Biology subjects

Hyser, J. M.

Publications and source records attributed to Hyser, J. M..

11 recordsLinked to original sources

bioRxiv2026

The L-type voltage-gated Ca2+ channel CaV1.2 regulates fibroblast-like synoviocytes invasiveness during rheumatoid arthritis

Fibroblast-like synoviocytes (FLS) are major drivers of synovial tissue inflammation and damage during rheumatoid arthritis (RA). A hallmark of the aggressiveness of RA-FLS is their invasiveness but the molecular mechanisms that regulate this invasiveness remain poorly understood. The KCa1.1 (BK, Slo1, MaxiK, KCNMA1) potassium channel is a major regulator of RA-FLS pathogenicity and inhibiting KCa1.1 expression or its activity with selective blockers reduces RA-FLS invasiveness and reduces disease severity and joint damage in rat models of RA. KCa1.1 blockade induces a Ca2+ influx in RA-FLS whose intracellular signaling results in increased cell adhesion to the extracellular matrix and thus a reduction in cell migration. However, the identity of the Ca2+-permeable channel responsible for the Ca2+ influx has remained elusive. Here, we demonstrate the co-expression of KCa1.1 with the L-type voltage-gated CaV1.2 channels at the plasma membrane of primary human and rat FLS. Selective blocking of the CaV1.2 channel blunts the Ca2+ influx induced by KCa1.1 blockade and abrogates the effects of KCa1.1 blockade on RA-FLS invasiveness, migration, cell shape, and podoplanin expression. Whereas KCa1.1 blockers reduce disease severity in rat models of RA, treating rats with the collagen-induced arthritis model of RA with both blockers of KCa1.1 and CaV1.2 eliminates the therapeutic benefits of treatment with KCa1.1 blockers alone. Together, these data demonstrate a physical and functional coupling of KCa1.1 and CaV1.2 channels in FLS during RA and its rat model that regulate the invasive phenotype of the FLS through modulation of intracellular Ca2+ homeostasis.

cell biology↗

bioRxiv2026

Development of fluorescent and affinity tagged variants of rotavirus nonstructural protein 4 using the reverse genetics system

Rotavirus (RV) nonstructural protein 4 (NSP4) is a multifunctional viroporin and enterotoxin that also serves an essential structural role in virion assembly, making it both a central virulence factor and a potential therapeutic target. However, studying NSP4 within infectious virus has been hindered by essential RNA packaging signals at the 5' end of gene segment 10 (gs10) and an DLP-binding domain at its C-terminus, both of which constrain conventional mutagenesis. Using a plasmid-based reverse genetics system, we generated six recombinant rotaviruses (rRVs) encoding tagged or reporter-fused NSP4 via three strategies: expression from an alternative segment (gs7) in a bicistronic arrangement, N-terminal tagging of gs10 with translation shifted downstream of the native packaging sequence, and retention of the gs10 5' UTR and fusion of the first 20 amino acids fused to a reporter followed by bicistronic full-length NSP4 expression. All six rRVs were replication competent, with gs10-based constructs closely matching wild-type replication kinetics while gs7-based constructs showed modest attenuation and, in one case, genetic instability upon passage. Engineered NSP4 proteins were robustly expressed, correctly glycosylated, and capable of oligomerization. Live-cell imaging showed reporter fluorescence reliably tracked NSP4 synthesis and preceded NSP4-dependent intercellular calcium signals. Affinity purification of tagged NSP4 recovered viroplasm-associated and structural viral proteins along with candidate host interactors, including ANP32A, ANP32E, PPM1G, and H2AC1. These rRVs constitute a validated toolkit for dissecting NSP4 function during bona fide infection and establish a generalizable strategy for engineering constrained rotavirus gene segments.

microbiology↗

bioRxiv2025

The Orai channel inhibitor BTP2 restricts Tulane virus and human norovirus replication independent of store-operated calcium entry

