Biology subjects

Dunn, S.

Publications and source records attributed to Dunn, S..

10 recordsLinked to original sources

bioRxiv2025

Pyocyanin produced by Pseudomonas aeruginosa Creates Legacy Effects That Boost Antibiotic Resistance Evolution in Enterococci

Polymicrobial infections are small communities of multiple interacting bacterial species. Interactions among constituent species may modify the growth of community members in the presence of antibiotics, for example via degradation of the antibiotic or induction of specific resistance mechanisms. However, for most polymicrobial infections the nature of such interactions is opaque, while they may affect both treatment efficacy and the evolution of antibiotic resistance. Here, we describe that past interaction of enterococci with Pseudomonas aeruginosa creates legacy effects that substantially alter their antibiotic tolerance and resistance evolution. Specifically, we find that the temporary exposure to pyocyanin, a secondary metabolite produced by P. aeruginosa, increases the efflux in enterococci. These tolerance legacy effects promote the evolution of antibiotic resistance of enterococci. This work shows that transient interactions in polymicrobial communities can alter the evolutionary fate of community members.

microbiology↗

bioRxiv2025

Key determinants of T cell epitope recognition revealed by TCR specificity profiles

Interactions between T-Cell Receptors (TCRs) and antigenic peptides presented on Major Histocompatibility Complex (MHC) molecules are central to the immune recognition of infected and malignant cells. The complexity of the TCR sequence space, the flexibility of the TCR-epitope interface and the lack of a standardized framework to visualize TCR specificity have hindered a comprehensive understanding of the principles governing TCR-epitope recognition across a broad range of epitopes. Here, we introduce a fully interpretable probabilistic framework, termed TCR specificity profiles (TSPs), which captures fundamental properties distinguishing epitope-specific from baseline TCR repertoires. We demonstrate that TSPs unravel key determinants of TCR-epitope recognition specificity. By identifying and analyzing TCRs recognizing dozens of epitope variants, we show that TSPs accurately predict cross-reactivity and reveal how TCR specificity evolves with epitope sequence, binding mode and MHC restriction. TSPs further enable interpretation of machine learning tools and reveal how AlphaFold3 can be used to decipher key determinants of TCR-epitope recognition specificity.

immunology↗

bioRxiv2025

Autophagy Suppresses CCL2 to Preserve Appetite and Prevent Lethal Cachexia

Macroautophagy (autophagy hereafter) captures intracellular components and delivers them to lysosomes for degradation and recycling1. In adult mice, autophagy sustains metabolism to prevent wasting by cachexia and to survive fasting, and also suppresses inflammation, liver steatosis, neurodegeneration, and lethality2,3. Defects in autophagy contribute to metabolic, inflammatory and degenerative diseases, however, the specific mechanisms involved were unclear 4. Here we profiled metabolism and inflammation in adult mice with conditional, whole-body deficiency in an essential autophagy gene and found that autophagy deficiency altered fuel usage, and reduced ambulatory activity, energy expenditure, and food intake, and elevated circulating GDF15, CXCL10, and CCL2. While deletion of Gdf15 or Cxcl10 provided no or mild benefit, deletion of Ccl2 restored food intake, suppressed cachexia and rescued lethality of autophagy-deficient mice. To test if appetite suppression by CCL2 was responsible for lethal cachexia we performed single nucleus RNA sequencing of the hypothalamus, the center of appetite control in the brain. Notably, we found that autophagy deficiency was specifically toxic to PMCH and HCRT neurons that produce orexigenic neuropeptides that promote food intake, which was rescued by deficiency in CCL2. Finally, the restoration of food intake via leptin deficiency prevented lethal cachexia in autophagy-deficient mice. Our findings demonstrate a novel mechanism where autophagy prevents induction of a cachexia factor, CCL2, which damages neurons that maintain appetite, the destruction of which may be central to degenerative wasting conditions. Key points of paper1) Autophagy-deficient mice have reduced food intake, systemic inflammation, and cachexia 2) CCL2, but not GDF15 or CXCL10, induces lethal cachexia caused by autophagy defect 3) Autophagy-deficient mice have CCL2-dependent destruction of appetite-promoting neurons in the hypothalamus 4) Leptin deficiency restores appetite and rescues lethal cachexia in autophagy-deficient mice 5) Autophagy-deficient mice die from cachexia mediated by appetite loss 6) Degenerative conditions due to impaired autophagy are caused by the inflammatory response to the damage 7) Targeting CCL2 may be a viable approach to prevent degenerative wasting disorders

molecular biology↗

bioRxiv2024

The RND efflux pump EefABC is highly conserved within lineages of E. coli commonly associated with infection.

