Biology subjects

Dolan, S. K.

Publications and source records attributed to Dolan, S. K..

4 recordsLinked to original sources

bioRxiv2023

Convergent evolution in toxin detection and resistance provides evidence for conserved bacterial-fungal interactions

Microbes rarely exist in isolation, and instead form complex polymicrobial communities. As a result, microbes have developed intricate offensive and defensive strategies that enhance their fitness in these complex communities. Thus, identifying and understanding the molecular mechanisms controlling polymicrobial interactions is critical for understanding the function of microbial communities. In this study, we show that the Gram-negative opportunistic human pathogen Pseudomonas aeruginosa, which frequently causes infection alongside a plethora of other microbes including fungi, encodes a genetic network which can detect, and defend against gliotoxin, a potent, disulfide-containing antimicrobial produced by the ubiquitous filamentous fungus Aspergillus fumigatus. We show that gliotoxin exposure disrupts P. aeruginosa zinc homeostasis, leading to transcriptional activation of a gene encoding a previously uncharacterized dithiol oxidase (DnoP), which detoxifies gliotoxin and structurally related toxins. While the enzymatic activity of DnoP is identical to that used by A. fumigatus to protect itself against gliotoxin, DnoP shares little homology to the A. fumigatus gliotoxin resistance protein. Thus, DnoP and its transcriptional induction by low zinc represent an example of both convergent evolution of toxin defense and environmental cue sensing across kingdoms. Collectively, these data support disulfide-containing natural products as mediators of inter-kingdom interactions and provide evidence that P. aeruginosa has evolved to survive exposure to these molecules in the natural environment. Significance statementBacteria and fungi frequently exist as complex, polymicrobial communities during infection. Reconstructing ecological structure in the laboratory is challenging and, consequently, the precise molecular mechanisms which underpin microbial interactions remain elusive. Using a pre-clinical model that mimics the cystic fibrosis lung, we discovered that the bacterium Pseudomonas aeruginosa detects and defends against a disulfide-containing toxin produced by the fungus Aspergillus fumigatus. In an example of both convergent evolution of toxin defense and environmental cue sensing across kingdoms, we discovered that these organisms use the same cue to produce/sense this toxin, and the same enzymatic mechanism to protect against toxicity. This discovery of convergent evolution provides strong evidence for P. aeruginosa exposure to microbially-produced disulfide-containing toxins in natural environments.

microbiology↗

bioRxiv2022

Systems-wide dissection of organic acid assimilation in Pseudomonas aeruginosa reveals a novel path to underground metabolism

The human pathogen Pseudomonas aeruginosa (Pa) is one of the most frequent and severe causes of nosocomial infection. This organism is also a major cause of airway infections in people with cystic fibrosis (CF). Pa is known to have a remarkable metabolic plasticity, allowing it to thrive in diverse environmental conditions and ecological niches, yet little is known about the central metabolic pathways which sustain its growth during infection, or precisely how these pathways operate. In this work, we used a combination of omics approaches (transcriptomics, proteomics, metabolomics and 13C-fluxomics) and reverse genetics to provide a systems-level insight into how the infection-relevant organic acids, succinate and propionate, are metabolized by Pa. Moreover, through structural and kinetic analysis of the 2-methylcitrate synthase (PrpC) and its paralogue, citrate synthase (GltA), we show how these two crucial enzymatic steps are interconnected in Pa organic acid assimilation. We found that Pa can rapidly adapt to the loss of GltA function by acquiring mutations in a transcriptional repressor, which then de-represses prpC expression. Our findings provide a clear example of how underground metabolism, facilitated by enzyme substrate promiscuity, "rewires" Pa metabolism, allowing it to overcome the loss of a crucial enzyme. This pathogen-specific knowledge is critical for the advancement of a model-driven framework to target bacterial central metabolism.

microbiology↗

bioRxiv2019

Contextual flexibility in Pseudomonas aeruginosa central carbon metabolism during growth in single carbon sources

Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge towards a common metabolic program as the organism adapts to the CF airway environment. However, at a systems level, we still have only a limited understanding of how these transcriptional changes impact on metabolic flux. To address this, we analysed the transcriptome, proteome and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in the CF airways. We show that the flux of carbon through central metabolism is radically different on each carbon source. For example, the newly-recognised EDEMP cycle plays an important role in supplying NADPH during growth on glycerol. By contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the isocitrate dehydrogenase(s)-catalyzed reaction. Perhaps more importantly, our proteomic and transcriptomic analyses reveal a global remodelling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; a plasticity which allows the organism to seamlessly segue between different carbon sources, maximising the energetic yield from each.\n\nImportancePseudomonas aeruginosa is an opportunistic human pathogen, well-known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well-adapted to life in the CF lung, little is currently known about how the organism metabolises the nutrients available in the airways. In this work, we use a combination of gene expression and isotope tracer (\"fluxomic\") analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We find that carbon is routed (\"fluxed\") through very different pathways during growth on these substrates, and that this is accompanied by an unexpected remodelling of the cells electron transfer pathways. Having access to this \"blueprint\" is important because the metabolism of P. aeruginosa is increasingly being recognised as a target for the development of much-needed antimicrobial agents.

microbiology↗

bioRxiv2018

Three enzymes and one substrate; regulation of flux through the glyoxylate shunt in the opportunistic pathogen, Pseudomonas aeruginosa.

The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for biosynthesis. The branchpoint between the TCA cycle and the glyoxylate shunt is therefore widely considered to be one of the most important junctions in the whole of microbial metabolism. In Escherichia coli, AceK-mediated phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), is necessary to redirect flux through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL). In contrast, Mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH) at the branchpoint. Flux partitioning here is controlled \"rheostatically\" through cross-activation of IDH by the product of ICL activity, glyoxylate. However, the opportunistic human pathogen, Pseudomonas aeruginosa, expresses IDH, ICD, ICL and AceK. Here, we present the structure, kinetics and regulation of each branchpoint enzyme. We show that flux partitioning is coordinated through reciprocal regulation of the enzymes involved, beautifully linking carbon flux with the availability of key gluconeogenic precursors in a way that cannot be extrapolated from an understanding of the branchpoint enzymes in other organisms.

biochemistry↗