Human norovirus is the leading cause of viral gastroenteritis across all age groups. While there is a need for human norovirus antivirals, therapeutic development has been hindered by a lack of cell culture systems and animal models of infection. Surrogate viruses, such as Tulane virus (TV), have provided tractable systems to screen potential antiviral compounds. Our previous work demonstrated that Tulane virus encodes a viral ion channel, which dysregulates cytosolic calcium signaling. We set out to investigate whether host pathways triggered by viral ion channel activity, including store-operated calcium entry (SOCE), play a role in virus replication. Using pharmacologic inhibitors and genetically engineered cell lines, we establish that the SOCE inhibitor, BTP2, reduces TV replication in an SOCE-independent manner. We observed a significant reduction in TV replication, protein expression, and RNA synthesis in cells with both pre- and post-infection BTP2 treatment. By serial passage and plaque isolation, we demonstrate that TV quasi-species have mixed susceptibility and resistance to BTP2. Sequence comparison of the quasi-species revealed that amino acid changes in the structural proteins were associated with drug resistance. We utilized reverse genetics to generate TV with the resistance-associated VP1 and VP2 amino acid changes and found that a single amino acid change in VP1 (I380M) conferred BTP2 resistance. Further, expression of resistant VP2 alone was sufficient to partially rescue the replication of susceptible virus. Together, this supports that TV structural proteins are the targets of BTP2. Finally, using human intestinal organoids, we demonstrate that BTP2 significantly reduces human norovirus replication. ImportanceOur work identifies BTP2 as a potential human norovirus antiviral pharmacophore and highlights the utility of targeting calicivirus structural proteins to restrict viral replication. Further, we establish a system whereby Tulane virus can be used to screen novel antiviral candidates and establish their mechanism of action. Together, this will facilitate rapid preclinical validation of other novel human norovirus therapeutics.

microbiology↗

bioRxiv2024

Viroporin activity from rotavirus nonstructural protein 4 induces intercellular calcium waves that contribute to pathogenesis

Acute gastroenteritis remains the second leading cause of death among children under the age of 5 worldwide. While enteric viruses are the most common etiology, the drivers of their virulence remain incompletely understood. We recently found that cells infected with rotavirus, the most prevalent enteric virus in infants and young children, initiate hundreds of intercellular calcium waves that enhance both fluid secretion and viral spread. Understanding how rotavirus triggers intercellular calcium waves may allow us to design safer, more effective vaccines and therapeutics, but we still lack a mechanistic understanding of this process. In this study, we used existing virulent and attenuated rotavirus strains, as well as reverse engineered recombinants, to investigate the role of rotavirus nonstructural protein 4 (NSP4) in intercellular calcium wave induction using in vitro, organoid, and in vivo model systems. We found that the capacity to induce purinergic intercellular calcium waves (ICWs) segregated with NSP4 in both simian and murine-like rotavirus backgrounds, and NSP4 expression alone was sufficient to induce ICWs. NSP4s ability to function as a viroporin, which conducts calcium out of the endoplasmic reticulum, was necessary for ICW induction. Furthermore, viroporin activity and the resulting ICWs drove transcriptional changes indicative of innate immune activation, which were lost upon attenuation of viroporin function. Multiple aspects of RV disease severity in vivo correlated with the generation of ICWs, identifying a critical link between viroporin function, intercellular calcium waves, and enteric viral virulence.

microbiology↗

bioRxiv2023

The Inositol Trisphosphate Receptor (IP3R) is Dispensable for Rotavirus-induced Ca2+ Signaling and Replication but Critical for Paracrine Ca2+ Signals that Prime Uninfected Cells for Rapid Virus Spread

Rotavirus is a leading cause of viral gastroenteritis. A hallmark of rotavirus infection is an increase in cytosolic Ca2+ caused by the nonstructural protein 4 (NSP4). NSP4 is a viral ion channel that releases Ca2+ from the endoplasmic reticulum (ER) and the increase in Ca2+ signaling is critical for rotavirus replication. In addition to NSP4 itself, host inositol 1,4,5- trisphosphate receptor (IP3R) ER Ca2+ channels may contribute to rotavirus-induced Ca2+ signaling and by extension, virus replication. Thus, we set out to determine the role of IP3R Ca2+ signaling during rotavirus infection using IP3R-knockout MA104-GCaMP6s cells (MA104- GCaMP6s-IP3R-KO), generated by CRISPR/Cas9 genome editing. Live Ca2+ imaging showed that IP3R-KO did not reduce Ca2+ signaling in infected cells but eliminated rotavirus-induced intercellular Ca2+ waves (ICWs) and therefore the increased Ca2+ signaling in surrounding, uninfected cells. Further, MA104-GCaMP6s-IP3R-TKO cells showed similar rotavirus susceptibility, single-cycle replication, and viral protein expression as parental MA104- GCaMP6s cells. However, MA104-GCaMP6s-IP3R-TKO cells exhibited significantly smaller rotavirus plaques, decreased multi-round replication kinetics, and delayed virus spread, suggesting that rotavirus-induced ICW Ca2+ signaling stimulates virus replication and spread. Inhibition of ICWs by blocking the P2Y1 receptor also resulted in decreased rotavirus plaque size. Conversely, exogenous expression of P2Y1 in LLC-MK2-GCaMP6s cells, which natively lack P2Y1 and rotavirus ICWs, rescued the generation of rotavirus-induced ICWs and enabled plaque formation. In conclusion, this study shows that NSP4 Ca2+ signals fully support rotavirus replication in individual cells; however, IP3R is critical for rotavirus-induced ICWs and virus spread by priming Ca2+-dependent pathways in surrounding cells. ImportanceMany viruses exploit host Ca2+ signaling to facilitate their replication; however, little is known about how distinct types of Ca2+ signals contribute to the overall dysregulation of Ca2+ signaling or promote virus replication. Using cells lacking IP3R, a host ER Ca2+ channel, we could differentiate between intracellular Ca2+ signals within virus-infected cells and intercellular Ca2+ waves (ICWs), which increase Ca2+ signaling in neighboring, uninfected cells. In infected cells, IP3R was dispensable for rotavirus-induced Ca2+ signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IP3R-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca2+ signals from the ICWs may prime nearby uninfected cells to better support virus replication upon eventual infection. This "pre-emptive priming" of uninfected cells by exploiting host intercellular pathways in the vicinity of virus-infected cells represents a novel mechanism for viral reprogramming of the host to gain a replication advantage.

