Biology subjects

Round, J.

Publications and source records attributed to Round, J..

4 recordsLinked to original sources

bioRxiv2024

Stress-driven emergence of heritable non-genetic drug resistance

Drug resistance is the chief cause of treatment failure for therapies targeting chronic and infectious diseases. Whether the emergence of resistance is accelerated by environmental exposure to low levels of therapeutics remains controversial. Here, we report a non-genetic mechanism of stress adaptation that promotes heritable resistance to the widely used antifungal drug fluconazole. In the human fungal pathogen Candida albicans, transient exposure to subtherapeutic fluconazole doses induces a protective response that we term para-resistance. Like conventional resistance mechanisms, para-resistance is heritable. However, it does not arise from genetic mutations and can revert spontaneously. Systematic analyses of para-resistant isolates suggest that its key regulators include the stress-activated MAP kinase Hog1, the histone deacetylase subunit Snt1, the chromatin regulator Rap1, and the Sko1 transcriptional factor. Notably, molecules that disrupt biomolecular condensation and prion propagation - crucial for the inheritance of protein assemblies - block the induction of para-resistance, whereas inhibiting histone deacetylases facilitates its induction. We find that para-resistance is common in clinical isolates and, remarkably, passage through the mammalian gut triggers its acquisition, compromising fluconazoles therapeutic efficacy. Our work defines a pervasive, prion-like epigenetic mechanism of stress adaptation and highlights potential strategies to mitigate the rapid emergence of drug resistance.

microbiology↗

bioRxiv2023

A Synthetic Cytotoxic T cell Platform for Rapidly Prototyping TCR Function

Current tools for functionally profiling T cell receptors with respect to cytotoxic potency and cross-reactivity are hampered by difficulties in establishing model systems to test these proteins in the contexts of different HLA alleles and against broad arrays of potential antigens. We have implemented and validated a granzyme-activatable sensor of T cell cytotoxicity in a novel universal prototyping platform which enables facile recombinant expression of any combination of TCR-, peptide-, and class I MHC-coding sequences and direct assessment of resultant responses. This system consists of an engineered cell platform based on the immortalized natural killer cell line, YT-Indy, and the MHC-null antigen-presenting cell line, K562. These cells were engineered using contemporary gene-editing techniques to furnish the YT-Indy/K562 pair with appropriate protein domains required for recombinant TCR expression and function in a non-T cell chassis, integrate a fluorescence-based target-centric early detection reporter of cytotoxic function, and deploy a set of protective genetic interventions designed to preserve antigen-presenting cells for subsequent capture and downstream characterization. Our data show successful reconstitution of the surface TCR complex in the YT-Indy cell line at biologically relevant levels. We also demonstrate successful induction and highly sensitive detection of antigen-specific response in multiple distinct model TCRs, with significant responses (p < 0.05 and Cohens d >1.9) in all cases. Additionally, we monitored destruction of targets in co-culture and found that our survival-optimized system allowed for complete preservation after 24-hour exposure to cytotoxic effectors. With this bioplatform, we anticipate investigators will be empowered to rapidly express and characterize T cell receptor responses, generate new knowledge regarding the patterns of T cell receptor recognition, and optimize novel therapeutic T cell receptors for improved cytotoxic potential and reduced cross-reactivity to undesired antigenic targets.

immunology↗

bioRxiv2023

Efficient encoding of large antigenic spaces by epitope prioritization with Dolphyn

We investigated a relatively underexplored component of the gut-immune axis by profiling the antibody response to gut phages using Phage Immunoprecipitation Sequencing (PhIP-Seq). To enhance this approach, we developed Dolphyn, a novel method that uses machine learning to select peptides from protein sets and compresses the proteome through epitope-stitching. Dolphyn improves the fraction of gut phage library peptides bound by antibodies from 10% to 31% in healthy individuals, while also reducing the number of synthesized peptides by 78%. In our study on gut phages, we discovered that the immune system develops antibodies to bacteria-infecting viruses in the human gut, particularly E.coli-infecting Myoviridae. Cost-effective PhIP-Seq libraries designed with Dolphyn enable the assessment of a wider range of proteins in a single experiment, thus facilitating the study of the gut-immune axis.

bioengineering↗

bioRxiv2023

Clec12a tempers inflammation while restricting expansion of a colitogenic commensal

SUMMARYRegulation of the microbiota is critical to intestinal health yet the mechanisms employed by innate immunity remain unclear. Here we show that mice deficient in the C-Type-lectin receptor, Clec12a developed severe colitis, which was dependent on the microbiota. Fecal-microbiota-transplantation (FMT) studies into germfree mice revealed a colitogenic microbiota formed within Clec12a-/- mice that was marked by expansion of the gram-positive organism, Faecalibaculum rodentium. Treatment with F. rodentium was sufficient to worsen colitis in wild-type mice. Macrophages within the gut express the highest levels of Clec12a. Cytokine and sequencing analysis in Clec12a-/- macrophages revealed heighten inflammation but marked reduction in genes associated with phagocytosis. Indeed, Clec12a-/- macrophages are impaired in their ability to uptake F. rodentium. Purified Clec12a had higher binding to gram-positive organisms such as F. rodentium. Thus, our data identifies Clec12a as an innate immune surveillance mechanism to control expansion of potentially harmful commensals without overt inflammation.

immunology↗