Biology subjects

Uthayakumar, D.

Publications and source records attributed to Uthayakumar, D..

4 recordsLinked to original sources

bioRxiv2026

Chronic stress promotes sex-divergent Type 1, 2 and 17 responses in brain resident lymphocytes.

T helper cytokines such as IFN{gamma} and IL-17 are dysregulated in a subset of patients with depression and are associated with poor responses to treatment. Both IFN{gamma} and IL-17 have been shown to regulate neural circuits involved in behavioral changes implicated in depression. However, the cellular source and the regulatory mechanisms governing these cytokines in this context remain incompletely understood. We analyzed brain lymphocytes in response to unpredictable chronic mild stress (UCMS), a model of depression with strong face and construct validity. We confirmed that UCMS induced depressive-like behavior in both male and female mice. Interestingly, UCMS preferentially reduced regional synaptic density in female and neuron counts in male hippocampi. Using single cell RNAseq, we identified T helper cytokine signatures to be enriched in distinct lymphocyte lineages expressing tissue resident markers and are regulated by both sex and chronic stress. Female brain-resident {gamma}{delta}T cells are significantly enriched for type 2 and type 17 signatures and have higher expression of inflammasome genes such as Nlrp3 and Il1b whereas male {beta}T cells and NKT cells are enriched for type 1 signatures and have elevated Cxcr3 and Il27ra expression. Chronic stress amplified the sex differences and preferentially increased the type 1 response including IFN{gamma} production in males at both the transcriptome and protein level. Type 17 response (e.g. IL-17A and IL-17F production) is also increased in response to UCMS, however with sex-divergent polarization: IL-22 is decreased in female and increased in male brain lymphocytes. Mechanistically, we found that UCMS suppressed P4ha1 in male and Zfp36 in female brain tissue resident lymphocytes. P4HA1 was previously shown to restrict mitochondrial function and limit type 1 responses in CD8 T cells, whereas ZFP36 is a suppressor of inflammatory cytokine translation. Consistent with this finding, ex vivo CD3-dependent stimulation of brain immune cells showed stress-dependent increase in cytokine production with sex-specific effects. Our study provides the cellular and molecular framework for chronic stress-induced tissue resident lymphocyte dysregulation and its sex bias in the brain, with implications for depressive disorders marked by inflammation.

immunology↗

bioRxiv2024

A role for the putative error-prone polymerase REV1 in DNA damage and antifungal drug resistance in Candida albicans

Antimicrobial-induced DNA damage, and subsequent repair via upregulation of DNA repair factors, including error-prone translesion polymerases, can lead to the increased accumulation of mutations in the microbial genome, and ultimately increased risk of acquired mutations associated with antimicrobial resistance. While this phenotype is well described in bacterial species, it is less thoroughly investigated amongst microbial fungi. Here, we monitor DNA damage induced by antifungal agents in the fungal pathogen Candida albicans, and find that commonly used antifungal drugs are able to induce DNA damage, leading to the upregulation of transcripts encoding predicted error-prone polymerases and related factors. We focus on REV1, encoding a putative error-prone polymerase, and find that while deleting this gene in C. albicans leads to increased sensitivity to DNA damage, it also unexpectedly renders cells more likely to incur mutations and evolve resistance to antifungal agents. We further find that deletion of REV1 leads to a significant depletion in the uncharacterized protein Shm1, which itself plays a role in fungal mutagenesis. Together, this work lends new insight into previously uncharacterized factors with important roles in the DNA damage response, mutagenesis, and the evolution of antifungal drug resistance.

microbiology↗

bioRxiv2022

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPRa to overexpress genes involved in pathogenesis and drug resistance and contribute towards the identification of novel phenotypes. Together, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

microbiology↗

bioRxiv2021

The SAGA and NuA4 component Tra1 regulates Candida albicans drug resistance and pathogenesis

Candida albicans is the most common cause of death from fungal infections. Emergence of resistant strains reducing the efficacy of first line therapy with echinocandins such as caspofungin calls for the identification of alternative therapeutic strategies. Tra1 is an essential component of the SAGA and NuA4 transcriptional co-activator complexes. As a PIKK family member, Tra1 is characterized by a C-terminal phosphoinositide 3-kinase domain. In Saccharomyces cerevisiae, the assembly and function of SAGA and NuA4 is compromised by a version of Tra1 (Tra1Q3) with three arginine residues in the putative ATP-binding cleft changed to glutamine, Whole transcriptome analysis of the S. cerevisiae tra1Q3 strain highlights Tra1s role in global transcription, stress response and cell wall integrity. As a result, tra1Q3 increases susceptibility to multiple stressors, including caspofungin. Moreover, the same tra1Q3 allele in the pathogenic yeast Candida albicans causes similar phenotypes, suggesting that Tra1 broadly mediates the antifungal response across yeast species. Transcriptional profiling in C. albicans identified 68 genes that were differentially expressed when the tra1Q3 strain was treated with caspofungin, as compared to gene expression changes induced by either tra1Q3 or caspofungin alone. Included in this set were genes involved in cell wall maintenance, adhesion and filamentous growth. Indeed, the tra1Q3 allele reduces filamentation and other pathogenesis traits in C. albicans. We identified EVP1, which encodes a putative plasma membrane protein, amongst the Tra1-regulated genes, Disrupting EVP1 results in reduced filamentation and infection capacity in C. albicans. Thus,Tra1 emerges as a promising therapeutic target for fungal infections. ImportanceFungal pathogens such as Candida albicans are important agents of infectious disease, with increasing rates of drug resistance, and limited available antifungal therapeutics. In this study, we characterize the role of C. albicans Tra1, a critical component of acetyltransferase complexes, involved in transcriptional regulation and responses to environmental stress. We find C. albicans genetic mutants with impaired Tra1 function have reduced tolerance to cell-wall targeting stressors, including the clinically-important antifungal caspofungin. We further use RNA-sequencing to profile the global fungal response to the tra1 mutation, and identify a previously uncharacterized C. albicans gene, EVP1. We find that both TRA1 and EVP1 play an important role in phenotypes associated with fungal pathogenesis, including cellular morphogenesis, biofilm formation, and toxicity towards host immune cells. Together, this work describes the key role for Tra1 in regulating fungal drug tolerance and pathogenesis, and positions this protein as a promising therapeutic target for fungal infections.

microbiology↗