Biology subjects

Price-Whelan, A.

Publications and source records attributed to Price-Whelan, A..

11 recordsLinked to original sources

bioRxiv2026

Pseudomonas aeruginosa R-bodies deliver an ADP deaminase toxin

Diverse bacteria produce proteinaceous polymeric coils called R-bodies (refractile bodies), which extend into ~10 m-long needles in response to environmental triggers. It has been proposed that R-bodies deliver toxins intracellularly after such bacteria are ingested by eukaryotes, but intra-host extension of R-bodies has not been directly observed and these toxins remain unidentified. We use Pseudomonas aeruginosa PA14 and Dictyostelium discoideum to show that R-bodies extend within amoebae. We also identify a novel toxin, DeaR (deaminase associated with the R-body), which contributes to predation resistance and forms a near-megadalton dodecamer with ADP deaminase activity that alters purine metabolism. Our findings elucidate the molecular basis of R-body-mediated toxicity during PA14 infection and provide a paradigm for understanding the potential of cytotoxic effectors delivered by R-bodies (CEDRs) in interkingdom interactions.

microbiology↗

bioRxiv2024

Oxygen Microenvironments in E. coli Biofilm Nutrient Transport Channels: Insights from Complementary Sensing Approaches

Chemical gradients and the emergence of distinct microenvironments in biofilms are vital to the stratification, maturation and overall function of microbial communities. These gradients have been well characterised throughout the biofilm mass but the microenvironment of recently discovered nutrient transporting channels in Escherichia coli biofilms remains unexplored. This study employs three different oxygen sensing approaches to provide a robust quantitative overview of the oxygen gradients and microenvironments throughout the biofilm transport channel networks formed by E. coli macrocolony biofilms. Oxygen nanosensing combined with confocal laser scanning microscopy established that the oxygen concentration changes along the length of biofilm transport channels. Electrochemical sensing provided precise quantification of the oxygen profile in the transport channels, showing similar anoxic profiles compared with the adjacent cells. Anoxic biosensing corroborated these approaches, providing an overview of the oxygen utilisation throughout the biomass. The discovery that transport channels maintain oxygen gradients contradicts the previous literature that channels are completely open to the environment along the apical surface of the biofilm. We provide a potential mechanism for the sustenance of channel microenvironments via orthogonal visualisations of biofilm thin sections showing thin layers of actively growing cells. This complete overview of the oxygen environment in biofilm transport channels primes future studies aiming to exploit these emergent structures for new bioremediation approaches.

microbiology↗

bioRxiv2024

The L-lactate dehydrogenases of Pseudomonas aeruginosa are conditionally regulated but both contribute to survival during macrophage infection

Pseudomonas aeruginosa is an opportunistic pathogen that thrives in environments associated with human activity, including soil and water altered by agriculture or pollution. Because L-lactate is a significant product of plant and animal metabolism, it is available to serve as a carbon source for P. aeruginosa in the diverse settings it inhabits. Here, we evaluate P. aeruginosas production and use of its redundant L-lactate dehydrogenases, termed LldD and LldA. We confirm that the protein LldR represses lldD and identify a new transcription factor, called LldS, that activates lldA; these distinct regulators and the genomic contexts of lldD and lldA contribute to their differential expression. We demonstrate that the lldD and lldA genes are conditionally controlled in response to lactate isomers as well as to glycolate and {square}-hydroxybutyrate, which, like lactate, are {square}-hydroxycarboxylates. We also show that lldA is induced when iron availability is low. Our examination of lldD and lldA expression across depth in biofilms indicates a complex pattern that is consistent with the effects of glycolate production, iron availability, and cross-regulation on enzyme preference. Finally, macrophage infection assays revealed that both lldD and lldA contribute to persistence within host cells, underscoring the potential role of L-lactate as a carbon source during P. aeruginosa-eukaryote interactions. Together, these findings help us understand the metabolism of a key resource that may promote P. aeruginosas success as a resident of contaminated environments and animal hosts. ImportancePseudomonas aeruginosa is a major cause of lung infections in people with cystic fibrosis, hospital-acquired infections, and wound infections. It consumes L-lactate, which is found at substantial levels in human blood and tissues. In this study, we investigated the spatial regulation of two redundant enzymes, called LldD and LldA, which enable L-lactate metabolism in P. aeruginosa biofilms. We uncovered mechanisms and identified compounds that control P. aeruginosas LldD/LldA preference. We also showed that both enzymes contribute to its ability to survive within macrophages, a behavior that is thought to augment the chronicity and recalcitrance of infections. Our findings shed light on a key metabolic strategy used by P. aeruginosa and have the potential to inform the development of therapies targeting bacterial metabolism during infection.