microbiology↗

bioRxiv2022

A bioengineered probiotic for the oral delivery of a peptide Kv1.3 channel blocker to treat rheumatoid arthritis

Engineered microbes for the delivery of biologics is a promising avenue for the treatment of various conditions such as chronic inflammatory disorders and metabolic disease. In this study, we developed a genetically engineered probiotic delivery system that delivers the small molecular biologic to the intestinal tract with high efficacy and minimized side effects. We constructed an inducible system in the probiotic Lactobacillus reuteri to secret functional Kv1.3 potassium blocker ShK-235 (LrS235). We show that LrS235 is capable of blocking Kv1.3 currents and preferentially inhibiting human T effector memory (TEM) cells proliferation in vitro. A single oral gavage of healthy rats with LrS235 resulted in adequate functional ShK-235 in the circulation to reduce inflammation in a delayed-type hypersensitivity model of atopic dermatitis mediated by TEM cells. Furthermore, the daily oral gavage of LrS235 dramatically reduced clinical signs of disease and joint inflammation in rats with a model of rheumatoid arthritis without eliciting immunogenicity against ShK-235. This work demonstrates the efficacy of using probiotic L. reuteri as a novel oral delivery platform for the small molecule ShK-235, and provides a efficacious strategy to deliver other biologics with great translational potential. Significance StatementNew therapeutics that combine efficacy with limited side effects and can be delivered non-invasively are needed to adequately treat patients with rheumatoid arthritis (RA) and other autoimmune diseases. Kv1.3 channel-expressing CCR7- effector memory T (TEM) lymphocytes are significant players in the pathogenesis of multiple autoimmune diseases and blocking Kv1.3 reduces disease severity in rat models of RA and patients with plaque psoriasis. However, peptide therapeutics require repeated injections, reducing patient compliance. We used a bioengineered Lactobacillus reuteri as an oral delivery method of a Kv1.3 blocker for immunomodulation in rat models of atopic dermatitis and RA. This study demonstrates a novel approach for the non-invasive delivery of peptide-based therapeutics for the oral treatment of chronic inflammatory diseases.

synthetic biology↗

bioRxiv2020

ROTAVIRUS INDUCES INTERCELLULAR CALCIUM WAVES THROUGH ADP SIGNALING

Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains incompletely characterized. Here we show that rotavirus-infected cells produce paracrine signals that manifest as intercellular calcium waves (ICWs); which are observed in both cell lines and human intestinal enteroids. Rotavirus ICWs are caused by the release of extracellular adenosine diphosphate (ADP) that activates P2Y1 purinergic receptors on neighboring cells and are blocked by P2Y1 antagonists or CRISPR/Cas9 knockout of P2Y1. Blocking the paracrine ADP signal reduces rotavirus replication, inhibits rotavirus-induced serotonin release and fluid secretion, and reduces diarrhea severity in neonatal mice. This is the first evidence that viruses exploit ICWs to amplify diarrheal signaling; a finding which has broad implications for gastrointestinal physiology. SummaryRotavirus triggers the extracellular release of ADP from infected cells to dysregulate nearby uninfected cells and activate pro-disease pathways.

