Biology subjects

Hogan, D.

Publications and source records attributed to Hogan, D..

9 recordsLinked to original sources

bioRxiv2026

Inflammasome Activation and IL-1β Release in Alveolar Macrophages Infected with Pseudomonas aeruginosa is Reduced in Hypoxia

Pseudomonas aeruginosa is an important agent of acute or chronic airway infections. A complex set of genetic and environmental factors determine the outcome of a P. aeruginosa airway infection. P. aeruginosa can undergo genetic adaptation to fine tune the expression of a type III secretion system (T3SS) and other virulence factors in response to selective pressures encountered during infection. Genetic and environmental factors predispose many patient groups for P. aeruginosa infections, including people with cystic fibrosis (pwCF). CF is a genetic disorder resulting in areas of hypoxia and thick mucus that fosters P. aeruginosa airway infection. Resident alveolar macrophages (AMs) help coordinate immune responses to P. aeruginosa in airways by producing proinflammatory cytokines such as IL-1{beta}, which can be important for infection resistance. AMs infected with laboratory strains of P. aeruginosa detect the T3SS and activate the NLRC4 inflammasome, resulting in IL-1{beta} release. Studies of inflammasome responses in AMs to a clinical CF isolate of P. aeruginosa have not been reported. Here, we characterized the CF clinical isolate DH1137 and found that it has a downregulated but functional T3SS, is adapted to grow in hypoxia, and induces significant production of the inflammasome cytokines IL-1{beta} and IL-1 during lung infection in a CF mouse model. Inflammasome gene expression was primed in AMs by LPS stimulation, and upon DH1137 infection these cells released IL-1[beta]. Intriguingly, we found that infection of AMs with DH1137 in hypoxia resulted in significantly dampened inflammasome activation compared to normoxia. These data describe a new mechanism that P. aeruginosa may exploit to evade immune detection by tissue-resident lung macrophages in the context of CF.

immunology↗

bioRxiv2024

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in Cystic Fibrosis

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lung, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection. New and noteworthyThe experiments in this report identify a novel mechanim whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet haves in OMVs secreted by P. aeruginiosa, which reduced the OMV-LPS induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF. Graphical abstractThe anti-inflammatory effect of tobramycin mediated by 5' tRNA-fMet halves secreted in P. aeruginosa OMVs. (A) P. aeruginosa colonizes the CF lungs and secrets OMVs. OMVs diffuse through the mucus layer overlying bronchial epithelial cells and induce IL-8 secretion, which recruits neutrophils that causes lung damage. (B) Tobramycin increases 5' tRNA-fMet halves in OMVs secreted by P. aeruginosa. 5' tRNA-fMet halves are delivered into host cells after OMVs fuse with lipid rafts in CF-HBEC and down-regulate protein expression of MAPK10, IKBKG, and EP300, which suppresses IL-8 secretion and neutrophils in the lungs. A reduction in neutrophils in CF BALF is predicted to improve lung function and decrease lung damage. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/578737v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@d9d5e7org.highwire.dtl.DTLVardef@16a5715org.highwire.dtl.DTLVardef@7b35ddorg.highwire.dtl.DTLVardef@10d4dc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Uncovering Protein Ensembles: Automated Multiconformer Model Building for X-ray Crystallography and Cryo-EM

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift towards modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g. Coot) and fit can be further improved by refinement using standard pipelines (e.g. Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

biophysics↗

bioRxiv2022

Community composition shapes microbial-specific phenotypes in a cystic fibrosis polymicrobial model system

Interspecies interactions can drive the emergence of unexpected microbial phenotypes that are not observed when studying monocultures. The cystic fibrosis (CF) lung consists of a complex environment where particular microbes, living as polymicrobial biofilm-like communities, are associated with negative clinical outcomes for persons with CF (pwCF). However, the current lack of in vitro models integrating the microbial diversity observed in the CF airway hampers our understanding of why polymicrobial communities are recalcitrant to therapy in this disease. Here, integrating computational approaches informed by clinical data, we built a mixed community of clinical relevance to the CF lung composed of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis and Prevotella melaninogenica. We developed and validated this model biofilm community with multiple isolates of these four genera. When challenged with tobramycin, a front-line antimicrobial used to treat pwCF, the microorganisms in the polymicrobial community show altered sensitivity to this antibiotic compared to monospecies biofilms. We observed that wild-type P. aeruginosa is sensitized to tobramycin in a mixed community versus monoculture, and this observation holds across a range of community relative abundances. We also report that LasR loss-of-function, a variant frequently detected in the CF airway, induces tolerance of P. aeruginosa to tobramycin specifically in the mixed community. The molecular basis of this community-specific recalcitrance to tobramycin for the LasR mutant variant is the increased production of redox-active phenazines. Our data support the importance of studying clinically-relevant model polymicrobial biofilms to understand community-specific traits relevant to infections. SIGNIFICANCE STATEMENTThe CF lung is colonized by biofilm-like microbial communities that exhibit both resistance and tolerance (collectively called "recalcitrance") to antimicrobials used in the clinic. Here, we leveraged clinical data from pwCF to inform our understanding of communities exhibiting recalcitrance. We developed and validated an in vitro model that revealed novel, community-specific phenotypes relevant to the clinic. We used this model to explore the underlying mechanism associated with a community-specific emergent behavior. We posit that in vitro models of polymicrobial communities may help in developing new antimicrobial strategies to improve patient outcomes, and that the approach used here can be applied to other polymicrobial models.