microbiology↗

bioRxiv2023

Cell arrangement impacts metabolic activity and antibiotic tolerance in Pseudomonas aeruginosa biofilms

Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided by internal conduits that allow substances to travel more readily through tissue than they would via diffusion. Microbes growing in multicellular structures, called biofilms, are also affected by differential access to resources and we hypothesized that this is influenced by the physical arrangement of the cells. In this study, we examined the microanatomy of biofilms formed by the pathogenic bacterium Pseudomonas aeruginosa and discovered that clonal cells form striations that are packed lengthwise across most of a mature biofilms depth. We identified mutants, including those defective in pilus function and in O-antigen attachment, that show alterations to this lengthwise packing phenotype. Consistent with the notion that cellular arrangement affects access to resources within the biofilm, we found that while the wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries. Furthermore, we found that altered cellular arrangement within biofilms affects the localization of metabolic activity, the survival of resident cells, and the susceptibility of subpopulations to antibiotic treatment. Our observations provide insight into cellular features that determine biofilm microanatomy, with consequences for physiological differentiation and drug sensitivity.

microbiology↗

bioRxiv2023

Cyanide-dependent control of terminal oxidase hybridization by Pseudomonas aeruginosa MpaR

Pseudomonas aeruginosa is a common, biofilm-forming pathogen that exhibits complex pathways of redox metabolism. It produces four different types of terminal oxidases for aerobic respiration, and for one of these-the cbb3-type terminal oxidases-it has the capacity to produce at least 16 isoforms encoded by partially redundant operons. It also produces small-molecule virulence factors that interact with the respiratory chain, including the poison cyanide. Previous studies had indicated a role for cyanide in activating expression of an "orphan" terminal oxidase subunit gene called ccoN4 and that the product contributes to P. aeruginosa cyanide resistance, fitness in biofilms, and virulence-but the mechanisms underlying this process had not been elucidated. Here, we show that the regulatory protein MpaR, which is predicted to be a pyridoxal phosphate-binding transcription factor and is encoded just upstream of ccoN4, controls ccoN4 expression in response to endogenous cyanide. Paradoxically, we find that cyanide production is required to support CcoN4s contribution to respiration in biofilms. We identify a palindromic motif required for cyanide- and MpaR-dependent expression of ccoN4 and co-expressed, adjacent loci. We also characterize the regulatory logic of this region of the chromosome. Finally, we identify residues in the putative cofactor-binding pocket of MpaR that are required for ccoN4 expression. Together, our findings illustrate a novel scenario in which the respiratory toxin cyanide acts as a signal to control gene expression in a bacterium that produces the compound endogenously. IMPORTANCECyanide is an inhibitor of heme-copper oxidases, which are required for aerobic respiration in all eukaryotes and many prokaryotes. This fast-acting poison can arise from diverse sources, but mechanisms by which bacteria sense it are poorly understood. We investigated the regulatory response to cyanide in the pathogenic bacterium Pseudomonas aeruginosa, which produces cyanide as a virulence factor. Although P. aeruginosa has the capacity to produce a cyanide-resistant oxidase, it relies primarily on heme-copper oxidases and even makes additional heme-copper oxidase proteins specifically under cyanide-producing conditions. We found that the protein MpaR controls expression of cyanide-inducible genes in P. aeruginosa and elucidated the molecular details of this regulation. MpaR contains a DNA-binding domain and a domain predicted to bind pyridoxal phosphate (vitamin B6), a compound that is known to react spontaneously with cyanide. These observations provide insight into the understudied phenomenon of cyanide-dependent regulation of gene expression in bacteria.

microbiology↗

bioRxiv2022

Light/dark and temperature cycling modulate metabolic electron flow in Pseudomonas aeruginosa biofilms