microbiology↗

bioRxiv2019

Recovirus NS1-2 has viroporin activity that induces aberrant cellular calcium signaling to facilitate virus replication

Enteric viruses in the Caliciviridae family cause acute gastroenteritis in humans and animals, but the cellular processes needed for virus replication and disease remain unknown. A common strategy among enteric viruses, including rotaviruses and enteroviruses, is to encode a viral ion channel (i.e., viroporin) that is targeted to the endoplasmic reticulum (ER) and disrupts host calcium (Ca2+) homeostasis. Previous reports have demonstrated genetic and functional similarities between the nonstructural proteins of caliciviruses and enteroviruses, including the calicivirus NS1-2 protein and the 2B viroporin of enteroviruses. However, it is unknown whether caliciviruses alter Ca2+ homeostasis for virus replication or whether the NS1-2 protein has viroporin activity like its enterovirus counterpart. To address these questions, we used Tulane virus (TV), a rhesus enteric calicivirus, to examine Ca2+ signaling during infection and determine whether NS1-2 has viroporin activity that disrupts Ca2+ homeostasis. We found that TV disrupts increases Ca2+ signaling during infection and increased cytoplasmic Ca2+ levels is important for efficient replication. Further, TV NS1-2 localizes to the endoplasmic reticulum (ER), the predominant intracellular Ca2+ store and the NS2 region has characteristics of a viroporin domain (VPD). NS1-2 had viroporin activity in a classic bacterial functional assay and caused aberrant Ca2+ signaling when expressed in mammalian cells, but truncation of the VPD abrogated these functions. Together, our data provide new mechanistic insights into the function of the NS2 region of NS1-2 and show that like many other enteric viruses, enteric caliciviruses also exploit host Ca2+ signaling to facilitate their replication.\n\nImportanceTulane virus is one of many enteric caliciviruses that cause acute gastroenteritis and diarrheal disease. Globally, enteric caliciviruses affect both humans and animals and result in >65 billion dollars per year in treatment and healthcare-associated costs, thus imposing an enormous economic burden. Recent progress has resulted in several cultivation systems (B cell, enteroid and zebrafish larvae) to study human noroviruses, but mechanistic insights into the viral factors and host pathways important for enteric calicivirus replication and infection are largely still lacking. Here we used Tulane virus, a calicivirus that is biologically similar to human noroviruses and can be cultivated in conventional cell culture, to identify and functionally validate NS1-2 as an enteric calicivirus viroporin. Viroporin-mediated calcium signaling may be a broadly utilized pathway for enteric virus replication, and its existence within caliciviruses provides a novel approach to developing antivirals and comprehensive therapeutics for enteric calicivirus diarrheal disease outbreaks.

microbiology↗

bioRxiv2019

Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum

Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating cytosolic calcium ([Ca2+]cyt) and decreasing endoplasmic reticulum (ER) stores. While an overall, monophasic increase in [Ca2+]cyt during RV infection has been shown, the nature of the RV-induced aberrant calcium signals and how they manifest over time at the single-cell level have not been characterized. Thus, we generated cell lines and human intestinal enteroids (HIEs) stably expressing cytosolic and/or ER-targeted genetically-encoded calcium indicators to characterize calcium signaling throughout RV infection by time-lapse imaging. We found that RV induces highly dynamic [Ca2+]cyt signaling that manifest as hundreds of discrete [Ca2+]cyt spikes, which increase during peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca2+]cyt spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced [Ca2+]cyt spikes were primarily from ER calcium release and were attenuated by inhibiting the store-operated calcium entry (SOCE) channel Orai1. RV-infected HIEs also exhibited prominent [Ca2+]cyt spikes that were attenuated by inhibiting SOCE, underlining the relevance of these [Ca2+]cyt spikes to gastrointestinal physiology and role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca2+]cyt by dynamic Ca2+ signaling, establishes a new, paradigm-shifting understanding of the spatial and temporal complexity of virus-induced Ca2+ signaling.

microbiology↗

bioRxiv2019

Human intestinal enteroids with inducible neurogenin-3 expression as a novel model of gut hormone secretion

