Biology subjects

Beatty, W.

Publications and source records attributed to Beatty, W..

16 recordsLinked to original sources

bioRxiv2025

Signal peptidase complex mediates rotavirus VP7 processing and virion assembly

For viruses that replicate in the proximity of or bud at the endoplasmic reticulum (ER) associated membranes, proper processing of their glycoproteins is critical for successful infection. Rotavirus outer capsid protein VP7 is an ER-resident protein. However, its N-terminal signal peptide is removed by an unknown proteolytic mechanism. In this study, we leveraged tandem affinity purification followed by high-resolution mass spectrometry to profile host proteins that interact with VP7. We identified members of the signal peptidase complex (SPC) family as important host factors that facilitate rotavirus infection. CRISPR knockout or siRNA knockdown of distinct SPC subunits resulted in significant decrease in infectious rotavirus titers in a viral strain- and cell type-independent manner. While viral transcription, translation, and replication were not altered in the absence of SPC, we observed formation of abnormal viral particles by transmission electron microscopy (TEM) in SPCS1 knockout cells. Mechanistically, loss of SPC proteins led to inefficient cleavage of VP7 signal peptide and severely impaired the final steps of virion maturation and assembly. Additionally, we identified residue E256 within VP7 as a key site for SPC binding. An E to R mutation abolished VP7 interaction with SPC and subsequently led to reduced viral infectivity. Taken together, these findings define SPC as a novel regulator of VP7 maturation and rotavirus assembly and highlight its role as a novel cellular target for potentially broad-spectrum antiviral therapeutic development. Author summaryPrevious reports demonstrated that SPC is involved in the replication cycles of several members of the flavivirus family and human T-cell leukemia virus type 1 (HTLV-1). As an ER-resident outer capsid protein, rotavirus VP7 must undergo proper post-translational modifications, including the cleavage of its N-terminal signal peptide, to be functionally incorporated into mature virions. However, the processing mechanism remains unknown. For the first time, our study identifies SPC as an essential regulator of rotavirus assembly by mediating the cleavage of the VP7 signal peptide. Loss of SPC impairs VP7 signal peptide cleavage and maturation, thereby disrupting correct virion assembly and reducing infectious particle production. Using Alphafold3, we predicted the VP7 residue E256 to be at the binding interface with SPC complex. Experimentally, mutation of glutamic acid to arginine (E256R) substantially weakens this interaction and results in reduced viral propagation. Our findings unveil a novel post-translational checkpoint in rotavirus regulated by SPC and underscore the promise of SPC as a broad-spectrum antiviral target, especially for rotavirus, flavivirus, and HTLV-1, whose viral glycoproteins and structural proteins require SPC processing for proper maturation.

microbiology↗

bioRxiv2025

Dual Membrane-spanning Anti-Sigma 2 Controls OMV biogenesis and Colonization Fitness in Bacteroides thetaiotaomicron

Bacteroides spp. are Gram-negative, gut commensals that shape the enteric landscape by producing outer membrane vesicles (OMVs) that degrade dietary fibers and traffic immunomodulatory biomolecules. Understanding the mechanism behind OMV biogenesis in Bacteroides spp. is necessary to determine their role in the gut. Recent studies showed that mutation of Dual Membrane-spanning Anti-sigma factor 1 increased OMV production in Bacteroides thetaiotaomicron (Bt) by regulating members of its downstream regulon. Additional members of the Dma family have been identified, but very little is known regarding their roles in Bt. Here, we investigate the role of Dual Membrane-spanning Anti-sigma factor 2 (Dma2) in controlling OMV biogenesis in Bt. We employ biochemical and proteomic analyses to show that mutation of dma2 increases OMV production in a manner that is dependent on the expression of its cognate sigma factor, das2. The precise mechanism by which dma2 increases OMV biogenesis remains elusive. However, transcriptome analyses revealed that{Delta} dma2 has decreased expression of select polysaccharide utilization loci (PULs) that primarily target host-associated glycans. Follow-up comparative proteomics showed that the PUL repertoire was most impacted in the OMV fraction. In vitro growth assessments showed that{Delta} dma2 exhibits delayed growth in the presence of select host-associated glycans. Colonization studies in mice revealed that{Delta} dma2 is outcompeted by the wild-type in the gut, which indicates that dma2 is a key determinant of colonization fitness in Bt. Altogether, these findings expand our knowledge of the Dma familys role in OMV biogenesis and demonstrates their importance in Bacteroides physiology. ImportanceDual membrane-spanning anti-sigma factors (Dma) are a novel class of regulatory system found solely amongst Bacteroidota. Previous studies have demonstrated the role of Dma1 in vesiculation, but the overall role of the Dma family in Bacteroides physiology remains poorly understood. Here, we demonstrate that Dma2 modulates vesiculation and regulates the expression of select polysaccharide utilization loci (PULs) that target host-associated glycans. In vivo studies revealed that Dma2 is an important fitness determinant when competing against kin bacteria. This work begins characterizing the multifaceted involvement of Dma2 in OMV biogenesis, PUL regulation, and colonization fitness.