Sunlight drives phototrophic metabolism, which affects redox conditions and produces substrates for non-phototrophs. These environmental parameters fluctuate daily due to Earths rotation, and non-phototrophic organisms can therefore benefit from the ability to respond to, or even anticipate, such changes. Circadian rhythms, such as daily changes in body temperature, in host organisms can also affect local conditions for colonizing bacteria. Here, we investigated the effects of light/dark and temperature cycling on biofilms of the opportunistic pathogen Pseudomonas aeruginosa PA14. We grew biofilms in the presence of a respiratory indicator dye and found that greater dye reduction occurred in biofilm zones that formed during dark intervals and at lower temperatures. This pattern formation occurred with cycling of blue, red, or far-red light, and a screen of mutants representing potential sensory proteins identified two with defects in pattern formation, specifically under red light cycling. We also found that the physiological states of biofilm subzones formed under specific light and temperature conditions were retained during subsequent condition cycling. Light/dark and temperature cycling affected expression of genes involved in primary metabolic pathways and redox homeostasis, including those encoding electron transport chain components. Consistent with this, we found that cbb3-type oxidases contribute to dye reduction under light/dark cycling conditions. Together, our results indicate that cyclic changes in light exposure and temperature have lasting effects on redox metabolism in biofilms formed by a non-phototrophic, pathogenic bacterium. IMPORTANCEOrganisms that do not obtain energy from light can nevertheless be affected by daily changes in light exposure. Many aspects of animal and fungal physiology fluctuate in response to these changes, including body temperature and the activities of antioxidant and other redox enzymes that play roles in metabolism. Whether redox metabolism is affected by light/dark and temperature cycling in bacteria that colonize such circadian organisms has not been studied in detail. Here we show that growth under light/dark and temperature cycling leads to rhythmic changes in redox metabolism in Pseudomonas aeruginosa and identify proteins involved in this response. P. aeruginosa is a major cause of healthcare-associated infections and designated as a serious threat by the CDC due to its recalcitrance during treatments. Our findings have the potential to inform therapeutic strategies that incorporate controlled light exposure or consider P. aeruginosas responses to conditions in the host.

microbiology↗

bioRxiv2020

Pseudomonas aeruginosa PA14 biofilms produce R-bodies, extendable protein polymers with roles in host colonization and virulence

Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes with the potential to confer production of R-bodies (i.e., a "reb cluster"). R-bodies are large, extendable protein polymers best known for their role in killing of paramecia by the bacterium Caedibacter taeniospiralis, and genes in the reb cluster have been implicated in PA14 virulence. Here, we present evidence that PA14 expresses reb cluster genes during colonization of plant and nematode hosts. We identify products of the reb cluster that are R-body-associated and that control stochastic expression of R-body structural genes. We also show that R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. These observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.

microbiology↗

bioRxiv2019

Light-mediated decreases in cyclic di-GMP levels are potentiated by pyocyanin and inhibit structure formation in Pseudomonas aeruginosa biofilms

Light is known to trigger regulatory responses in diverse organisms including slime molds, animals, plants, and phototrophic bacteria. However, light-dependent processes in non-phototrophic bacteria, and those of pathogens in particular, have received comparatively little research attention. In this study, we examined the impact of light on multicellular development in Pseudomonas aeruginosa, a leading cause of biofilm-based bacterial infections, using a colony morphology assay. In this assay, P. aeruginosa strain PA14 grown in the dark forms vertical structures (i.e., \"wrinkles\") on the third day of incubation. We found that growth in blue light inhibited wrinkle formation until the fifth day and that this required the phenazine pyocyanin, a redox-active metabolite produced by PA14. Light-dependent inhibition of wrinkling was also correlated with low levels of cyclic di-GMP (c-di-GMP), consistent with the role of this signal in stimulating biofilm matrix production. Though phenazine-null biofilms also showed lower levels of c-di-GMP and subtle effects on wrinkling when grown in the light, their overall levels of c-di-GMP were higher than those of the wild type. This indicates that phenazines and light simultaneously promote c-di-GMP degradation such that c-di-GMP is pushed to a minimum level, yielding a pronounced macroscopic phenotype. A screen of enzymes with the potential to catalyze c-di-GMP synthesis or degradation identified four proteins that contribute to light-dependent inhibition of biofilm wrinkling. Together, these results provide a foundation for understanding the significance of light-dependent regulation in P. aeruginosa.\n\nImportanceThe response to light in non-phototrophic bacteria (i.e., chemotrophs) is relatively understudied in comparison to light-mediated behavior in eukaryotes and phototrophic bacteria. Though they do not depend on light for growth, chemotrophic bacteria could benefit from sensing this cue when it correlates with other parameters that are important for metabolism. In this paper, we describe light-dependent effects on a cellular signal that controls the development of multicellular assemblages, called biofilms, in Pseudomonas aeruginosa. We found that light at intensities that are not harmful to human cells inhibited biofilm maturation. P. aeruginosa is a leading cause of chronic lung infections in people with cystic fibrosis and of hospital-acquired infections. As P. aeruginosas recalcitrance to treatment is attributed in part to its facile formation of biofilms, this study provides insight into a mechanism that could be inhibited via new therapeutic tools, such as photodynamic therapy.

microbiology↗

bioRxiv2019

Interdependency of respiratory metabolism and phenazine-associated physiology in Pseudomonas aeruginosa PA14

