Biology subjects

Crabbe, A.

Publications and source records attributed to Crabbe, A..

6 recordsLinked to original sources

bioRxiv2026

Genomic Potential vs. Pathogenic Reality: Functional Virulence Variation in Pseudomonas aeruginosa

Despite the rapid expansion of bacterial genome sequencing, predicting pathogen virulence from genomic information remains a major challenge. To study this association, we integrate comparative genomics, functional phenotyping, host-pathogen interaction assays, and infection outcomes across a diverse collection of 28 Pseudomonas aeruginosa isolates, representing clinically relevant diversity in serotype, type IV pilus group, and flagellar type. While virulence-associated systems were broadly retained across the strain collection, isolates displayed substantial variation in phenotypic behavior and pathogenicity. Genotypic classifications based on pilus, flagellar, siderophore, or secretion system characteristics were poor predictors of virulence-associated phenotypes. Pathogenicity in a Galleria mellonella model varied markedly among strains and was most strongly associated with surface-associated motility, whereas other virulence traits and quorum-sensing genotypes showed limited predictive value. In a three-dimensional lung epithelial cell model, inflammatory responses were negatively correlated with activity of the type III secretion system effector ExoS, suggesting a role in modulation of host immune signaling. Together, these findings demonstrate that virulence emerges from functional deployment of conserved virulence repertoires, rather than their mere presence. By linking genomic diversity to phenotypic expression, pathogenicity, and host responses within a single strain collection, this study highlights fundamental limitations of gene-content-based virulence prediction and underscores the importance of integrating genomic and functional data to better understand P. aeruginosa virulence.

microbiology↗

bioRxiv2025

Identification of DksA as a novel pro-inflammatory mediator of Pseudomonas aeruginosa under conditions mimicking chronic cystic fibrosis lung infection

Chronic infection with Pseudomonas aeruginosa is a major driver of airway inflammation, which plays a central role in the progression of cystic fibrosis (CF) lung disease. During long-term colonization, P. aeruginosa adapts to the CF lung by downregulating virulence factors and adopting a biofilm-associated, mucoid lifestyle. Despite the expected reduction in immune activation due to these adaptations, excessive inflammation persists, a paradox that remains poorly understood. Our objective was to identify novel bacterial mediators sustaining persistent inflammation by P. aeruginosa in the CF lung. To this end, we analyzed clinical P. aeruginosa CF isolates, cultured them in synthetic CF sputum medium, and exposed 3D lung epithelial cell cultures to the resulting cell-free supernatants. There was considerable variability in pro-inflammatory activity among the isolates, with a subset of the isolates inducing strong IL-8 secretion by the 3D cells despite low production of known virulence factors. Comparative proteomics analysis of the cell-free supernatants of pro- and anti-inflammatory isolates revealed several mediators not previously linked to inflammation. Thirteen of these candidate pro-inflammatory mediators were selected for further analysis. Using P. aeruginosa transposon mutants lacking the respective mediators, DksA (a transcription factor) was confirmed as an immunomodulatory mediator in the 3D lung model. Finally, analysis of existing transcriptomes of P. aeruginosa in CF sputum, revealed that dksA was found to be one of the most strongly expressed genes in this patient population, highlighting the relevance of our findings. In conclusion, we identified a novel P. aeruginosa mediator that may contribute to CF airway inflammation.

microbiology↗

bioRxiv2024

Identification of echinacoside as a tobramycin potentiator against Pseudomonas aeruginosa aggregates

Cyclic diguanylate (c-di-GMP) is a central biofilm regulator, where increased intracellular levels promote biofilm formation and antibiotic tolerance. Targeting the c-di-GMP network is a promising anti-biofilm approach. Most agents reported previously decreased c-di-GMP to eliminate surface-attached biofilms, which did not recapitulate in vivo biofilms well and may thus impede their clinical impact. Here, the expression profile of genes encoding proteins associated with c-di-GMP metabolism was analysed among 32 Pseudomonas aeruginosa strains grown as suspended aggregates in synthetic sputum or planktonic cells. A diguanylate cyclase, SiaD, proved essential for auto-aggregation under in vivo-like conditions. Virtual screening against SiaD identified echinacoside as an inhibitor, which reduced intracellular c-di-GMP levels and aggregate sizes and potentiated the efficacy of tobramycin against aggregates established by >80% of tested strains. This synergistic effect was also observed for in vivo-like 3-D alveolar cells infected by cytotoxic P. aeruginosa, demonstrating its high potential as an adjunctive therapy for recalcitrant P. aeruginosa infections.