Tripartite resistance-nodulation-division (RND) efflux pumps confer multidrug resistance (MDR) in Gram-negative bacteria and are critical for many physiological functions including virulence and biofilm formation. The common laboratory strain of E. coli, K-12 MG1655 has six recognised RND transporters participating in tripartite pump formation (AcrB, AcrD, AcrF, CusA, MdtBC, and MdtF). However, by studying >20,000 E. coli genomes we show that E. coli belonging to phylogroups B2, D, E, F and G, which are commonly associated with infection, possess an additional, seventh RND transporter, EefB. It is found in a five gene operon, eefRABCD, which also encodes a TetR family transcription factor, a periplasmic adapter protein, an outer membrane factor and major facilitator superfamily pump. In contrast, E. coli from phylogroups A, B1 and C, generally containing environmental and commensal strains, do not encode the operon and instead encode an uncharacterised ORF, ycjD. In phylogroups where the eefRABCD operon is present it was very highly conserved. In fact, conservation levels were comparable to that of the major E. coli RND efflux system AcrAB-TolC, suggesting a critical biological function. Protein modelling shows that this pump is highly divergent from endogenous E. coli RND systems with unique structural features, while showing similarities to efflux systems found in Pseudomonas aeruginosa. However, unlike other major RND efflux systems, EefABC does not appear to transport antimicrobials and instead may be important for infection or survival in the host environment. ImportanceEfflux pumps are molecular machines that export molecules out of bacterial cells. The efflux pumps belonging to the RND family are particularly important as they export antibiotics out of Gram-negative bacterial cells, contributing to antibiotic resistance. The important human pathogen, E. coli, has been previously reported to have six RND pumps. However, we show that phylogroups of E. coli commonly associated with infection encode a seventh RND pump, EefABC which is highly conserved, suggesting an important biological function. While the function of EefABC in E. coli remains to be resolved, it does not seem to transport antimicrobial compounds. These findings are important because they reveal a new RND pump, potentially involved in virulence and survival in the host, that could represent a new therapeutic target. Additionally, it again shows that laboratory type strains of common bacterial pathogens are not representative of those that are infection causing.

microbiology↗

bioRxiv2024

Phage display profiling of CDR3β loops enables machine learning predictions of NY-ESO-1 specific TCRs

T cells targeting epitopes in infectious diseases or cancer play a central role in spontaneous and therapy-induced immune responses. T-cell epitope recognition is mediated by the binding of the T-Cell Receptor (TCR) and TCRs recognizing clinically relevant epitopes are promising for T-cell based therapies. Starting from one of the few known TCRs targeting the cancer-testis antigen NY-ESO-1157-165 epitope, we built large phage display libraries of TCRs with randomized Complementary Determining Region 3 of the {beta} chain. The TCR libraries were panned against the NY-ESO-1 epitope, which enabled us to collect thousands of epitope-specific TCR sequences. We then trained a machine learning TCR-epitope interaction predictor with this data and could identify several epitope-specific TCRs directly from TCR repertoires. Cellular binding and functional assays revealed that the predicted TCRs displayed activity towards the NY-ESO-1 epitope and no detectable cross-reactivity with self-peptides. Overall, our work demonstrates how display technologies combined with machine learning models of TCR-epitope recognition can effectively leverage large TCR repertoires for TCR discovery.

bioinformatics↗

bioRxiv2024

Bone marrow by day, lymph nodes by night: Undernutrition imparts a distinct circadian rhythm to T cells in mice

In well-nourished organisms, T cells migrate between the blood and secondary lymphoid organs, conducting surveillance for invading pathogens. T cell surveillance is under circadian control via diurnal fluctuations in corticosterone levels and undernutrition is associated with increased corticosterone. Therefore, we hypothesized that undernutrition disrupts the circadian migratory patterns of T cells. We report that compared to well-nourished controls, undernourished mice demonstrate enhanced T cell relocation to the bone marrow throughout the 24-hour period, but especially during the light phase, and diminished T cell migration to the lymph nodes only during the light phase. Undernutrition-related changes in T cell expression of key migration proteins are also mostly limited to the light phase. For example, undernourished naive CD4+ T cells exhibited higher levels of CXCR4 and CCR7 as well as reduced levels of S1P1 compared to controls; with all changes, except for CXCR4 expression, being restricted to the light phase. These results suggest that naive CD4+ T cells in the lymph nodes upregulate CXCR4 during the dark phase, enabling their migration to the bone marrow where they remain for the light phase. Once there, CCR7 is upregulated, presumably sending them back to the lymph node, thereby preserving immunosurveillance during the dark phase. Naive CD4+ T cell disengagement from S1P1-related egress signals may further contribute to increased retention of cells within each compartment during the appropriate phase. Undernutrition-related increases in T cell residency of the bone marrow likely preserve T cell numbers until nutrition is restored.

immunology↗

bioRxiv2023

Wiskott-Aldrich Syndrome Protein Regulates Nucleolar Organization and Function in Innate Immune Response

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by the dysfunction of the WAS protein (WASP). Using an isogenic macrophage model derived from genome edited induced pluripotent stem cells we demonstrated that WASP functions in the nucleolus, which plays important roles in immune regulation. The absence of WASP resulted in smaller and misshapen nucleoli, decreased fibrillar center territory, and impaired ribosomal RNA (rRNA) transcription. The nucleolar and rRNA phenotypes were confirmed in WAS patient samples. Furthermore, WASP interacts with nucleolar proteins, including nucleophosmin 1 (NPM1) and fibrillarin (FBL). NPM1 deficiency is known to cause elevated cytokine expression following lipopolysaccharide (LPS) stimulation. Consistently, WASP deficient cells displayed lower levels of NPM1 and a heightened inflammatory cytokine response to LPS, which was rescued by overexpressing NPM1. Together, our research provides novel insights into the critical role of WASP in nucleolar function and the modulation of inflammatory cytokine production.