Extracellular electron transfer (EET), the reduction of compounds that shuttle electrons to distal oxidants, can support bacterial survival when preferred oxidants are not directly accessible. EET has been shown to contribute to virulence in some pathogenic organisms and is required for current generation in mediator-based fuel cells. In several species, components of the electron transport chain (ETC) have been implicated in electron shuttle reduction, raising the question of how shuttling-based metabolism is integrated with primary routes of metabolic electron flow. The clinically relevant bacterium Pseudomonas aeruginosa can utilize carbon sources (i.e., electron donors) covering a broad range of reducing potentials and possesses a branched ETC that can be modulated to optimize respiratory efficiency. It also produces electron shuttles called phenazines that facilitate intracellular redox balancing, increasing the complexity of its metabolic potential. In this study, we investigated the reciprocal influence of respiratory metabolism and phenazine-associated physiology in Pseudomonas aeruginosa PA14. We found that phenazine production affects respiratory activity and terminal oxidase gene expression, and that carbon source identity influences the mechanisms enabling phenazine reduction. Furthermore, we found that growth in biofilms, a condition for which phenazine metabolism is critical to normal development and redox balancing, dramatically affects the composition of the P. aeruginosa phenazine pool. Together, these findings can aid interpretation of P. aeruginosa behavior during host infection and provide inroads to understanding the crosstalk between primary metabolism and shuttling-based physiology in the diverse bacteria that carry out EET.\n\nIMPORTANCEPseudomonas aeruginosa is a major cause of healthcare-associated infections and long-term lung infections in people with cystic fibrosis. It can use diverse organic compounds as electron donors and possesses multiple enzymes that can transfer electrons from central metabolism to O2. These pathways support a balanced intracellular redox state and the production of cellular energy. Under hypoxic conditions, P. aeruginosa can reduce phenazines, secondary metabolites that also promote redox homeostasis and that contribute to virulence. We asked how these primary and secondary routes of electron flow influence each other. We found that phenazines affect respiratory function, that the roles of respiratory enzymes in phenazine reduction are highly condition-dependent, and that the complement of phenazines produced is strongly affected by growth in assemblages called biofilms. These results provide a more nuanced understanding of P. aeruginosa redox metabolism and may inform strategies for treating persistent infections caused by this bacterium.

microbiology↗

bioRxiv2018

The Pseudomonas aeruginosa complement of lactate dehydrogenases enables use of D- and L-lactate and metabolic crossfeeding

Pseudomonas aeruginosa is the most common cause of chronic, biofilm-based lung infections in patients with cystic fibrosis (CF). Sputum from patients with CF has been shown to contain oxic and hypoxic subzones as well as millimolar concentrations of lactate. Here, we describe the physiological roles and expression patterns of P. aeruginosa lactate dehydrogenases in the contexts of different growth regimes. P. aeruginosa produces four enzymes annotated as lactate dehydrogenases, three of which are known to contribute to anaerobic or aerobic metabolism in liquid cultures. These three are LdhA, which reduces pyruvate to D-lactate during anaerobic survival, and LldE and LldD, which oxidize D-lactate and L-lactate, respectively, during aerobic growth. We demonstrate that the fourth enzyme, LldA, performs redundant L-lactate oxidation during growth in aerobic cultures in both a defined MOPS-based medium and synthetic CF sputum medium. However, LldA differs from LldD in that its expression is induced specifically by the L-enantiomer of lactate. We also show that all four enzymes perform functions in colony biofilms that are similar to their functions in liquid cultures. Finally, we provide evidence that the enzymes LdhA and LldE have the potential to support metabolic cross-feeding in biofilms, where LdhA can catalyze the production of D-lactate in the anaerobic zone that is then used as a substrate in the aerobic zone. Together, these observations further our understanding of the metabolic pathways that can contribute to P. aeruginosa growth and survival during CF lung infection.\n\nIMPORTANCELactate is thought to serve as a carbon and energy source during chronic infections. Sites of bacterial colonization can contain two enantiomers of lactate: the L-form, generally produced by the host, and the D-form, which is usually produced by bacteria including the pulmonary pathogen Pseudomonas aeruginosa. Here, we characterize P. aeruginosas set of four enzymes that it can use to interconvert pyruvate and lactate, the functions of which depend on the availability of oxygen and specific enantiomers of lactate. We also show that anaerobic pyruvate fermentation triggers production of the aerobic D-lactate dehydrogenase in both liquid cultures and biofilms, thereby enabling metabolic cross-feeding of lactate over time and space between subpopulations of cells. These metabolic pathways could contribute to P. aeruginosa growth and survival in the lung.

microbiology↗

bioRxiv2017

An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e., \"orphan\") operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen P. aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets.

microbiology↗