this is the first example of anti-phage defence mediated by replacement of a bacterial cell surface component with a phage-encoded protein.

microbiology↗

bioRxiv2024

Prophages block cell surface receptors to ensure survival of their viral progeny

In microbial communities, viruses compete for host cells to infect, and thus evolved diverse ways to inhibit their competitors. One mechanism is Superinfection exclusion (Sie), whereby a virus that has established an infection prevents a secondary infection. We identified a Pseudomonas prophage Sie protein that alters pilus dynamics through the pilus assembly chaperone, PilZ. This protein, known as Zip for PilZ interacting protein, does not abrogate pilus activity, but fine tunes it, providing strong phage resistance without a fitness cost. This tuning is modulated through quorum sensing, which coordinates Zip production in concert with bacterial cell density to ensure maximal protection when bacterial populations are at the highest risk of phage infection. Most notably, Zip activity prevents internalization and destruction of phage progeny. We refer to this as the "anti-Kronos effect" after the Greek god who devoured his own children and show that it is a conserved feature of diverse prophage-encoded Sie systems.

microbiology↗

bioRxiv2022

Taxonomy of interventions at academic institutions to improve research quality

Research institutions and researchers have become increasingly concerned about poor research reproducibility and replicability, and research waste more broadly. Research institutions play an important role and understanding their intervention options is important. This review aims to identify and classify possible interventions to improve research quality, reduce waste, and improve reproducibility and replicability within research-performing institutions. Taxonomy development steps: 1) use of an exemplar paper of journal-level research quality improvement interventions, 2) 2-stage search in PubMed using seed and exemplar articles, and forward and backward citation searching to identify articles evaluating or describing research quality improvement, 3) elicited draft taxonomy feedback from researchers at an open-sciences conference workshop, and 4) cycles of revisions from the research team. The search identified 11 peer-reviewed articles on relevant interventions. Overall, 93 interventions were identified from peer-review literature and researcher reporting. Interventions covered before, during, and after study conduct research stages and whole of institution. Types of intervention included: Tools, Education & Training, Incentives, Modelling & Mentoring, Review & Feedback, Expert involvement, and Policies & Procedures. Identified areas for research institutions to focus on to improve research quality and for further research includes improving incentives to implement quality research practices, evaluating current interventions, encourage no- or low-cost/high-benefit interventions, examine institution research culture, and encourage mentor-mentee relationships.

scientific communication and education↗

bioRxiv2022

Core Defense Hotspots within Pseudomonas aeruginosa are a consistent and rich source of anti-phage defense systems

Bacteria use a diverse arsenal of anti-phage immune systems, including CRISPR-Cas and restriction enzymes. Identifying the full defense repertoire of a given species is still challenging, however. Here, we developed a computational tool to broadly identify anti-phage systems, which was applied to >180,000 genomes available on NCBI, revealing Pseudomonas aeruginosa to possess the most diverse anti-phage arsenal of any species with >200 sequenced genomes. Using network analysis to identify the common neighbors of anti-phage systems, we surprisingly identified two highly conserved core defense hotspot loci (cDHS1 and cDHS2). Across more than 1,000 P. aeruginosa strains, cDHS1 is up to 224 kb (mean: 34 kb) with varied arrangements of at least 31 immune systems while cDHS2 has 24 distinct systems (mean: 15.4 kb). cDHS1/2 are present in most P. aeruginosa isolates, in contrast to highly variable mobile DHSs. Most cDHS genes are of unknown function potentially representing new anti-phage systems, which we validated by identifying a novel anti-phage system (Shango) commonly encoded in cDHS1. Identification of core gene markers that flank immune islands could be a simple approach for immune system discovery and may represent popular landing spots for diverse MGEs carrying anti-phage systems.

genomics↗

bioRxiv2022

A contractile injection system mediates developmentally regulated intra-strain cell death in Streptomyces coelicolor

