Biology subjects

Washington, E. J.

Publications and source records attributed to Washington, E. J..

6 recordsLinked to original sources

bioRxiv2026

Trehalose-6-phosphate synthase promotes thermotolerance by governing glycolytic flux in Cryptococcus deneoformans

Growth at physiologically relevant temperatures is essential for fungal pathogenesis and is controlled by several cellular factors. The evolution of fungal thermotolerance is concerning as warming environments may promote the emergence of new pathogens. Trehalose, a disaccharide absent in mammals, plays a central role in thermotolerance by stabilizing proteins and membranes during heat stress. Trehalose is synthesized from glucose-6-phosphate (G6P) and uridine-diphosphate-glucose (UDPG) in two steps catalyzed by trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2). Here, we investigated genetic suppression of Tps1 function in Cryptococcus deneoformans, a species in the Cryptococcus pathogenic species complex. Tps1 is essential for growth at 37{degrees}C in C. deneoformans and spontaneous suppressor mutations restored the growth of tps1{Delta} mutants at 37{degrees}C. Whole-genome sequencing followed by variant calling analysis primarily identified loss-of-function mutations in the gene encoding hexokinase 1 (Hxk1). Targeted gene deletion mutants further showed that loss of either HXK1 or HXK2 can bypass tps1{Delta} in a carbon source-dependent manner. The tps1{Delta} mutant exhibited elevated hexokinase activity, accumulation of G6P and glycogen, and ATP depletion after heat shock. Deletion of HXK1 or HXK2 restored hexokinase activity and partially restored G6P and ATP levels in the tps1{Delta} mutant, while glycogen remained elevated, indicating that excess glycolytic flux underlies the tps1{Delta} high-temperature growth defect. Overall, our study uncovers a previously unappreciated mechanism of Tps1-mediated heat adaptation in C. deneoformans, by revealing that Tps1 functions as a critical metabolic gatekeeper that safeguards glycolytic flux to sustain growth at elevated temperatures. Article summaryTrehalose is crucial for fungal thermal adaptation and mutants lacking trehalose are inviable at 37{degrees}C. This study examined how genetic suppressors restore viability at 37{degrees}C in mutants lacking TPS1, which encodes trehalose-6-phosphate synthase. Through whole-genome sequencing of spontaneous suppressor isolates and variant calling analysis, mutations were identified in HXK1. Gene deletion mutants and biochemical assays show these mutations alter glycolytic flux. We show that tps1{Delta} mutants exhibit unbridled glycolysis, and their growth at 37{degrees}C is restored by hxk1{Delta} mutations that reduce glycolytic flux. This study highlights the interdependence between Tps1 and Hxk1, which may have broader relevance across organisms.

genetics↗

bioRxiv2025

Inhibitors of trehalose-6-phosphate synthase activity in fungal pathogens compromise thermal tolerance pathways

Infections caused by fungal pathogens such as Candida and Cryptococcus are associated with high mortality rates, partly due to limitations in the current antifungal arsenal. This highlights the need for antifungal drug targets with novel mechanisms of action. The trehalose biosynthesis pathway is a promising antifungal drug target because trehalose biosynthesis is essential for virulence in Cryptococcus neoformans and Candida albicans and is also a mediator of fungal stress responses, such as thermotolerance. To exploit its untapped antifungal potentials, we screened the St. Jude 3-point pharmacophore library to identify small molecule inhibitors of the first enzyme in the trehalose biosynthesis pathway, trehalose-6-phosphate synthase (Tps1). Structure-guided optimization of a potent hit, SJ6675, yielded a water-soluble inhibitor named 4456dh. Employing biochemical, structural and cell-based assays, we demonstrate that 4456dh inhibits Tps1 enzymatic activity, suppresses trehalose synthesis and exerts a fungicidal effect. Notably, the structure of Tps1 in complex with 4456 reveals that 4456 occupies the substrate binding pocket. Importantly, 4456dh renders normally thermotolerant fungal pathogens unable to survive at elevated temperatures, which is critical as we investigate the emergence of fungi from the environment due to a warming climate. Overall, this work develops the water-soluble 4456dh as an early-stage antifungal drug that has a distinct mechanism of action compared to existing clinical antifungals. ImportanceThe rise of fungal infections in recent years is alarming due to an increase in the vulnerable immunocompromised population, global temperature increase and limited antifungal treatment options. One of the major hurdles in developing new drugs is the identification of fungal-specific antifungal drug targets due to highly conserved cellular machinery between fungi and humans. Here, we describe a small molecule inhibitor, 4456dh, of the trehalose biosynthesis pathway. This pathway is present in fungi but not humans. Trehalose plays a critical role in stress responses such as thermotolerance in fungal pathogens and is essential for their virulence. We show that treatment with 4456dh blocks production of trehalose and renders fungal cells inviable. Thus far, 4456dh is active against two fungal pathogens of critical importance suggesting a broad-spectrum activity.

microbiology↗

bioRxiv2024

Redox control of the deubiquitinating enzyme Ubp2 regulates translation during stress