microbiology↗

bioRxiv2025

Induction of TFEB promotes Kupffer cell survival and reduces lipid accumulation and inflammation in MASLD

Kupffer cells (KCs) are the tissue-resident macrophage of the liver where they serve a critical role in maintaining liver tissue homeostasis and as a filter for circulation. The composition of liver macrophages changes during metabolic dysfunction-associated liver disease (MASLD), with the loss of resident KCs being a hallmark of disease progression. The mechanism(s) and consequences of KC death in metabolic liver disease have yet to be defined. Transcription factor EB (TFEB) is a master regulator of lysosome function and lipid metabolism which has been shown to protect macrophages from lipid stress in atherosclerosis. We hypothesized that TFEB would improve KC fitness in MASLD. To investigate this possibility, we created a transgenic mice in which TFEB was induced specifically in KCs. We found that TFEB induction protected KCs from cell death in two mouse models of MASLD. KC preservation through TFEB induction reduced liver steatosis via a mechanism that was dependent on macrophage lysosomal lipolysis and mitochondrial fatty acid oxidation. The protection from cell death in TFEB KCs was the result of reduced oxidative stress and ferroptosis through a mechanism that involved enhanced NADPH levels. Together, we provide evidence that TFEB promotes KC fitness during MASLD and orchestrates beneficial effects on liver pathology, thus providing potential targets to develop cell-specific therapeutics.

immunology↗

bioRxiv2025

Stathmin 1 regulates mitophagy and cellular function in hematopoietic stem cells

Stathmin 1 is a cytoplasmic phosphoprotein that regulates microtubule dynamics via promotion of microtubule catastrophe and sequestration of free tubulin heterodimers. Stathmin 1 is highly expressed in hematopoietic stem cells (HSCs), and overexpressed in leukemic cells, however its role in HSCs is not known. Herein, we found that loss of Stathmin 1 is associated with altered microtubule architecture in HSCs, and markedly impaired HSC function. Transcriptomic studies suggested alterations in oxidative phosphorylation in Stmn1-/- HSCs, and further mechanistic studies revealed defective mitochondrial structure and function in the absence of Stathmin 1 with increased ROS production. Microtubules associate with mitochondria and lysosomes to facilitate autophagosome formation and mitophagy, and indeed we found that this critical mitochondrial quality control process is impaired in Stathmin 1-deficient HSCs. Finally, stimulation of autophagy improved the colony forming ability of Stmn1-/- hematopoietic stem and progenitor cells. Together, our data identify Stathmin 1 as a novel regulator of mitophagy and mitochondrial health in HSCs. Key PointsO_LIThe microtubule regulating protein Stathmin 1 is highly expressed in HSPCs and promotes normal microtubule architecture. C_LIO_LILoss of Stathmin 1 in HSPCs leads to impaired autophagy with abnormal mitochondrial morphology, decreased respiratory capacity, and impaired cellular function. C_LI

cell biology↗

bioRxiv2024

Covalently linked adenovirus-AAV complexes as a novel platform technology for gene therapy

Adeno-associated virus (AAV) has found immense success as a delivery system for gene therapy, yet the small 4.7 kb packaging capacity of the AAV sharply limits the scope of its application. In addition, high doses of AAV are frequently required to facilitate therapeutic effects, leading to acute toxicity issues. While dual and triple AAV approaches have been developed to mitigate the packaging capacity problem, these necessitate even higher doses to ensure that co-infection occurs at sufficient frequency. To address these challenges, we herein describe a novel delivery system consisting of adenovirus (Ad) covalently linked to multiple adeno-associated virus capsids as a new way of more efficiently co-infecting cells with lower overall amounts of AAVs. We utilize the DogTag-DogCatcher (DgT-DgC) molecular glue system to construct our AdAAVs and we demonstrate that these hybrid virus complexes achieve enhanced co-transduction of cultured cells, including physiologically relevant primary cells. On this basis, AdAAV technology may eventually facilitate therapeutic co-delivery of multiple transgenes at low virus doses for treating complex ailments.