Extracellular contractile injection systems (eCIS) are encoded in diverse clades of bacterial species. Although closely related to contractile phage tails, these entities can inject toxic proteins into eukaryotic cells. The roles of eCIS in mediating cytotoxic activities has led to a view of them as defense mechanisms that are not central to normal bacterial lifecycles. Here, we provide evidence that eCIS play an entirely distinct role in Streptomyces coelicolor (Sco), where they appear to participate in the complex developmental process of this species. In particular, we have shown that Sco produces eCIS particles as a part of its normal growth cycle and that strains lacking functional eCIS particles exhibit pronounced alterations in their developmental program. Most intriguingly, eCIS-deficient mutants display significantly reduced levels of cell death and altered morphology during liquid growth. These data suggest that Sco eCIS function by inducing intra-strain lethality rather than by attacking foreign species.

microbiology↗

bioRxiv2022

Diverse innate immune factors protect yeast from lethal viral pathogenesis

In recent years, newly characterized anti-viral systems have proven to be remarkably conserved from bacteria to mammals, demonstrating that unique insights into these systems can be gained by studying microbial organisms. Despite the enthusiasm generated by these findings, the key microbial model organism Saccharomyces cerevisiae (budding yeast) has been minimally exploited for studies of viral defense, primarily because it is not infected with exogenously transmitted viruses. However, most yeast strains are infected with an endogenous double stranded RNA (dsRNA) virus called L-A, and previous studies identified conserved antiviral systems that attenuate L-A replication. Although these systems do not completely eradicate L-A, we show here that they do prevent proteostatic stress and lethality caused by L-A over-proliferation. Exploiting this new finding, we demonstrate that the genetic screening methods available in yeast can be used to identify additional conserved antiviral systems. Using these approaches, we discovered antiviral functions for the yeast homologs of polyA-binding protein (PABPC1) and the La-domain containing protein Larp1, which are both involved in viral innate immunity in humans. We also identified new antiviral functions for the RNA exonucleases REX2 and MYG1, both of which have distinct but poorly characterized human and bacterial homologs. These findings highlight the potential of yeast as a powerful model system for the discovery and characterization of conserved antiviral systems. Significance StatementThe budding yeast Saccharomyces cerevisiae has been minimally exploited for investigation of host-virus interactions despite its chronic infection with a double-stranded RNA virus called L-A. Controverting its presumed harmless nature, we show here that L-A causes pathogenesis in cells lacking parallel-acting viral attenuation pathways. Taking advantage of the genetic tools available in budding yeast, we identify several highly conserved proteins to play a role in antiviral defense. Some of these have been recently identified in humans to be involved in viral innate immunity, thus highlighting the potential of budding yeast as a model organism to identify and investigate new antiviral systems.

microbiology↗

bioRxiv2021

Structural and mechanistic insight into CRISPR-Cas9 inhibition by anti-CRISPR protein AcrIIC4

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPRs have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal struture of AcrIIC4 and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4 likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

biochemistry↗

bioRxiv2021

F-type Pyocins are Diverse Non-Contractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or non-contractile phage tails known as R-type and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3-end of the F-type pyocin cluster are divergent in sequence, and likely determine bactericidal specificity. We use sequence similarities among these proteins to define 11 distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly re-assorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins emerged earlier or have been subject to distinct evolutionary pressures. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as anti-bacterial therapeutics. IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes a broad spectrum of antibiotic resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great interest in the development of alternative therapeutics. One alternative is protein-based antimicrobials called bacteriocins, which are produced by one strain of bacteria to kill other strains. In this study, we investigate F-type pyocins, bacteriocins naturally produced by P. aeruginosa that resemble non-contractile phage tails. We show that they are potent killers of P. aeruginosa, and distinct pyocin groups display different killing specificities. We have identified the probable specificity determinants of F-type pyocins, which opens up the potential to engineer them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well characterized phage tails will greatly facilitate their development into effective antibacterials.

microbiology↗

bioRxiv2020

An anti-CRISPR protein induces strong non-specific DNA binding activity in a CRISPR-Cas complex

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complemetarity to the CRISPR RNA. We also demonstrate that induction of non-specific DNA-binding is a conserved property of diverse AcrIF9 homologues, implying that this activity contributes the biologically relevant function of this Acr family. AcrIF9 provides another example of the surprising variety of mechanisms by which Acrs inhibit CRISPR-Cas systems.

microbiology↗

bioRxiv2019

Early life stress decreases the proliferation and numbers of adult hypothalamic neural stem cells.

