Biology subjects

O'Toole, G. A.

Publications and source records attributed to O'Toole, G. A..

14 recordsLinked to original sources

bioRxiv2026

Ileitis abolishes tolerogenic functions of the enterohepatic bile acid pool

Bile acids (BAs) regulate lipid uptake, epithelial integrity, and immune responses in the gut. Hepatocytes synthesize primary BAs, which microbiota metabolize into secondary metabolites. Together, these species form a composite pool that circulates enterohepatically between the liver and intestines. Here, we show that immunoregulatory outputs of the enterohepatic BA pools involve competition between multiple BA species for individual nuclear receptors and are dependent on intestinal health. In healthy mice, the primary BA, tauro {beta} muricholic acid (t{beta}MCA) antagonized ROR{gamma}t-mediated Th17 function in the presence of two secondary BAs (tDCA, tLCA) which activated ROR{gamma}t. Conversely, ileitis in Crohns disease patients and Tnf{delta}ARE mice depleted primary BAs by reducing the number and function of BA transporting enterocytes. In mice, ileitis driven t{beta}MCA depletion enhanced tDCA and tLCA mediated ROR{gamma}t activation and supported Th17 cell function. Replenishing t{beta}MCA in Tnf{delta}ARE mice reestablished BA dependent Th17 cell regulation. Thus, intra pool competition underpins BA immunoregulatory functions and reveals new opportunities for precision Crohns disease therapy.

immunology↗

bioRxiv2026

Structural basis of biofilm formation mediated by the Pseudomonas aeruginosa fibrillar adhesin CdrA

Many bacteria, including the important human pathogen Pseudomonas aeruginosa, are naturally found in antibiotic-tolerant, multicellular biofilms. Cell-cell interactions within P. aeruginosa biofilms are mediated by a large fibrillar adhesin called CdrA in an extracellular polysaccharide-dependent manner. Here, we report an electron cryomicroscopy structure of the 60 kDa CdrA adhesive N-terminus, which combined with electron cryotomography of focused-ion beam milled specimens, allows us to derive a complete in situ model of the native adhesin. Our structure reveals a small adhesive domain (called ADEPT) at the distal tip of CdrA that is nearly perfectly conserved across the P. aeruginosa pangenome, with structural similarity to previously reported sugar-binding domains in multiple bacterial species. Inhibitory nanobodies targeting CdrA that reduce biofilm formation bind to epitopes in, or close to, the ADEPT on bacterial cells. Furthermore, structure-guided mutagenesis of residues within the ADEPT abolishes bacterial aggregation, and genomic deletion of the whole ADEPT leads to strong attenuation of biofilm formation. Our data forms a rational basis for future targeted inhibition of pathogenic P. aeruginosa biofilms and elucidates the mechanism of biofilm formation mediated by fibrillar adhesins that are widespread in bacteria.

microbiology↗

bioRxiv2025

Targeted disruption of phage liquid crystalline droplets abolishes antibiotic tolerance of bacterial biofilms

All bacterial biofilms contain an extracellular matrix rich in filamentous molecules 1 that self-associate 2-4, conferring emergent properties to bacteria 5, including antibiotic tolerance 6. Pseudomonas aeruginosa is a human pathogen that forms biofilms in diverse infectious settings 7,8, where the upregulation of a filamentous bacteriophage Pf4, has been shown to be a key virulence factor that protects bacteria from antibiotics 9,10. Here, we modelled biophysical characteristics of biofilm-linked liquid crystalline droplets formed by Pf4, which predicted that sub-stoichiometric phage binders had the ability to disrupt liquid crystals by changing the surface properties of the phage. We tested this prediction by developing nanobodies targeting the outer surface of the Pf4 phage, which disrupted in vitro reconstituted droplets, promoted antibiotic diffusion into bacteria, disrupted P. aeruginosa biofilm formation under a variety of conditions, and abolished antibiotic tolerance of biofilms. The inhibition strategy illustrated in this study could be extended to biofilms of other pathogenic bacteria, where filamentous molecules are pervasive in the extracellular matrix. Furthermore, our findings exemplify how targeting a biophysical mechanism, rather than a defined biochemical target, is a promising avenue for intervention, with the potential of applying this concept to other disease-related contexts.

