Biology subjects

Mast, F. D.

Publications and source records attributed to Mast, F. D..

15 recordsLinked to original sources

bioRxiv2025

Host oxidative stress primes mycobacteria for rapid antibiotic resistance evolution

The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) threatens global TB control, yet the mechanisms enabling rapid evolution of drug resistance in Mtb remain poorly understood. Here we reveal that pre-existing mutations in oxidative stress response genes create permissive genomic backgrounds that accelerate high-level isoniazid resistance (INHR) without fitness costs, challenging the paradigm that resistance mutations always precede their fitness compensatory adaptations. Using M. smegmatis mc2155 (Msm) as a model, we show that brief exposure to sublethal INH (2x IC50) enriches for "low-level resistance and tolerance" (LLRT) mutants in a single step. These LLRT mutants, particularly those with ohrR loss-of-function mutations, acquire high-level resistance (> 500x IC50) at 6-fold higher rates than wildtype, primarily through otherwise-deleterious mycothiol biosynthesis mutations that become tolerable in the oxidative stress-buffered background. Crucially, we demonstrate that sublethal oxidative stress alone, mimicking host immune pressure, nearly tripled the rate of INH resistance evolution in Msm. Bayesian analysis of 1,578 clinical Mtb isolates from Vietnam confirmed that mutations in oxidative stress response genes were significantly associated with the emergence of INHR strains (p-value = 1.09x10-7). Independently, reanalysis of genome-wide CRISPRi screens revealed that the OSR network and high Bayes probability genes are functionally associated with treatment escape and survival with multiple antibiotics, including isoniazid, rifampicin, ethambutol, bedaquiline, vancomycin, clarithromycin, linezolid, and streptomycin. Our findings that host-imposed oxidative stress and inadequate drug penetration may synergistically prime Mtb populations for rapid resistance evolution suggest that targeting pre-resistance mechanisms, such as oxidative stress defenses, could help slow the emergence of antibiotic resistance in tuberculosis.

microbiology↗

bioRxiv2025

A Conditional Cas9 System for Stage-Specific Gene Editing in P. falciparum

The malaria parasite has a complex lifecycle involving various host cell environments in both human and mosquito hosts. The parasite must tightly regulate gene expression at each stage in order to adapt to its current environment while continuing development. However, it is challenging to study gene function and regulation of essential genes across the parasites multi-host lifecycle. Thus, we adapted a recently developed a single-plasmid dimerizable Cre recombinase system for rapamycin-controllable expression of Cas9, allowing for conditional introduction of mutations. We explored rates of gene deletion using varying repair template lengths, showing functionality of donor templates under 250bp for homology-directed repair. As a proof of concept, we conditionally disrupted two uncharacterized genes in blood and gametocyte stages, identifying new stage-specific phenotypes. ImportanceAs progress towards eliminating malaria has stalled, there is a pressing need for new antimalarials and vaccines. Genes essential to multiple stages of development represent ideal candidates for both antimalarials and vaccines. However, much of the parasite genome remains uncharacterized. Conditional gene perturbation approaches are needed in order to study gene function across the lifecycle. Currently available tools are limited in their ability to perturb genes at the scale required for large screens. We describe a tool that allows for conditional introduction of desired mutations by controlling Cas9 with the DiCre-loxP system. We demonstrate the accessibility of this approach by designing gRNA-donor pairs that can be commercially synthesized. This toolkit provides a scalable system for identifying new drug and vaccine candidates targeting multiple stages of the parasite lifecycle

microbiology↗

bioRxiv2025

TOR and heat shock response pathways regulate peroxisome biogenesis during proteotoxic stress

Peroxisomes are versatile organelles mediating energy homeostasis and redox balance. While peroxisome dysfunction is linked to numerous diseases, the molecular mechanisms and signaling pathways regulating peroxisomes during cellular stress remain elusive. Using yeast, we show that perturbations disrupting protein homeostasis including loss of ER or cytosolic chaperone function, impairments in ER protein translocation, blocking ER N-glycosylation, or reductive stress, cause peroxisome proliferation. This proliferation is driven by increased de novo biogenesis from the ER as well as increased fission of pre-existing peroxisomes, rather than impaired pexophagy. Notably, peroxisome biogenesis is essential for cellular recovery from proteotoxic stress. Through comprehensive testing of major signaling pathways, we determine this response to be mediated by activation of the heat shock response and inhibition of Target of Rapamycin (TOR) signaling. Finally, the effects of proteotoxic stress and TOR inhibition on peroxisomes are also captured in human fibroblasts. Overall, our findings reveal a critical and conserved role of peroxisomes in cellular response to proteotoxic stress.

