Biology subjects

Brennan, R. G.

Publications and source records attributed to Brennan, R. G..

7 recordsLinked to original sources

bioRxiv2026

Mitochondrial cardiolipin sequestration of caspofungin underlies Cryptococcus neoformans inherent resistance and may contribute to cardiotoxicity

Cryptococcus exhibits inherent resistance to the echinocandin, caspofungin, which inhibits the synthesis of (1,3)-{beta}-D-glucan, a key component of the polysaccharide cell wall. The essential FKS1 gene encodes the catalytic subunit of (1,3)-{beta}-D-glucan synthase and caspofungin effectively inhibits its activity in vitro, yet the drug remains ineffective against Cryptococcus, suggesting mechanisms beyond target insensitivity. The underlying mechanisms of caspofungin resistance remain unknown, although altered regulation of cell-wall remodeling genes, plasma membrane modifications, drug efflux pathways, and melanin biosynthesis have been suggested. Using boron dipyrromethene (BD-) and fluorescein (F-) labelled caspofungin, we demonstrate that caspofungin enters the cryptococcal cell and primarily accumulates in the mitochondrial inner membrane rather than the plasma membrane. We further establish that this mitochondrial accumulation is driven by a specific interaction between caspofungin and cardiolipin, a phospholipid found in mitochondrial membranes. We demonstrate that this unforeseen localization indicates that mitochondrial sequestration diminishes the effective drug concentrations at the intended target. Notably, the interaction between caspofungin and cardiolipin also takes place in human cells, establishing a mechanistic connection to caspofungin-related cardiotoxicity. Our findings reveal a previously unrecognized mechanism of echinocandin resistance in Cryptococcus and emphasize cardiolipin as an important factor in caspofungin effectiveness.

microbiology↗

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

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

Hormonal steroids bind the Neisseria gonorrhoeae multidrug resistance regulator, MtrR, to induce a multidrug binding efflux pump and stress-response sigma factor

Overexpression of the multidrug efflux pump MtrCDE, a critical factor of multidrug-resistance in Neisseria gonorrhoeae, the causative agent of gonorrheae, is repressed by the transcriptional regulator, MtrR (multiple transferable resistance repressor). Here, we report the results from a series of in vitro experiments to identify innate, human inducers of MtrR and to understand the biochemical and structural mechanisms of the gene regulatory function of MtrR. Isothermal titration calorimetry experiments reveal that MtrR binds the hormonal steroids progesterone, {beta}-estradiol, and testosterone, all of which are present at significant concentrations at urogenital infection sites as well as ethinyl estrogen, a component of some birth control pills. Binding of these steroids results in decreased affinity of MtrR for cognate DNA, as demonstrated by fluorescence polarization-based assays. The crystal structures of MtrR bound to each steroid provided insight into the flexibility of the binding pocket, elucidated specific residue-ligand interactions, and revealed the conformational consequences of the induction mechanism of MtrR. Three residues, D171, W136 and R176 are key to the specific binding of these gonadal steroids. These studies provide a molecular understanding of the transcriptional regulation by MtrR that promotes N. gonorrhoeae survival in its human host.

biochemistry↗

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↗

bioRxiv2020

The nucleotide messenger (p)ppGpp is a co-repressor of the purine synthesis transcription regulator PurR in Firmicutes

The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Yet despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria. Here we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes from soil bacteria to pathogens: (p)ppGpp serves as a co-repressor of the transcription factor PurR to downregulate purine biosynthesis. We identified PurR as a receptor of (p)ppGpp in Bacillus anthracis and revealed that (p)ppGpp strongly enhances PurR binding to its regulon in the Bacillus subtilis genome. A co-structure reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of PRT enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant fails to downregulate purine synthesis genes upon starvation. Our work establishes precedent of (p)ppGpp as a classical transcription co-repressor and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from the human intestinal tract to host-pathogen interfaces.

microbiology↗