microbiology↗

bioRxiv2024

Interplay of Virulence Factors and Signaling Molecules: Albumin and Calcium-Mediated Biofilm Regulation in Bordetella bronchiseptica

Bordetella bronchiseptica, a respiratory pathogen capable of infecting various mammals, including humans, is associated with chronic infections, contrasting with the acute infections caused by Bordetella pertussis. Both pathogens can form biofilm-like structures in vivo, providing tolerance against environmental stresses. Biofilm formation by B. bronchiseptica is regulated by the BvgAS two-component system, with intermediate concentrations of certain modulators inducing a phase favoring biofilm formation. Recent studies have highlighted the role of cyclic diguanylate monophosphate (c-di-GMP) in this process: elevated c-di-GMP levels stimulate biofilm formation, whereas phosphodiesterase (PDE) activation reduces biofilms. Respiratory secretions, which contain albumin and calcium at higher concentrations than standard growth media, promote an increase in the amount and extracellular localization of the adenylate cyclase toxin (ACT), an important Bordetella spp. virulence factor. Secreted ACT present in the extracellular media or attached to the outer membrane inhibits biofilm formation. Based on these observations, we hypothesized that serum albumin and calcium together inhibit biofilm formation and explored the potential role of c-di-GMP in this process. Our findings demonstrate that serum albumin and calcium inhibit B. bronchiseptica biofilm formation by two apparently independent mechanisms, increasing AC secretion and inducing c-di-GMP degradation. This study contributes to the understanding of the mechanisms governing B. bronchiseptica biofilm formation and its modulation by host factors.

microbiology↗

bioRxiv2024

Quantifying forms and functions of intestinal bile acid pools in mice

Bile acids (BAs) are gastrointestinal metabolites that serve dual functions in lipid absorption and cell signaling. BAs circulate actively between the liver and distal small intestine (i.e., ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with intestinal cells in vivo remain ill-defined. Through multi-site sampling of nearly 100 BA species in individual wild type mice, as well as mice lacking the ileal BA transporter, Asbt/Slc10a2, we calculate the ileal BA pool in fasting C57BL/6J mice to be [~]0.3 moles/g. Asbt-mediated transport accounts for [~]80% of this pool and amplifies size, whereas passive absorption explains the remaining [~]20%, and generates diversity. Accordingly, ileal BA pools in mice lacking Asbt are [~]5-fold smaller than in wild type controls, enriched in secondary BA species normally found in the colon, and elicit unique transcriptional responses in cultured ileal explants. This work quantitatively defines ileal BA pools in mice and reveals how BA dysmetabolism can impinge on intestinal physiology.

physiology↗

bioRxiv2023

Persistent delay in maturation of the developing gut microbiota in infants with cystic fibrosis

The healthy human infant gut microbiome undergoes stereotypical changes in taxonomic composition between birth and maturation to an adult-like stable state. During this time, extensive communication between microbiota and the host immune system contributes to health status later in life. Although there are many reported associations between microbiota compositional alterations and disease in adults, less is known about how microbiome development is altered in pediatric diseases. One pediatric disease linked to altered gut microbiota composition is cystic fibrosis (CF), a multi-organ genetic disease involving impaired chloride secretion across epithelia and heightened inflammation both in the gut and at other body sites. Here, we use shotgun metagenomics to profile the strain-level composition and developmental dynamics of the infant fecal microbiota from several CF and non-CF longitudinal cohorts spanning from birth to greater than 36 months of life. We identify a set of keystone species whose prevalence and abundance reproducibly define microbiota development in early life in non-CF infants, but are missing or decreased in relative abundance in infants with CF. The consequences of these CF-specific differences in gut microbiota composition and dynamics are a delayed pattern of microbiota maturation, persistent entrenchment in a transitional developmental phase, and subsequent failure to attain an adult-like stable microbiota. We also detect the increased relative abundance of oral-derived bacteria and higher levels of fungi in CF, features that are associated with decreased gut bacterial density in inflammatory bowel diseases. Our results define key differences in the gut microbiota during ontogeny in CF and suggest the potential for directed therapies to overcome developmental delays in microbiota maturation.