cell biology↗

bioRxiv2024

Systems-level reconstruction of kinase phosphosignaling networks regulating endothelial barrier integrity using temporal data

Phosphosignaling networks control cellular processes. We built kinase-mediated regulatory networks elicited by thrombin stimulation of brain endothelial cells using two computational strategies: Temporal Pathway Synthesizer (TPS), which uses phosphoproteomics data as input, and Temporally REsolved KInase Network Generation (TREKING), which uses kinase inhibitor screens. TPS and TREKING predicted overlapping barrier-regulatory kinases connected with unique network topology. Each strategy effectively describes regulatory signaling networks and is broadly applicable across biological systems.

systems biology↗

bioRxiv2024

Automated, image-based quantification of peroxisome characteristics with perox-per-cell

perox-per-cell automates cumbersome, image-based data collection tasks often encountered in peroxisome research. The software processes microscopy images to quantify peroxisome features in yeast cells. It uses off-the-shelf image processing tools to automatically segment cells and peroxisomes and then outputs quantitative metrics including peroxisome counts per cell and spatial areas. In validation tests, we found that perox-per-cell output agrees well with manually-quantified peroxisomal counts and cell instances, thereby enabling high-throughput quantification of peroxisomal characteristics. The software is available at https://github.com/AitchisonLab/perox-per-cell

bioinformatics↗

bioRxiv2023

Predicting host-based, synthetic lethal antiviral targets from omics data

Traditional antiviral therapies often have limited effectiveness due to toxicity and development of drug resistance. Host-based antivirals, while an alternative, may lead to non-specific effects. Recent evidence shows that virus-infected cells can be selectively eliminated by targeting synthetic lethal (SL) partners of proteins disrupted by viral infection. Thus, we hypothesized that genes depleted in CRISPR KO screens of virus-infected cells may be enriched in SL partners of proteins altered by infection. To investigate this, we established a computational pipeline predicting SL drug targets of viral infections. First, we identified SARS-CoV-2-induced changes in gene products via a large compendium of omics data. Second, we identified SL partners for each altered gene product. Last, we screened CRISPR KO data for SL partners required for cell viability in infected cells. Despite differences in virus-induced alterations detected by various omics data, they share many predicted SL targets, with significant enrichment in CRISPR KO-depleted datasets. Comparing data from SARS-CoV-2 and influenza infections, we found possible broad-spectrum, host-based antiviral SL targets. This suggests that CRISPR KO data are replete with common antiviral targets due to their SL relationship with virus-altered states and that such targets can be revealed from analysis of omics datasets and SL predictions.

bioinformatics↗

bioRxiv2023

Multiple Receptor Tyrosine Kinases Regulate Dengue Infection of Hepatocytes

Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 300 kinases are regulating DENV infection. Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of ERBB2, FGFR2 and IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.

systems biology↗

bioRxiv2023

Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast, Fridy et al. 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.

biochemistry↗

bioRxiv2022

The nucleoporin Nup170 mediates subtelomeric gene silencing through the Ctf18-RFC complex and PCNA

The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interaction, genetic interaction, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170. The Ctf18-RFC complex is recruited to a subpopulation of NPCs that lack the nuclear basket proteins Mlp1 and Mlp2. In the absence of Nup170, PCNA levels on DNA are reduced, resulting in the loss of silencing of subtelomeric genes. Increasing PCNA levels on DNA by removing Elg1, which is required for PCNA unloading, rescues subtelomeric silencing defects in nup170{Delta}. The NPC therefore mediates subtelomeric gene silencing by regulating PCNA levels on DNA.

cell biology↗

bioRxiv2021

Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2

Despite the great promise of vaccines, the COVID-19 pandemic is ongoing and future serious outbreaks are highly likely, so that multi-pronged containment strategies will be required for many years. Nanobodies are the smallest naturally occurring single domain antigen binding proteins identified to date, possessing numerous properties advantageous to their production and use. We present a large repertoire of high affinity nanobodies against SARS-CoV-2 Spike protein with excellent kinetic and viral neutralization properties, which can be strongly enhanced with oligomerization. This repertoire samples the epitope landscape of the Spike ectodomain inside and outside the receptor binding domain, recognizing a multitude of distinct epitopes and revealing multiple neutralization targets of pseudoviruses and authentic SARS-CoV-2, including in primary human airway epithelial cells. Combinatorial nanobody mixtures show highly synergistic activities, and are resistant to mutational escape and emerging viral variants of concern. These nanobodies establish an exceptional resource for superior COVID-19 prophylactics and therapeutics.