microbiology↗

bioRxiv2023

Genome-Scale Modeling of Rothia mucilaginosa Reveals Insights into Metabolic Capabilities and Therapeutic Strategies for Cystic Fibrosis

BackgroundCystic fibrosis (CF) is an inherited genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in the production of sticky and thick mucosal fluids. This leads to an environment that facilitates the colonization of various microorganisms, some of which can cause acute and chronic lung infections, while others may have a positive influence on the disease process. Rothia mucilaginosa, an oral commensal, is relatively abundant in the lungs of CF patients. Recent studies have unveiled the anti-inflammatory properties of R. mucilaginosa using in vitro three-dimensional (3-D) lung epithelial cell cultures and in vivo mouse models relevant to chronic lung diseases. Apart from a potentially beneficial anti-inflammatory role in chronic lung diseases, R. mucilaginosa has been associated with severe infections. This dual nature highlights the bacteriums complexity and diverse impact on health and disease. However, its metabolic capabilities and genotype-phenotype relationships remain largely unknown. ResultsTo gain insights into the cellular metabolism and genetic content of R. mucilaginosa, we developed the first manually curated genome-scale metabolic model, iRM23NL. Through growth kinetic experiments and high-throughput phenotypic microarray testings, we defined its complete catabolic phenome. Subsequently, we assessed the models effectiveness in accurately predicting growth behaviors and utilizing multiple substrates. We used constraint-based modeling techniques to formulate novel hypotheses that could expedite the development of antimicrobial strategies. More specifically, we detected putative essential genes and assessed their effect on metabolism under varying nutritional conditions. These predictions could offer novel potential antimicrobial targets without laborious large-scale screening of knock-outs and mutant transposon libraries. ConclusionOverall, iRM23NL demonstrates a solid capability to predict cellular phenotypes and holds immense potential as a valuable resource for accurate predictions in advancing antimicrobial therapies. Moreover, it can guide metabolic engineering to tailor R. mucilaginosas metabolism for desired performance.

systems biology↗

bioRxiv2019

Non-hierarchical, RhlR-regulated acyl-homoserine lactone quorum sensing in a cystic fibrosis isolate of Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy, and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often \"rewired\" such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this \"rewired\" background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations to that of PAO1. Unlike PAO1, cytotoxicity by E90 in a three-dimensional lung epithelium cell model is also RhlR-regulated. These data illuminate a \"rewired\" LasR-independent RhlR regulon in chronic infection isolates and suggest that RhlR may be a target for therapeutic development in chronic infections.\n\nAUTHOR SUMMARYPseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Some isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR gene regulation in chronic P. aeruginosa infections.

microbiology↗

bioRxiv2018

Various evolutionary trajectories lead to loss of the tobramycin-potentiating activity of the quorum sensing inhibitor baicalin hydrate in Burkholderia cenocepacia biofilms

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant and -tolerant bacteria. As these potentiators typically do not interfere with essential processes of bacteria, it has been hypothesized that they are less likely to induce resistance than conventional antibiotics. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop resistance towards one such adjuvant, baicalin hydrate (BH), a quorum sensing inhibitor known to increase antibiotic-induced oxidative stress. Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After each cycle of treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to treatments with TOB and TOB+BH, compared to the start population, and the potentiating effect of BH towards TOB was lost. Whole genome sequencing was performed to probe which changes were involved in the reduced effect of BH and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter), as well as a partial deletion of two larger regions. No changes in the minimal inhibitory or minimal bactericidal concentration of TOB or changes in the number of persister cells were observed in the evolved populations. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that resistance towards antibiotic-potentiating activity can develop rapidly in B. cenocepacia J2315 biofilms and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as potential mechanisms.\n\nImportanceBacteria show a markedly reduced susceptibility to antibiotics when growing in a biofilm, which hampers effective treatment of biofilm-related infections. The use of potentiators that increase the activity of antibiotics against biofilms has been proposed as a solution to this problem, but it is unclear whether resistance to these potentiators could develop. Using an experimental evolution approach, we convincingly demonstrate that Burkholderia cenocepacia biofilms rapidly develop resistance towards the tobramycin-potentiating activity of baicalin hydrate. Whole genome sequencing revealed that there are different mechanisms that lead to this resistance, including mutations resulting in metabolic changes, changes in production of intracellular levels of reactive oxygen species, and differences in transporter-mediated tobramycin uptake. Our study suggests that this form of combination therapy is not evolution-proof and highlights the usefulness of experimental evolution to identify mechanisms of resistance and tolerance in biofilm-grown bacteria.

microbiology↗