Biology subjects

Whiteley, M.

Publications and source records attributed to Whiteley, M..

7 recordsLinked to original sources

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

Experimental evolution of gene essentiality in bacteria

Essential gene products carry out fundamental cellular activities in interaction with other components. However, the lack of essential gene mutants and appropriate methodologies to link essential gene functions with their partners poses significant challenges. Here, we have generated deletion mutants in 32 genes previously identified as essential, with 23 mutants showing extremely slow growth in the SK36 strain of Streptococcus sanguinis. The 23 genes corresponding to these mutants encode components of diverse pathways, are widely conserved among bacteria, and are essential in many other bacterial species. Whole-genome sequencing of 243 independently evolved populations of these mutants has identified >1000 spontaneous suppressor mutations in experimental evolution. Many of these mutations define new gene and pathway relationships, such as F1Fo-ATPase/V1Vo-ATPase/TrkA1-H1 that were demonstrated across multiple Streptococcus species. Patterns of spontaneous mutations occurring in essential gene mutants differed from those found in wildtype. While gene duplications occurred rarely and appeared most often at later stages of evolution, substitutions, deletions, and insertions were prevalent in evolved populations. These essential gene deletion mutants and spontaneous mutations fixed in the mutant populations during evolution establish a foundation for understanding gene essentiality and the interaction of essential genes in networks.

evolutionary biology↗

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

Volatile hydrogen cyanide released by Pseudomonas aeruginosa provides a competitive advantage over Staphylococcus aureus in biofilm and in vivo lung environments

Diverse bacterial volatile compounds alter bacterial stress responses and physiology, but their contribution to population dynamics in polymicrobial communities is not well known. In this study, we showed that airborne volatile hydrogen cyanide (HCN) produced by a wide range of Pseudomonas aeruginosa clinical strains leads to at-a-distance inhibition of the growth of a wide array of Staphylococcus aureus strains. We determined that low oxygen level environments not only enhance P. aeruginosa HCN production but also increase S. aureus sensitivity to HCN, which impacts P. aeruginosa-S. aureus competition in microaerobic in vitro mixed biofilms as well as in an in vitro cystic fibrosis lung sputum medium. Consistently, we demonstrated that production of HCN provides a competitive advantage to P. aeruginosa in a mouse model of airways co-infected by P. aeruginosa and S. aureus. Our study therefore demonstrates that P. aeruginosa HCN contributes to local and distant airborne competition against S. aureus and potentially other HCN-sensitive bacteria in contexts relevant to cystic fibrosis and other polymicrobial infectious diseases. IMPORTANCEAirborne volatile compounds produced by bacteria are often only considered as attractive or repulsive scents, but they also directly contribute to bacterial physiology. Here we showed that volatile hydrogen cyanide (HCN) released by a wide range of Pseudomonas aeruginosa clinical strains inhibits Staphylococcus aureus growth in low oxygen in vitro biofilms or aggregates and in vivo lung environments. These results are of pathophysiological relevance, since lungs of cystic fibrosis patients are known to present microaerophilic areas and to be commonly associated with the presence of S. aureus and P. aeruginosa in polymicrobial communities. Our study therefore provides insights into how a bacterial volatile compound can contribute to the exclusion of S. aureus and other HCN-sensitive competitors from P. aeruginosa ecological niches. It opens new perspectives for the management or monitoring of P. aeruginosa infections in lower lung airway infections and other polymicrobial disease contexts.

microbiology↗

bioRxiv2021

The SARS-CoV-2 variants associated with infections in India, B.1.617, show enhanced spike cleavage by furin

The spike (S) glycoprotein of the SARS-CoV-2 virus that emerged in 2019 contained a suboptimal furin cleavage site at the S1/S2 junction with the sequence 681PRRAR/S686. This cleavage site is required for efficient airway replication, transmission, and pathogenicity of the virus. The B.1.617 lineage has recently emerged in India, coinciding with substantial disease burden across the country. Early evidence suggests that B.1.617.2 (a sublineage of B.1.617) is more highly transmissible than contemporary lineages. B.1.617 and its sublineages contain a constellation of S mutations including the substitution P681R predicted to further optimise this furin cleavage site. We provide experimental evidence that virus of the B.1.617 lineage has enhanced S cleavage, that enhanced processing of an expressed B.1.617 S protein in cells is due to P681R, and that this mutation enables more efficient cleavage of a peptide mimetic of the B.1.617 S1/S2 cleavage site by recombinant furin. Together, these data demonstrate viruses in this emerging lineage have enhanced S cleavage by furin which we hypothesise could be enhancing transmissibility and pathogenicity.

microbiology↗

bioRxiv2021

Altered Sub-Genomic RNA Expression in SARS-CoV-2 B.1.1.7 Infections

SARS-CoV-2 lineage B.1.1.7 viruses are more transmissible, may lead to greater clinical severity, and result in modest reductions in antibody neutralization. subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome and is a crucial step in the SARS-CoV-2 life cycle. Applying our tool (periscope) to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA expression profiles are significantly increased in B.1.1.7 infections (n=879). This increase is seen over the previous dominant circulating lineage in the UK, B.1.177 (n=943), which is independent of genomic reads, E gene cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median expression. We hypothesise that this is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT>CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3 of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging potential variants of concern. One Sentence SummaryThe recently emerged and more transmissible SARS-CoV-2 lineage B.1.1.7 shows greater subgenomic RNA expression in clinical infections and enhanced expression of a noncanonical subgenomic RNA near ORF9b.

genomics↗

bioRxiv2018

The Pseudomonas aeruginosa Wsp pathway undergoes positive evolutionary selection during chronic infection

Author Contributions Author Contributions Introductory paragraph Results Discussion Materials and Methods References ESG and CWM performed the experimental work. JSH performed the colony PCR. SBC infected and sampled the porcine burn wounds. DS generated the sequence library. MW and GRL quantified the strain frequency in the wounds. ESG, CWM, MW, VSC, and DJW conceptualized the project and wrote the manuscript.\n\nIntroductory paragraph Author Contributions Introductory paragraph Results Discussion Materials and Methods References Pathogens experience pressure in an infection to adapt, with selection favoring mutants that persist. Pseudomonas aeruginosa commonly ad ...

microbiology↗