Protein ubiquitination is essential to govern cells ability to cope with harmful environments by regulating many aspects of protein dynamics from synthesis to degradation. As important as the ubiquitination process, the reversal of ubiquitin chains mediated by deubiquitinating enzymes (DUBs) is critical for proper recovery from stress and re-establishment of proteostasis. Although it is known that ribosomes are decorated with K63-linked polyubiquitin (K63-ub) chains that control protein synthesis under stress, the mechanisms by which these ubiquitin chains are reversed and regulate proteostasis during stress recovery are still illusive. Here, we showed in budding yeast that the DUB Ubp2 is redox regulated during oxidative stress in a reversible manner, which determines the levels of K63-ub chains present on ribosomes. We also demonstrate that Ubp2 is a processive enzyme whose activity is modulated by a series of repeated domains and the formation of important disulfide bonds. By combining, cellular, biochemical, and proteomics analyses, we showed that Ubp2 is crucial for restoring translation after stress cessation, indicating an important role in determining cellular response to oxidative stress. Our work demonstrates a novel role for Ubp2, revealing that a range of signaling pathways can be controlled by redox regulation of DUB activity in eukaryotes, which in turn will define cellular states of health and diseases.

biochemistry↗

bioRxiv2024

A single amino acid in the Salmonella effector SarA/SteE triggers supraphysiological activation of STAT3 for anti-inflammatory target gene expression

Non-typhoidal Salmonella enterica cause an estimated 1 million cases of gastroenteritis annually in the United States. These serovars use secreted protein effectors to mimic and reprogram host cellular functions. We previously discovered that the secreted effector SarA (Salmonella anti-inflammatory response activator; also known as SteE) was required for increased intracellular replication of S. Typhimurium and production of the anti-inflammatory cytokine interleukin-10 (IL-10). SarA facilitates phosphorylation of STAT3 through a region of homology with the host cytokine receptor gp130. Here, we demonstrate that a single amino acid difference between SarA and gp130 is critical for the anti-inflammatory bias of SarA-STAT3 signaling. An isoleucine at the pY+1 position of the YxxQ motif in SarA (which binds the SH2 domain in STAT3) causes increased STAT3 phosphorylation and expression of anti-inflammatory target genes. This isoleucine, completely conserved in [~]4000 Salmonella isolates, renders SarA a better substrate for tyrosine phosphorylation by GSK-3. GSK-3 is canonically a serine/threonine kinase that nonetheless undergoes tyrosine autophosphorylation at a motif that has an invariant isoleucine at the pY+1 position. Our results provide a molecular basis for how a Salmonella secreted effector achieves supraphysiological levels of STAT3 activation to control host genes during infection.

molecular biology↗

bioRxiv2023

Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans: a target for novel antifungals

Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of novel drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Cryptococcus neoformans Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement towards the catalytic pocket by the N-terminus upon ligand binding and identifies residues required for substrate-binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved amongst Cryptococcal species and closely related Basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in density maps. We determined that the IDD is not required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. In toto, these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes. Significance StatementFungal infections are responsible for over a million deaths worldwide each year. Biosynthesis of a disaccharide, trehalose, is required for multiple pathogenic fungi to transition from the environment to the human host. Enzymes in the trehalose biosynthesis pathway are absent in humans and, therefore, are potentially significant targets for novel antifungal therapeutics. One enzyme in the trehalose biosynthesis is trehalose-6-phosphate synthase (Tps1). Here, we describe the cryo-electron microscopy structures of the CnTps1 homo-tetramer in the unliganded form and in complex with a substrate and a product. These structures and subsequent biochemical analysis reveal key details of substrate-binding residues and substrate specificity. These structures should facilitate structure-guided design of inhibitors against CnTps1.

biochemistry↗

bioRxiv2022

Single-cell genome-wide association reveals a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus

Diversity in the human genome is one factor that confers resistance and susceptibility to infectious diseases. This is observed most dramatically during pandemics, where individuals exhibit large differences in risk and clinical outcomes against a pathogen infecting large portions of the worlds populations. Here, we developed scHi-HOST (single cell High-throughput Human in vitrO Susceptibility Testing), a method for rapidly identifying genetic variants that confer resistance and susceptibility to pathogens. scHi-HOST leverages scRNA-seq (single-cell RNA-sequencing) to simultaneously assign genetic identity to individual cells in mixed infections of cell lines of European, African, and Asian origin, reveal associated genetic variants for viral entry and replication, and identify expression quantitative trait loci (eQTLs). Applying scHi-HOST to influenza A virus (IAV), we identified eQTLs at baseline and in genes that are induced by IAV infection. Integration of scHi-HOST with a human IAV challenge study (Prometheus) revealed that a missense variant in ERAP1 (Endoplasmic reticulum aminopeptidase 1; rs27895) was associated with IAV burden in cells and human volunteers. Functional studies using RNA interference, ERAP1 inhibitor, and overexpression of alternative alleles demonstrated that ERAP1 is exploited by IAV to promote infection. Specifically, the nonsynonymous substitution, which results in a glycine to aspartate substitution at ERAP1 residue 348, would disrupt the substrate binding pocket of ERAP1, likely resulting in a significantly altered preference for substrates, poorer catalytic efficiency, or both. Finally, rs27895 exhibits substantial population differentiation, with the higher frequency of the minor T allele in two African populations likely contributing to the greater permissivity of cells from these populations to IAV infection. scHi-HOST is an important resource for understanding susceptibility to influenza and is a broadly applicable method for decoding human genetics of infectious disease.

genetics↗