Early life stress (ELS) is a potent environmental factor that can confer enduring effects on brain structure and function. Exposure to stress during early life has been linked to a wide range of physiopathological consequences later in life. In particular, ELS has been shown to have lasting effects on neurogenesis in the adult brain, suggesting that ELS is a significant regulator of adult neural stem cell function. Here, we investigated the effect of ELS on the numbers and proliferation of neural stem cells in the hypothalamus of adult mice. We show that ELS has long term negative effects on hypothalamic neural stem cell numbers and on their proliferation. Specifically, ELS reduced the total numbers of PCNA+ cells present in hypothalamic areas surrounding the 3rd ventricle; the numbers of PCNA+/Sox2+/Nestin-GFP+ cells present in the medial eminence at the base of the 3rd ventricle; and the number of {beta}-tanycytes around the ventral 3rd ventricle, without affecting the numbers of -tanycytes in more dorsal areas. These results suggest that a reduction of proliferation and tanycyte numbers contributes to the effects of ELS on the hypothalamus and its consequent physiological alterations.

neuroscience↗

bioRxiv2019

Listeria phages induce Cas9 degradation to protect lysogenic genomes

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy foreign DNA. Bacteriophages, in turn, have evolved diverse \"anti-CRISPR\" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode 2-3 distinct anti-Cas9 proteins, with acrIIA1 always present; however, its mechanism is unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain and, in Listeria, triggers Cas9 degradation. AcrIIA1 displays broad-spectrum inhibition of Type II-A and II-C Cas9s, including an additional highly-diverged Listeria Cas9. During lytic infection, AcrIIA1 is insufficient for rapid Cas9 inactivation, thus phages require an additional \"partner\" Acr that rapidly blocks Cas9-DNA-binding. The AcrIIA1 N-terminal domain (AcrIIA1NTD) is dispensable for anti-CRISPR activity; instead it is required for optimal phage replication through direct transcriptional repression of the anti-CRISPR locus. AcrIIA1NTD is widespread amongst Firmicutes, can repress anti-CRISPR deployment by other phages, and has been co-opted by hosts potentially as an \"anti-anti-CRISPR.\" In summary, Listeria phages utilize narrow-spectrum inhibitors of DNA binding to rapidly inactivate Cas9 in lytic growth and the broad-spectrum AcrIIA1 to stimulate Cas9 degradation for protection of the Listeria genome in lysogeny.

microbiology↗

bioRxiv2018

Potent Cas9 inhibition in bacterial and human cells by new anti-CRISPR protein families

CRISPR-Cas systems are widely used for genome engineering technologies, and in their natural setting, they play crucial roles in bacterial and archaeal adaptive immunity, protecting against phages and other mobile genetic elements. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems1. We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature2-4. We have identified new anti-CRISPRs using the bioinformatic approach that successfully identified previous Acr proteins1 against Neisseria meningitidis Cas9 (NmeCas9). In this work we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems5. We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect co-evolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.

molecular biology↗

bioRxiv2017

Bacteria defend against phages through type IV pilus glycosylation

Bacterial surface structures such as type IV pili are common receptors for phage. Strains of the opportunistic pathogen Pseudomonas aeruginosa express one of five different major type IV pilin alleles, two of which are glycosylated with either lipopolysaccharide O-antigen units or polymers of D-arabinofuranose. Here we show that both these post-translational modifications protect P. aeruginosa from a variety of pilus-specific phages. We identified a phage capable of infecting strains expressing both non-glycosylated and glycosylated pilins, and through construction of a chimeric phage, traced this ability to its unique tail proteins. Alteration of pilin sequence, or masking of binding sites by glycosylation, both block phage infection. The energy invested by prokaryotes in glycosylating thousands of pilin subunits is thus explained by the protection against phage predation provided by these common decorations.\n\nSIGNIFICANCEPost-translational modification of bacterial and archaeal surface structures such as pili and flagella is widespread, but the function of these decorations is not clear. We propose that predation by bacteriophages that use these structures as receptors selects for strains that mask potential phage binding sites using glycosylation. Phages are of significant interest as alternative treatments for antibiotic-resistant pathogens, but the ways in which phage interact with host receptors are not well understood. We show that specific phage tail proteins allow for infection of strains with glycosylated pili, providing a foundation for the creation of designer phages that can circumvent first-line bacterial defenses.

microbiology↗