synthetic biology↗

bioRxiv2024

Host-derived CEACAM-laden vesicles engage enterotoxigenic E. coli for elimination and toxin neutralization.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections. Significance statementEnterotoxigenic E. coli, characterized by the production of heat-labile (LT) and heat-stable (ST) toxins, are a very common cause of diarrhea in low-income regions responsible for hundreds of millions of infections each year, and the major cause of diarrhea in travelers to endemic areas. Although these infections may be severe and cholera-like, they are typically self-limited. These studies demonstrate that extracellular vesicles produced by host intestinal cells can capture the bacteria and its secreted toxins at a distance from the cell surface, potentially acting as molecular decoys to neutralize the enterotoxins and extinguish the infection.

microbiology↗

bioRxiv2023

An inducible genetic tool for tracking and manipulating specific microglial states in development and disease

Recent single-cell RNA sequencing studies have revealed distinct microglial states in development and disease. These include proliferative region-associated microglia (PAM) in developing white matter and disease-associated microglia (DAM) prevalent in various neurodegenerative conditions. PAM and DAM share a similar core gene signature and other functional properties. However, the extent of the dynamism and plasticity of these microglial states, as well as their functional significance, remains elusive, partly due to the lack of specific tools. Here, we report the generation of an inducible Cre driver line, Clec7a-CreERT2, designed to target PAM and DAM in the brain parenchyma. Utilizing this tool, we profile labeled cells during development and in several disease models, uncovering convergence and context-dependent differences in PAM/DAM gene expression. Through long-term tracking, we demonstrate surprising levels of plasticity in these microglial states. Lastly, we specifically depleted DAM in cuprizone-induced demyelination, revealing their roles in disease progression and recovery.

neuroscience↗

bioRxiv2023

Encapsulation of AAV into protein vault nanoparticles as a novel solution to gene therapy's neutralizing antibody problem

Although adeno-associated virus (AAV) has enjoyed enormous success as a delivery modality for gene therapy, it continues to suffer from the high prevalence of preexisting neutralizing antibodies in human populations, limiting who can receive potentially life-saving treatments. In this regard, AAV therapies generally also must be administered as a single dose since neutralizing antibodies develop in patients who receive the virus. Strategies for circumventing these issues remain limited. As a novel solution, we employed SpyTag-SpyCatcher molecular glue technology to facilitate packaging of AAVs inside of recombinant protein vault nanoparticles. Vaults are endogenous particles produced by mammalian cells. We therefore hypothesized that they may shield packaged molecules from neutralizing antibodies. Vaults have previously been utilized to deliver drugs and proteins into cells, but our study represents the first time anyone has packaged an entire virus inside of a vault. We showed that our vaultAAV (VAAV) delivery vehicle transduces cells in the presence of anti-AAV neutralizing serum. VAAV is positioned as a new gene therapy delivery platform with potential to overcome the neutralizing antibody problem and perhaps even allow administration of multiple doses, expanding the scope of AAV treatments.

bioengineering↗

bioRxiv2023

Dual Membrane-Sspanning Anti-Sigma Factors Regulate Vesiculation in Gut Bacteroidota

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are at least partially mediated by Outer Membrane Vesicles (OMVs). In this work, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). Our screening led us to the identification of a novel family of Dual Membrane-spanning Anti-sigma factors (Dma), which regulate OMV biogenesis in Bt. We employed molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, results in hypervesiculation by modulating the expression of NigD1, which belongs to a family of uncharacterized proteins found throughout Bacteroidota. Dma1 has an unprecedented domain organization: it contains a C-terminal {beta}-barrel embedded in the OM; its N-terminal domain interacts with its cognate sigma factor in the cytoplasm, and both domains are tethered via an intrinsically disordered region that traverses the periplasm. Phylogenetic analyses reveal that the Dma family is a unique feature of Bacteroidota. This study provides the first mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

microbiology↗

bioRxiv2023

Multiple pathways for glucose phosphate transport and utilization support growth of Cryptosporidium parvum

