Biology subjects

O'Toole, G.

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

7 recordsLinked to original sources

bioRxiv2018

Ethanol decreases Pseudomonas aeruginosa flagellar motility through a cyclic-di-GMP- and stator-dependent pathway

Pseudomonas aeruginosa frequently encounters microbes that produce bioactive metabolites including ethanol. At concentrations that do not affect growth, we found that ethanol reduces P. aeruginosa motility by 30% in a swim agar assay and this decrease is accompanied by a 2.5-fold increase in levels of cyclic diguanylate (c-di-GMP), a second messenger that represses motility, in planktonic cells. A screen of mutants lacking genes involved in c-di-GMP metabolism identified SadC and GcbA as diguanylate cyclases involved in swim zone reduction by ethanol and ethanol-induced c-di-GMP production. The reduction of swimming in response to ethanol also required the stator set, MotAB, two PilZ-domain proteins (FlgZ and PilZ), PilY1-a proposed surface-sensing protein, and PilMNOP, which comprises the pilus alignment complex and these proteins have been previously implicated in the control of motility on agar surfaces. Microscopic analysis of the fraction of quiescent cells in swim medium showed that ethanol decreased the portion of motile cells in the wild type, but had opposite effects in the {triangleup}pilY1, {triangleup}pilMNOP, {triangleup}motAB, and {triangleup}pilZ{triangleup}flgZ mutants. Together, these data indicate ethanol induces a regulated change in motility in planktonic cells at concentrations similar to those produced by other microbes. We propose that this ethanol-responsiveness may contribute to the co-localization of P. aeruginosa with ethanol-producing microbes.

microbiology↗

bioRxiv2018

Coopting the Lap system of Pseudomonas fluorescens to reversibly customize bacterial cell surfaces

Initialattachment to a surface is a key and highly regulated step in biofilm formation. In this study, we present a platform for reversibly functionalizing bacterial cell surfaces, with an emphasis on designing biofilms. We engineered the Lap system of Pseudomonas fluorescens Pf0-1, which is normally used to regulate initial cell surface attachment, to display various protein cargo at the bacterial cell surface and control extracellular release of the cargo in response to changing levels of the second messenger cdi-GMP. To accomplish this goal, we fused the protein cargo between the N-terminal retention module and C-terminal secretion signal of LapA, and controlled surface localization of the cargo with natural signals known to stimulate or deplete c-di-GMP levels in P. fluorescens Pf0-1. We show this system can tolerate large cargo in excess of 500 amino acids, direct P. fluorescens Pf0-1 to surfaces it does not typically colonize, and program this microbe to sequester the toxic medal cadmium.

bioengineering↗

bioRxiv2018

Altered Stool Microbiota of Infants with Cystic Fibrosis Shows Reduction in Genera Associated with Immune Programming

Previous work from our group indicated a connection between the gastrointestinal microbiota of infants and children with cystic fibrosis (CF) and airway disease in this population. Here we examine the stool microbiota of infants with CF and from the general population who did not have CF over the first year of life. CF children had reduced gastrointestinal Bacteroides and Bifidobacterium beginning in infancy, even after adjusting for antibiotic treatment. We also identify several metabolic pathways that are enriched or under represented among the microbial communities in the stool of these young patients with CF as compared to children without CF. In vitro studies demonstrated that exposure of the apical face of a polarized Intestinal cell line to Bacteroides thetaiotaomicron significantly reduced production of IL-8 secreted from both the apical and basolateral face of these cells, suggesting a mechanism whereby changes in the intestinal microflora could impact systemic inflammation. This work further establishes a link between gastrointestinal microbiota, systemic inflammation and airway disease, and presents the opportunity for therapeutic probiotic interventions.\n\nSignificance statementThere is a surprising link between gastrointestinal microbial communities and airway disease progression in CF. Here we show that infants with CF [≤]1 year of age show a distinct stool microbiota compared with children of a comparable age from a general population cohort. We detect associations between stool microbes and airway exacerbation events in the cohort of infants with CF, and in vitro studies provide a possible mechanism for this observation. These data argue that current therapeutics do not establish a healthy-like gastrointestinal microbiota in young patients with CF, and we suggest that interventions that direct the gastrointestinal microbiota closer to a healthy state may provide benefit to these patients.

microbiology↗

bioRxiv2018

Pseudomonas aeruginosa Can Inhibit Growth of Streptococcal Species via Iron Sequestration

Cystic Fibrosis (CF) is a genetic disease that causes patients to accumulate thick, dehydrated mucus in the lung and develop chronic, polymicrobial infections due to reduced mucociliary clearance. These chronic polymicrobial infections and subsequent decline in lung function are significant factors in the morbidity and mortality of CF. Pseudomonas aeruginosa and Streptococcus spp. are among the most prevalent organisms in the CF lung; the presence of P. aeruginosa correlates with lung function decline and the Streptococcus milleri group (SMG), a subgroup of the viridans streptococci, is associated with exacerbations in patients with CF. Here we characterize the interspecies interactions that occur between these two genera. We demonstrated that multiple P. aeruginosa laboratory strains and clinical CF isolates promote the growth of multiple SMG strains and oral streptococci in an in vitro coculture system. We investigated the mechanism by which P. aeruginosa enhances growth of streptococci by screening for mutants of P. aeruginosa PA14 unable to enhance Streptococcus growth, and we identified the P. aeruginosa pqsL::TnM mutant, which failed to promote growth of S. constellatus and S. sanguinis. Characterization of the P. aeruginosa {Delta}pqsL mutant revealed that this strain cannot promote Streptococcus growth. Our genetic data and growth studies support a model whereby the P. aeruginosa {Delta}pqsL mutant overproduces siderophores, and thus likely outcompetes Streptococcus sanguinis for limited iron. We propose a model whereby competition for iron represents one important means of interaction between P. aeruginosa and Streptococcus spp.\n\nImportanceCystic fibrosis (CF) lung infections are increasingly recognized for their polymicrobial nature. These polymicrobial infections may alter the biology of the organisms involved in CF-related infections, leading to changes in growth, virulence and/or antibiotic tolerance, and could thereby affect patient health and response to treatment. In this study, we demonstrate interactions between P. aeruginosa and streptococci using a coculture model, and show that one interaction between these microbes is likely competition for iron. Thus, these data indicate that one CF pathogen may influence the growth of another and add to our limited knowledge of polymicrobial interactions in the CF airway.