microbiology↗

bioRxiv2023

How individual P. aeruginosa cells with diverse stator distributions collectively form a heterogeneous macroscopic swarming population

Swarming is a macroscopic phenomenon in which surface bacteria organize into a motile population. The flagellar motor that drives swarming in Pseudomonas aeruginosa is powered by stators MotAB and MotCD. Deletion of the MotCD stator eliminates swarming, whereas deletion of the MotAB stator enhances swarming. Interestingly, we measured a strongly asymmetric stator availability in the WT strain, with MotAB stators produced [~]40-fold more than MotCD stators. However, recruitment of MotCD stators in free swimming cells requires higher liquid viscosities, while MotAB stators are readily recruited at low viscosities. Importantly, we find that cells with MotCD stators are [~]10x more likely to have an active motor compared to cells without, so wild-type, WT, populations are intrinsically heterogeneous and not reducible to MotAB-dominant or MotCD-dominant behavior. The spectrum of motility intermittency can either cooperatively shut down or promote flagellum motility in WT populations. In P. aeruginosa, transition from a static solid-like biofilm to a dynamic liquid-like swarm is not achieved at a single critical value of flagellum torque or stator fraction but is collectively controlled by diverse combinations of flagellum activities and motor intermittencies via dynamic stator recruitment. Experimental and computational results indicate that the initiation or arrest of flagellum-driven swarming motility does not occur from individual fitness or motility performance but rather related to concepts from the jamming transition in active granular matter. ImportanceAfter extensive study, it is now known that there exist multifactorial influences on swarming motility in P. aeruginosa, but it is not clear precisely why stator selection in the flagellum motor is so important or how this process is collectively initiated or arrested. Here, we show that for P. aeruginosa PA14, MotAB stators are produced [~]40-fold more than MotCD stators, but recruitment of MotCD over MotAB stators requires higher liquid viscosities. Moreover, we find the unanticipated result that the two motor configurations have significantly different motor intermittencies, the fraction of flagellum-active cells in a population on average, with MotCD active [~]10x more often than MotAB. What emerges from this complex landscape of stator recruitment and resultant motor output is an intrinsically heterogeneous population of motile cells. We show how consequences of stator recruitment led to swarming motility, and how they potentially relate to surface sensing circuitry.

microbiology↗

bioRxiv2023

Bacterial mechanosensing of surface stiffness promotes signaling and growth leading to biofilm formation by Pseudomonas aeruginosa

The attachment of bacteria onto a surface, consequent signaling, and the accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that the stiffness of a surface may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of and response to surface stiffness are unknown. Furthermore, whether, and how, the surface stiffness impacts biofilm development, after initial accumulation, is not known. We use thin and thick hydrogels to create stiff and soft composite materials, respectively, with the same surface chemistry. Using quantitative microscopy, we find that the accumulation, motility, and growth of the opportunistic human pathogen Pseudomonas aeruginosa respond to surface stiffness, and that these are linked through cyclic-di-GMP signaling that depends on surface stiffness. The mechanical cue stemming from surface stiffness is elucidated using finite-element modeling combined with experiments - adhesion to stiffer surfaces results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to softer surfaces with identical surface chemistry. The cell-surface-exposed protein PilY1 acts as a mechanosensor, that upon surface engagement, results in higher cyclic-di-GMP levels, lower motility, and greater accumulation on stiffer surfaces. PilY1 impacts the biofilm lag phase, which is extended for bacteria attaching to stiffer surfaces. This study shows clear evidence that bacteria actively respond to different stiffness of surfaces where they adhere via perceiving varied mechanical stress and strain upon surface engagement. ImportanceBacteria colonize many types of biological and medical surfaces with a large range of stiffnesses. Colonization leads to the formation of biofilms, which cause costly and life-impairing chronic infections. However, whether and how bacteria can sense and respond to the mechanical cue provided by surface stiffness has remained unknown. We find that bacteria do indeed respond to surface stiffness in a way that is both consistent with expectations based on equilibrium continuum mechanics and that quantitatively impacts multiple aspects of early biofilm formation. This is a new understanding for the nascent field of bacterial mechanobiology. Furthermore, this finding suggests the possibility of a new category of approaches to hindering biofilm development by tuning the mechanical properties of biomedical surfaces.