Cryptosporidium parvum is an obligate intracellular parasite with a highly reduced mitochondrion that lacks the TCA cycle and the ability to generate ATP, making the parasite reliant on glycolysis. Genetic ablation experiments demonstrated that neither of the two putative glucose transporters CpGT1 and CpGT2 were essential for growth. Surprisingly, hexokinase was also dispensable for parasite growth while the downstream enzyme aldolase was required, suggesting the parasite has an alternative way of obtaining phosphorylated hexose. Complementation studies in E. coli support a role for direct transport of glucose-6-phosphate from the host cell by the parasite transporters CpGT1 and CpGT2, thus bypassing a requirement for hexokinase. Additionally, the parasite obtains phosphorylated glucose from amylopectin stores that are released by the action of the essential enzyme glycogen phosphorylase. Collectively, these findings reveal that C. parvum relies on multiple pathways to obtain phosphorylated glucose both for glycolysis and to restore carbohydrate reserves.

microbiology↗

bioRxiv2023

The combined impact of killer whale consumptive and non-consumptive effects on northern sea otter population viability in the Western Aleutians Archipelago, Alaska

Predators can alter the abundance, distribution, and behavior of prey populations through consumptive and non-consumptive effects. In the Aleutian Archipelago of Alaska, killer whales (Orcinus orca) are considered the most probable cause of northern sea otter (Enhydra lutris kenyoni) population declines in the southwestern Alaska stock, which led to their listing as a threatened distinct population segment under the Endangered Species Act. Much of the research attention in the Aleutian Archipelago region has focused on the consumptive effects of killer whales on sea otter population dynamics. Here, we explore non-consumptive effects by accounting for restricted sea otter habitat use within discrete predation refuges characterized by areas of shallow, complex reef habitats close to shore. We constructed Population Viability Analysis (PVA) models that incorporated sea otter count data collected by aerial and skiff-based methods over six decades (1959 - 2021) to inform uplisting (to endangered) and downlisting (delisting from ESA) criteria. Our models incorporated both density-dependent effects and density-independent effects on sex and age structure, which we termed predation hazards. Prior to 1990, predation hazards were negligible, fluctuated at high values between 1990 and the early 2000s, and then declined as sea otter populations reached low densities. We estimated a current regional abundance of 2,405 sea otters (95%CI = 1,734 - 3,238) in the Western Aleutians Management Unit. Our base PVA model that considered only inter-island fragmentation indicated the risk of the regional sea otter population becoming endangered was <5% when there were at least 1,500 otters (95%CI = 1,200 - 2,100), and provided a delisting threshold of 2,100 sea otters. A PVA model that accounted for restricted habitat use of sea otters within discrete predation refuges (i.e. inter-island and intra-island fragmentation) indicated a less encouraging potential for sea otter recovery. The probability of the population becoming endangered increased to >10% and the delisting threshold increased to >10,000 sea otters (nearly 5x higher). Our results indicate sea otters within fragmented predation refuges could be more susceptible to the effects of stochastic processes with potentially limited ability for rescue effects. Overall, our research reveals the importance of evaluating both consumptive and non-consumptive effects when considering conservation and management plans for at-risk populations thought to be limited by a predator.

ecology↗

bioRxiv2022

Type I IFN signaling mediates NET release to promote Mycobacterium tuberculosis replication and granuloma caseation

Neutrophils are the most abundant cell type in airways of tuberculosis patients. Recent investigations reported induction of neutrophil extracellular traps (NETs) during Mycobacterium tuberculosis (Mtb) infection, however, the molecular regulation and impact of NETosis on Mtb pathogenesis is unknown. We find that in response to Mtb infection in neutrophils, PAD4 citrullinates histones to decondense chromatin that gets packaged into vesicles for release as NETs in a manner that can maintain neutrophil viability and promote Mtb replication. Type I interferon, which has been associated with NETosis in numerous contexts but without a known mechanism, promotes formation of chromatin-containing vesicles and NET release. Analysis of nonhuman primate granulomas supports a model where neutrophils are exposed to type I interferon from macrophages as they migrate into the granuloma, where they release NETs that contribute to necrosis and caseation. Our data reveals NETosis as a promising target to inhibit Mtb replication and granuloma caseation.