biochemistry↗

bioRxiv2020

A synthetic lethal interaction with a viral-induced hypomorph of GBF1

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal partners of cancer-specific mutations. Synthetic lethal interactions of viral-induced hypomorphs have the potential to be similarly targeted for the development of host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for synthetic lethal partners of GBF1 revealed ARF1 as the top hit, disruption of which, selectively killed cells that synthesize poliovirus 3A. Thus, viral protein interactions can induce hypomorphs that render host cells vulnerable to perturbations that leave uninfected cells intact. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2. SummaryUsing a viral-induced hypomorph of GBF1, Navare et al., demonstrate that the principle of synthetic lethality is a mechanism to selectively kill virus-infected cells.

molecular biology↗

bioRxiv2020

Dengue activates mTORC2 signaling to counteract apoptosis and maximize viral replication

The mechanistic target of rapamycin (mTOR) functions in at least two distinct complexes: mTORC1, which regulates cellular anabolic-catabolic homeostasis, and mTORC2, which is an important regulator of cell survival and cytoskeletal maintenance. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. In contrast, the role of mTORC2 in viral pathogenesis is unknown. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication.

microbiology↗

bioRxiv2020

A Genome-wide CRISPR-Cas9 Screen Identifies Host Factors Essential for Optimal Plasmodium Liver Stage Development.

Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources. We show that host microtubules dynamically reorganize around the developing liver stage (LS) parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, ER/Golgi stress and lipid biogenesis that regulate Plasmodium LS development. These novel regulators of infection, including Centromere Protein J (CENPJ), led us to interrogate how microtubule organizing centers (MTOCs) are regulated during infection. Foci of {gamma}-tubulin localized to the parasite periphery; depletion of CENPJ exacerbated this re-localization and increased infection. Further, we show that the Golgi acts as a non-centrosomal MTOC by organizing {gamma}-tubulin and stimulating microtubule nucleation at the parasite periphery. Collectively, we show that the Plasmodium LS recruits the host Golgi to form MT mediated conduits along which host organelles are recruited to the PVM, to support liver stage development. Our findings suggest many host-targeted pharmacological inhibitors may inhibit LS infection.

cell biology↗

bioRxiv2019

Plasmodium secretion induces hepatocyte lysosome exocytosis and promotes parasite entry

The invasion of a suitable host hepatocyte by Plasmodium sporozoites is an essential step in malaria infection. We demonstrate that in infected hepatocytes, lysosomes are redistributed away from the nucleus, and surface exposure of lysosomal-associated membrane protein (LAMP1) is increased. Lysosome exocytosis in infected cells occurs independently of sporozoite traversal. Instead, a sporozoite-secreted factor is sufficient for the process. Knockdown of the SNARE proteins involved in lysosome-plasma membrane fusion reduces lysosome exocytosis and Plasmodium infection. In contrast, promoting fusion between the lysosome and plasma membrane dramatically increases infection. Our work demonstrates new parallels between Plasmodium sporozoite entry of hepatocytes and infection by the excavate pathogen, Trypanosoma cruzi and raises the question of whether convergent evolution has shaped host cell invasion by divergent pathogens.

cell biology↗

bioRxiv2017

ESCRT-III acts in scissioning new peroxisomes from the ER

Dynamic control of peroxisome proliferation is integral to the peroxisomes many functions. A breakdown in the ability of cells to form peroxisomes is linked to many human health issues, including defense against infectious agents, cancer, aging, heart disease, obesity and diabetes, and forms the basis of a spectrum of peroxisomal genetic disorders that cause severe neuropathologies. The ER serves as a source for preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and to support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. Here we show that the evolutionarily ancient endosomal sorting complexes required for transport (ESCRT)-III are peroxisome biogenesis factors that function to cleave PPVs budding from the ER into the cytosol. Using comprehensive morphological and genetic assays of peroxisome formation and function we find that absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay we show that ESCRT-III proteins Vps20 and Snf7 are required to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and towards the cytosol, a finding that has important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.

cell biology↗