microbiology↗

bioRxiv2022

Porous marine snow differentially benefits chemotactic, motile, and non-motile bacteria

Particulate organic carbon settling through the marine water column is a key process that regulates global climate by sequestering atmospheric carbon. The initial colonization of marine particles by heterotrophic bacteria represents the first step in recycling this carbon back to inorganic constituents - setting the magnitude of vertical carbon transport to the abyss. Here, we demonstrate experimentally that bacterial motility is required for particle colonization and chemotaxis specifically benefits at higher settling velocities. We further explore the role of particle microstructure on the colonization efficiency of bacteria with different motility traits. We highlight that non-motile cells benefit disproportionally from the porous microstructure and are relatively enriched in the particle wake due to the efficient particle colonization of chemotactic and motile cells. Our results imply that although the chemotactic and motile bacteria benefit from the high nutrient availability when colonizing the particles, scavenging of these cells benefits the often oligotrophic, non-motile cells common among the planktonic community. Significance statementBacteria in the ocean rely on ephemeral nutrient patches from sinking marine particles, but attaching to these structures is challenging as particle settling rates often exceed bacterial swimming velocities and the numerically dominant marine bacteria are non-motile - posing an interesting paradox about the prominence of particle foraging. Here, we quantify the importance of chemotaxis and motility for the efficient colonization of marine particles and find that although chemotaxis provides a clear advantage, motility is the basic requirement for particle colonization. We expand this analysis to consider highly heterogeneous particle structures and find a disproportionate benefit for non-motile cells by facilitating a direct encounter with the particle surface and enriching non-motile microbes in the nutrient-rich particle plume.

microbiology↗

bioRxiv2021

Non-motile subpopulations of Pseudomonas aeruginosa repress flagellar motility in motile cells through a type IV pili- and Pel-dependent mechanism

The downregulation of Pseudomonas aeruginosa flagellar motility is a key event in biofilm formation, host-colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that under swarming conditions, swarming motility can be repressed by cells that are non-motile due to the absence of a flagellum or flagellar rotation. Non-swarming cells, due to mutations that prevent either flagellum biosynthesis or rotation, present at 5% of the total population suppressed swarming of wild-type cells under the conditions tested in this study. Non-swarming cells required functional type IV pili and the ability to produce Pel exopolysaccharide to suppress swarming by the flagellated wild type. In contrast, flagellated cells required only type IV pili, but not Pel production, in order for swarming to be repressed by non-flagellated cells. We hypothesize that interactions between motile and non-motile cells may enhance the formation of sessile communities including those involving multiple genotypes, phenotypically-diverse cells, and perhaps other species. ImportanceOur study shows that, under the conditions tested, a small population of non-swarming cells can impact the motility behavior of the larger population. The interactions that lead to the suppression of swarming motility require type IV pili and a secreted polysaccharide, two factors with known roles in biofilm formation. These data suggest that interactions between motile and non-motile cells may enhance the transition to sessile growth in populations and promote interactions between cells with different genotypes.

microbiology↗

bioRxiv2021

Biochemical and Structural Characterization of Two Cif-Like Epoxide Hydrolases from Burkholderia cenocepacia

Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes typically utilize a pair of tyrosine residues to orient the substrate epoxide ring in the active site and stabilize the hydrolysis intermediate. A new subclass of epoxide hydrolases that utilize a histidine in place of one of the tyrosines was established with the discovery of the CFTR Inhibitory Factor (Cif) from Pseudomonas aeruginosa. Although the presence of such Cif-like epoxide hydrolases was predicted in other opportunistic pathogens based on sequence analyses, only Cif and its homologue aCif from Acinetobacter nosocomialis have been characterized. Here we report the biochemical and structural characteristics of Cfl1 and Cfl2, two Cif-like epoxide hydrolases from Burkholderia cenocepacia. Cfl1 is able to hydrolyze xenobiotic as well as biological epoxides that might be encountered in the environment or during infection. In contrast, Cfl2 shows very low activity against a diverse set of epoxides. The crystal structures of the two proteins reveal quaternary structures that build on the well-known dimeric assembly of the /{beta} hydrolase domain, but broaden our understanding of the structural diversity encoded in novel oligomer interfaces. Analysis of the interfaces reveals both similarities and key differences in sequence conservation between the two assemblies, and between the canonical dimer and the novel oligomer interfaces of each assembly. Finally, we discuss the effects of these higher-order assemblies on the intra-monomer flexibility of Cfl1 and Cfl2 and their possible roles in regulating enzymatic activity.

biochemistry↗

bioRxiv2020

Discovering the Molecular Determinants of Phaeobacter inhibens susceptibility to Phaeobacter phage MD18

Bacteriophage technologies have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. Here, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium Phaeobacter inhibens and dissected its mechanism of infection. Phaeobacter phage MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. Consistent with this finding, the sequence of the MD18 revealed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, GO term enrichment, and protein associations reveals that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens. ImportanceBacteriophages are useful non-antibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identifies Phaeobacter phage MD18, the first documented phage of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggests that the P haeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identifies genes that modulate host susceptibility to phage MD18 and implicates the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

microbiology↗

bioRxiv2019

Phenotypic differences in reversible attachment behavior reveal distinct P. aeruginosa surface colonization strategies

Despite possessing the machinery to sense, adhere to, and proliferate on surfaces, it is commonly observed that bacteria initially have a difficult time attaching to a surface. Before forming a bacterial biofilm, planktonic bacteria exhibit a random period of transient surface attachment known as \"reversible attachment\" which is poorly understood. Using community tracking methods at single-cell resolution, we examine how reversible attachment progresses during initial stages of surface sensing. Pseudomonas aeruginosa strains PAO1 and PA14, which exhibit similar exponential trends of surface cell population increase, show unanticipated differences when the behavior of each cell was considered at the full lineage level and interpreted using the unifying quantitative framework of an exactly solvable stochastic model. Reversible attachment comprises two regimes of behavior, processive and nonprocessive, corresponding to whether cells of the lineage stay on the surface long enough to divide, or not, before detaching. Stark differences between PAO1 and PA14 in the processive regime of reversible attachment suggest the existence of two complementary surface colonization strategies, which are roughly analogous to \"immediate-\" vs \"deferred-gratification\" in a prototypical cognitive-affective processing system. PAO1 lineages commit relatively quickly to a surface compared to PA14 lineages. PA14 lineages allow detaching cells to retain memory of the surface so that they are primed for improved subsequent surface attachment. In fact, it is possible to identify motility suppression events in PA14 lineages in the process of surface commitment. We hypothesize that these contrasting strategies are rooted in downstream differences between Wsp-based and Pil-Chp-based surface sensing systems.

microbiology↗

bioRxiv2019

Modeling the control of bacterial infections via antibiotic-induced proviruses

Most bacteria and archaea are infected by latent viruses that change their physiology and responses to environmental stress. We use a population model of the bacteria-phage relationship to examine the role that latent phage play on the bacterial population over time in response to antibiotic treatment. We demonstrate that the stress induced by antibiotic administration, even if bacteria are resistant to killing by antibiotics, is sufficient to control the infection under certain conditions. This work expands the breadth of understanding of phage-antibiotic synergy to include both temperate and chronic viruses persisting in their latent form in bacterial populations.\n\nImportanceAntibiotic-resistance is a growing concern for management of common bacterial infections. Here we show that antibiotics can be effective at sub-inhibitory levels when bacteria carry latent phage. Our findings suggest that specific treatment strategies based on the identification of latent viruses in individual bacterial strains may be an effective personalized medicine approach to antibiotic stewardship.

ecology↗