microbiology↗

bioRxiv2022

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa

Lung infection by antibiotic resistant strains of Pseudomonas aeruginosa is a well-known concern for immunocompromised hosts including people with lung diseases such as cystic fibrosis. We have previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AEC) deliver miRNA let-7b-5p to P. aeruginosa where it suppresses biofilm formation and increases sensitivity to beta-lactam antibiotics. In this study we used RNA-seq to characterize the small RNA (sRNA) content of EVs secreted by AEC and demonstrate transfer of multiple distinct RNA fragments from EVs to P. aeruginosa. Bioinformatic predictions reveal that several sRNAs may target all three subunits of the fluoroquinolone efflux pump MexHI-OpmD, an effect predicted to increase antibiotic sensitivity to fluoroquinolone antibiotics. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%) and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S rRNA external transcribed spacer that was transferred to P. aeruginosa by EVs was sufficient to reduce planktonic growth of P. aeruginosa in the presence of ciprofloxacin, to reduce the minimum inhibitory concentration (MIC) of P. aeruginosa for ciprofloxacin by over 50%, and to significantly reduce protein levels of MexH and OpmD. In conclusion, an rRNA fragment secreted by AEC in EVs increases the ciprofloxacin sensitivity of P. aeruginosa by targeting and down-regulating the fluoroquinolone efflux pump MexHI-OpmD. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections. Author SummaryAccording to the World Health Organization and the U.S. Centers for Disease Control and Prevention the development of antibiotic resistant strains of bacteria, including Pseudomonas aeruginosa, are a significant global threat to human health. Thus, development of new approaches to eliminate antibiotic resistant infections is required. In this study, we report that lung epithelial cells secrete extracellular vesicles (EVs) that fuse with and deliver small rRNAs to P. aeruginosa, and that the rRNAs increase the sensitivity of P. aeruginosa to the antibiotic ciprofloxacin by reducing protein levels of the drug efflux pump MexHI-OpmD. We identified one rRNA fragment that by itself significantly reduced the protein levels of MexH and OpmD and increased the ability of ciprofloxacin to kill P. aeruginosa. We propose that developing synthetic vesicles containing a combination of the rRNA that inhibits antibiotic efflux pumps and ciprofloxacin would benefit patients with antibiotic resistant P. aeruginosa infections.

microbiology↗

bioRxiv2022

Metabolic basis for the evolution of a common pathogenic Pseudomonas aeruginosa variant

Microbes frequently evolve in reproducible ways. Here, we show that differences in specific metabolic regulation explain the frequent presence of lasR loss-of-function mutations in the bacterial pathogen Pseudomonas aeruginosa. While LasR contributes to virulence, lasR mutants have been associated with more severe disease. A model based on the intrinsic growth kinetics for a wild type strain and its LasR- derivative, in combination with an experimental evolution based genetic screen and further genetics analyses, indicated that differences in metabolism were sufficient to explain the rise of these common mutant types. The evolution of LasR- lineages in laboratory and clinical isolates depended on activity of the two-component system CbrAB, which modulates substrate prioritization through the catabolite repression control pathway. LasR- lineages frequently arise in cystic fibrosis lung infections and their detection correlates with disease severity. Our analysis of bronchoalveolar lavage fluid metabolomes identified compounds that negatively correlate with lung function, and we show that these compounds support enhanced growth of LasR- cells in a CbrB-controlled manner. We propose that in vivo metabolomes are a major driver of pathogen evolution, which may influence the progression of disease and its treatment.

microbiology↗

bioRxiv2020

qFit 3: Protein and ligand multiconformer modeling for X-ray crystallographic and single-particle cryo-EM density maps

New X-ray crystallography and cryo-electron microscopy (cryo-EM) approaches yield vast amounts of structural data from dynamic proteins and their complexes. Modeling the full conformational ensemble can provide important biological insights, but identifying and modeling an internally consistent set of alternate conformations remains a formidable challenge. qFit efficiently automates this process by generating a parsimonious multiconformer model. We refactored qFit from a distributed application into software that runs efficiently on a small server, desktop, or laptop. We describe the new qFit 3 software and provide some examples. qFit 3 is open-source under the MIT license, and is available at https://github.com/ExcitedStates/qfit-3.0.

biophysics↗

bioRxiv2017

WeSeqMiner: A Weka package for building machine-learning models for sequence data

In most cases, the application of machine learning techniques to biological sequence data requires a vector representation of the sequences. Extracting the numerical features from sequence data can be time consuming, especially if the user lacks programming skills. To this end, we propose a Weka package called WeSeqMiner, which provides several useful filters for extracting numerical features from sequence data for use in the Weka machine learning workbench. Motivated with an example, we show that the WeSeqMiner package integrates well with the Weka API, allowing transformations to be incorporated into Weka workflows for predictive model generation. WeSeqMiner can be installed by pointing the Weka package manager to the URL github.com/djhogan/WeSeqMiner/raw/master/WeSeqMiner.zip. The Javadoc for WeSeqMiner classes can be accessed at djhogan.github.io/seqminer.

bioinformatics↗

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↗