cell biology↗

bioRxiv2023

Loss of type VI secretion systems in multi-drug resistant Escherichia coli clones

The repeated emergence of multi-drug resistant (MDR) Escherichia coli clones is a threat to public health globally. In recent work, drug resistant E. coli were shown to be capable of displacing commensal E. coli in the human gut. Given the rapid colonisation observed in travel studies, it is possible that the presence of a type VI secretion system (T6SS) may be responsible for the rapid competitive advantage of drug resistant E. coli clones. We employed large scale genomic approaches to investigate this hypothesis. First, we searched for T6SS genes across a curated dataset of over 20,000 genomes representing the full phylogenetic diversity of E. coli. This revealed large, non-phylogenetic variation in the presence of T6SS genes. No association was found between T6SS gene carriage and MDR lineages. However, multiple clades containing MDR clones have lost essential structural T6SS genes. We characterised the T6SS loci of ST410 and ST131 and identified specific recombination and insertion events responsible for the parallel loss of essential T6SS genes in two MDR clones. Data SummaryThe genome sequence data generated in this study is publicly available from NCBI under BioProject PRJNA943186, alongside a complete assembly in GenBank under accessions CP120633-CP120634. All other sequence data used in this paper has been taken from ENA with the appropriate accession numbers listed within the methods section. The E. coli genome data sets used in this work are from a previous publication, the details of which can be found in the corresponding supplementary data files 10.6084/m9.figshare.21360108 [1]. Impact StatementEscherichia coli is a globally significant pathogen that causes the majority of urinary tract infections. Treatment of these infections is exacerbated by increasing levels of drug resistance. Pandemic multi-drug resistant (MDR) clones, such as ST131-C2/H30Rx, contribute significantly to global disease burden. MDR E. coli clones are able to colonise the human gut and displace the resident commensal E. coli. It is important to understand how this process occurs to better understand why these pathogens are so successful. Type VI secretion systems may be one of the antagonistic systems employed by E. coli in this process. Our findings provide the first detailed characterisation of the T6SS loci in ST410 and ST131 and shed light on events in the evolutionary pathways of the prominent MDR pathogens ST410-B4/H42RxC and ST131-C2/H30Rx.

microbiology↗

bioRxiv2022

RND pumps across the Acinetobacter genus; AdeIJK is the ancestral efflux system.

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is extensively drug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes 6 RND efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, however the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-three species making up the Acinetobacter genus were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter, and through genomic, structural and phenotypic work we show that these genes are actually orthologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter, such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the Acinetobacter genus. AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH were only found in a subset of Acinetobacter, that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter, treatments for infections can avoid efflux-mediated resistance and improve patient outcomes. Impact statementEfflux pumps extrude antibiotics from within bacterial cells directly conferring antibiotic resistance and underpinning other mechanisms of resistance. By understanding the exact complement of efflux pumps and their roles across infection-causing organisms such as those within the Acinetobacter genus, it is possible to understand how cells become resistant to antibiotics and how this might be tackled. Efflux is an attractive target for inhibition to increase susceptibility to existing drugs and therefore, knowing which pumps are present in each species is important. Furthermore, we present a novel method using conserved RND residues to predict the total number of RND proteins including currently novel systems, within bacterial genomes. Data SummaryThis study made use of publicly available datasets downloaded from NCBIs GenBank. A full list of accession numbers can be found in supplementary text 3. Bioinformatics software used in this study was previously published and listed in the methods section. The BLASTp conserved residue files are in S1 text 1 and 2. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2022

Evolutionary responses to acquiring a multidrug resistance plasmid are dominated by metabolic functions across diverse Escherichia coli lineages

Multidrug resistance (MDR) plasmids drive the spread of antibiotic resistance between bacterial lineages. The immediate impact of MDR plasmid acquisition on fitness and cellular processes varies among bacterial lineages, but how the evolutionary processes enabling the genomic integration of MDR plasmids vary is less well understood, particularly in clinical pathogens. Using diverse Escherichia coli lineages experimentally evolved for [~]700 generations, we show that the evolutionary response to gaining the MDR plasmid pLL35 was dominated by chromosomal mutations affecting metabolic and regulatory functions, with both strain-specific and shared mutational targets. The expression of several of these functions, such as anaerobic metabolism, is known to be altered upon acquisition of pLL35. Interactions with resident mobile genetic elements, notably several IS-elements, potentiated parallel mutations, including insertions upstream of hns that were associated with its upregulation and the downregulation of the plasmid-encoded extended-spectrum beta-lactamase gene. Plasmid parallel mutations targeted conjugation-related genes, whose expression was also commonly downregulated in evolved clones. Beyond their role in horizontal gene transfer, plasmids can be an important selective force shaping the evolution of bacterial chromosomes and core cellular functions.

evolutionary biology↗