microbiology↗

bioRxiv2022

Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small 1intestinal epithelia

Enterotoxigenic E. coli (ETEC), produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, and are a common cause of diarrhea in children of resource-poor regions. ETEC have also been linked repeatedly to poorly understood sequelae including enteropathy, malnutrition, and growth impairment. While the cellular actions of ETEC enterotoxins leading to diarrhea are well-established, their potential contribution to subsequent pathology is unclear. LT stimulates cellular cAMP production to activate protein kinase A (PKA) which phosphorylates cellular ion channels that drive export of salt and water into the intestinal lumen resulting in diarrhea. However, as PKA exhibits broad kinase activity and its activated catalytic subunits modulate transcription of many genes, we interrogated the transcriptional profiles of LT-treated small intestinal epithelia. These studies demonstrated toxin-induced changes in hundreds of genes including those required for biogenesis and function of the brush border, the major site absorption of nutrients, and suppression of a key transcription factors, HNF4 and SMAD4, critical to differentiation of intestinal epithelia. Accordingly, LT treatment of intestinal epithelial cells significantly disrupted the absorptive microvillus architecture and altered transport of essential nutrients. In addition, challenge of neonatal mice with LT-producing ETEC recapitulated the architectural derangement of the brush border while maternal vaccination with LT prevented brush border disruption in ETEC-challenged neonatal mice. Finally, mice repeatedly challenged with toxigenic ETEC exhibited impaired growth recapitulating the multiplicative impact of recurring ETEC infections in children. These findings highlight impacts of ETEC enterotoxins beyond acute diarrheal illness and may inform approaches to mitigate and prevent major sequelae including malnutrition that impact millions of young children.

microbiology↗

bioRxiv2022

Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions

For decades, investigators have studied the interaction of Mycobacterium tuberculosis (Mtb) with macrophages, which serve as a major cellular niche for the bacilli. Because Mtb are prone to aggregation, investigators rely on varied methods to disaggregate the bacteria for these studies. Here, we examined the impact of routinely used preparation methods on bacterial cell envelop integrity, macrophage inflammatory responses, and intracellular Mtb survival. We found that both gentle sonication and filtering damaged the mycobacterial cell envelope and markedly impacted the outcome of macrophage infections. Unexpectedly, sonicated bacilli were hyperinflammatory, eliciting dramatically higher TLR2-dependent gene expression and elevated secretion of IL-1{beta} and TNF-. Despite evoking enhanced inflammatory responses, sonicated bacilli replicated normally in macrophages. In contrast, Mtb that had been passed through a filter induced little inflammatory response, and they were attenuated in macrophages. Previous work suggests that the mycobacterial cell envelope lipid, phthiocerol dimycocerosate (PDIM), dampens macrophage inflammatory responses to Mtb. However, we found that the impact of PDIM depended on the method used to prepare Mtb. In conclusion, widely used methodologies to disaggregate Mtb may introduce experimental artifacts in Mtb-host interaction studies, including alteration of host inflammatory signaling, intracellular bacterial survival, and interpretation of bacterial mutants.

microbiology↗

bioRxiv2021

Modern Acinetobacter baumannii clinical isolates replicate inside spacious vacuoles and egress from macrophages

Multidrug-resistant Acinetobacter baumannii infections are increasing at alarming rates. Therefore, novel antibiotic-sparing treatments to combat these A. baumannii infections are urgently needed. The development of these interventions would benefit from a better understanding of this bacteriums pathobiology, which remains poorly understood. A. baumannii is regarded as an extracellular opportunistic pathogen. However, research on Acinetobacter has largely focused on common lab strains, such as ATCC 19606, that have been isolated several decades ago. These strains exhibit reduced virulence when compared to recently isolated clinical strains. In this work, we demonstrate that, unlike ATCC 19606, several modern A. baumannii clinical isolates, including the recent clinical urinary isolate UPAB1, persist and replicate inside macrophages within spacious vacuoles. We show that intracellular replication of UPAB1 is dependent on a functional type I secretion system (T1SS) and pAB5, a large conjugative plasmid that controls the expression of several chromosomally-encoded genes. Finally, we show that UPAB1 escapes from the infected macrophages by a lytic process. To our knowledge, this is the first report of intracellular growth and replication of A. baumannii. We suggest that intracellular replication within macrophages may contribute to evasion of the immune response, dissemination, and antibiotic tolerance of A. baumannii.

microbiology↗

bioRxiv2019

Encephalitic alphaviruses exploit caveolae-mediated transcytosis at the blood-brain barrier for CNS entry

Venezuelan and Western equine encephalitis viruses (VEEV and WEEV) invade the CNS early during infection, via neuronal and hematogenous routes (1, 2). While viral replication mediates host-shut off, including expression of type I interferons (IFN) (3, 4), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin (Cav)-1-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and non-replicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1-association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS, and that IFN differentially restricts this process at the BBB.\n\nImportanceVEEV, WEEV and EEEV are emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the blood-stream, however the underlying molecular mechanisms have remained largely unknown. Here we demonstrate that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPAses within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses.

microbiology↗