BackgroundEnteroendocrine cells (EECs) are specialized epithelial cells that produce molecules vital for intestinal homeostasis, but due to their limited numbers, in-depth functional studies have remained challenging. Human intestinal enteroids (HIEs) that are derived from intestinal crypt stem cells are a biologically relevant in vitro model of the intestinal epithelium. HIEs contain all intestinal epithelial cell types; however, like the intestine, HIEs spontaneously produce few EECs, which limits their study.\n\nMethodsTo increase the number of EECs in HIEs, we used lentivirus transduction to stably engineer jejunal HIEs with doxycycline-inducible expression of neurogenin-3 (NGN3), a transcription factor that drives EEC differentiation (tetNGN3-HIEs). We examined the impact of NGN3 induction on EECs by quantifying the increase in the enterochromaffin cells and other EEC subtypes. We functionally assessed secretion of serotonin and EEC hormones in response to norepinephrine and rotavirus infection.\n\nResultsTreating tetNGN3-HIEs with doxycycline induced a dose-dependent increase of chromogranin A (ChgA)-positive and serotonin-positive cells, demonstrating increased enterochromaffin cell differentiation. Despite increased ChgA-positive cells, other differentiated cell types of the epithelium remained largely unchanged by gene expression and immunostaining. RNA sequencing of doxycycline-induced tetNGN3- HIEs identified increased expression of key hormones and enzymes associated with several other EEC subtypes. Doxycycline-induced tetNGN3-HIEs secreted serotonin, monocyte chemoattractant protein-1, glucose-dependent insulinotropic peptide, peptide YY, and ghrelin in response to norepinephrine and rotavirus infection, further supporting the presence of multiple EEC types.\n\nConclusionsWe have combined HIEs and inducible-NGN3 expression to establish a flexible in vitro model system for functional studies of EECs in enteroids and advance the molecular and physiological investigation of EECs.\n\nSynopsisEnteroendocrine cells have low abundance but exert widespread effects on gastrointestinal physiology. We engineered human intestinal enteroids with inducible expression of neurogenin-3, resulting in increased enteroendocrine cells and facilitating investigations of host responses to the dynamic intestinal environment.

microbiology↗

bioRxiv2018

Discovery of a bacterial peptide as a modulator of GLP-1 and metabolic disease

Early work in germ-free rodents highlighted the gut microbiotas importance in metabolic disease, including Type II Diabetes Mellitus (T2DM) and obesity. Glucagon-like peptide-1 (GLP-1) is an incretin secreted by enteroendocrine L-cells lining the gastrointestinal epithelium. GLP-1 has important functions including promoting insulin secretion, insulin sensitivity, and {beta}-cell mass, while inhibiting gastric emptying and appetite. We set out to elucidate how the microbiota can modulate GLP-1 secretion, with the goal to identify microbial strains with GLP-1 stimulatory activity as a metabolic disease therapeutic. Over 1500 human-derived strains were isolated from fecal, breast milk, and colon and intestinal biopsy samples from healthy individuals. In vitro screening for GLP-1 modulation was performed by incubating bacterial cell-free supernatants with NCI H716 human L-cells. Approximately 45 strains capable of increasing GLP-1 levels, measured by ELISA, were discovered. Interestingly, all positive strains were identified as Staphylococcus epidermidis by 16S rRNA sequencing. Non-GLP-1 stimulatory S. epidermidis strains were also identified. Mass spectrometry analysis identified a 3 kDa peptide, termed GLP-1 stimulating peptide (GspA), present in GLP-1 positive but absent in GLP-1 neutral S. epidermidis. Studies in human L-cells and intestinal enteroids demonstrated that GspA alone is sufficient to enhance GLP-1 secretion. When administered in high-fat-fed mice, GspA-producing S. epidermidis significantly reduced markers associated with obesity and T2DM, including adiposity and hyperinsulinemia. Further characterization of GspA suggests a GLP-1 stimulatory action via calcium signaling. The presented results identify a novel host-microbe interaction which may ultimately lead to the development of a microbial peptide-based therapeutic for obesity and T2DM.\n\nImportanceThe human gastrointestinal microbiota has been shown to modulate metabolic disease, including Type II Diabetes Mellitus and obesity, through mechanisms involving gut hormone secretion. We initiated this study to identify bacterial strains that can stimulate one of these hormones, glucagon-like peptide-1. We first identified that some strains of Staphylococcus epidermidis have such stimulatory activity. We then found that these strains could be used in a mouse model of high-fat feeding to reduce markers associated with metabolic disease, including adiposity and elevated insulin levels. We also identified the peptide from S. epidermidis that stimulates glucagon-like peptide-1 and propose a mode of action through calcium signaling. This newly identified microbial-derived peptide and host-microbe interaction provide a promising therapeutic approach against Type II Diabetes Mellitus and obesity.

microbiology↗