microbiology↗

bioRxiv2017

Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction Between a Diguanylate Cyclase and Its Receptor

The second messenger, cyclic dimeric GMP (c-di-GMP) regulates biofilm formation for many bacteria. The binding of c-d{inverted exclamation}-GMP by the inner-membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbCs calcium channel chemotaxis (CACHE) domain. We provide evidence that biofilm formation is stimulated by the environmentally relevant organic acid citrate (and a related compound, isocitrate) in a GcbC-dependent manner through enhanced GcbC-LapD interaction, which results in increased LapA localization to the cell surface. Furthermore, GcbC shows little ability to synthesize c-di-GMP in isolation. However, when LapD is present GcbC activity is significantly enhanced ~8-fold, indicating that engaging the LapD receptor stimulates the activity of this DGC; citrate-enhanced GcbC-LapD interaction further stimulates c-di-GMP synthesis. We propose that the l-site of GcbC serves two roles beyond allosteric control of this enzyme: promoting GcbC-LapD interaction and stabilizing the active conformation of GcbC in the GcbC-LapD complex. Finally, given that LapD can interact with a dozen different DGCs of P. fluorescens, many of which have ligand-binding domains, the ligand-mediated enhanced signaling via LapD-GcbC interaction described here is likely a conserved mechanism of signaling in this network. Consistent with this idea, we identify a second example of ligand-mediated enhancement of DGC-LapD interaction that promotes biofilm formation.\n\nImportanceIn many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP, however it is unclear how undesired crosstalk is mitigated in the context of this soluble signal, and how c-di-GMP signaling is regulated by environmental inputs. We demonstrate that GcbC, a DGC, shows little ability to synthesize c-d{inverted exclamation}-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the l-site of GcbC. We further show evidence for a ligand-mediated mechanism of signaling specificity via increased physical interaction of a DGC with its cognate receptor. We envision a scenario wherein a \"cloud\" of weakly active DGCs can increase their activity by specific interaction with their receptor in response to appropriate environmental signals, concomitantly boosting c-di-GMP production, ligand-specific signaling and biofilm formation.

microbiology↗

bioRxiv2017

Cell surface associated LapA of Pseudomonas fluorescens is anchored inside its Type-1 Secretion TolC-like Pore

The type-1 secretion system (T1SS) of gram-negative bacteria enables a one-step translocation strategy known to move functionally diverse proteins from the cytoplasm into the extracellular environment without a periplasmic intermediate. LapA of Pseudomonas fluorescens Pf0-1 is a giant type-1 secreted (T1S) adhesin that facilitates biofilm formation only when displayed at the cell surface. A LapA-targeting periplasmic protease, LapG, connects intracellular cyclic diguanylate (c-di-GMP) levels with cell surface-associated LapA by cleaving and absolving LapA from the cell surface under conditions unsuitable for biofilm formation. Here, we demonstrate that LapA contains a novel N-terminal element, called the retention module (RM), which prohibits classical one-step T1S of LapA. We provide evidence that the RM of LapA tethers LapA at the cell surface through its outer membrane TolC-like pore, LapE, where LapA is accessible to the periplasmic protease LapG. We also demonstrate that this unusual retention strategy is likely conserved among LapA-like proteins and represents a new subclass of T1SS ABC transporters exclusively involved in transporting LapA-like adhesins.\n\nSignificance StatementBacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface associated adhesins is often key for initiating contact with a preferred substrate to facilitate biofilm formation. Our work demonstrates that P. fluorescens uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal Proteobacteria and provide phylogenetic evidence these adhesins are secreted by a new subclass of T1SS ABC transporters.

microbiology↗

bioRxiv2017

A Multimodal Strategy Used By A Large c-di-GMP Network

The Pseudomonas fluorescens genome encodes for 50+ proteins involved in -di-GMP signaling. Here, we demonstrate that when tested across 188 nutrients, these enzymes and effectors appear capable of impacting biofilm formation. Transcriptional analysis of network members across [~]50 nutrient conditions indicates that altered gene expression can explain a subset, but not all, of biofilm-formation responses to the nutrients. Additional organization of the network is likely achieved through physical interaction, as determined via probing [~]2000 interactions by bacterial two-hybrid assays. Our analysis revealed a multimodal regulatory strategy, using combinations of ligand-mediated signals, protein-protein interaction and/or transcriptional regulation to further fine-tune c-di-GMP-mediated responses. These results create a profile of a large c-di-GMP network that is used to make important cellular decisions, opening the door to future model building and the ability to engineer this complex circuitry in other bacteria.\n\nAbstract ImportanceCyclic diguanylate (c-di-GMP) is a key signalling molecule regulating bacterial biofilm formation, and many microbes have up to dozens of proteins that make, break or bind this dinucleotide. Thus, a major open question in the field is how signalling specificity is conferred in this context with a soluble signalling molecule. Here, we take a systems approach, using mutational analysis, transcriptional studies and bacterial two-hybrid analysis to interrogate this network. We find that the network typically combines two or more modes of regulation (i.e., transcriptional control with protein-protein interaction) to generate an observed output.

microbiology↗