Biology subjects

Sanchez, E. L.

Publications and source records attributed to Sanchez, E. L..

7 recordsLinked to original sources

bioRxiv2026

Impact of Neurons on Group B Streptococcus Interactions at the Blood Brain Barrier

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive opportunistic pathogen and the leading cause of neonatal bacterial meningitis, a life-threatening infection of the central nervous system (CNS) that occurs when bacteria cross the blood-brain barrier (BBB). GBS commonly colonizes the maternal genital tract and is a major cause of invasive neonatal disease, including bacteremia and meningitis. Despite treatment advances, GBS meningitis remains associated with substantial mortality and long-term neurological sequelae. The BBB is a highly specialized barrier formed by brain endothelial cells (BECs) that restrict microbial entry into the CNS through tightly regulated intercellular junctions. The BBB exists within the neurovascular unit, where neurons and other CNS cell types actively regulate endothelial barrier properties through intercellular signaling. However, the contribution of neuronal-endothelial interactions to BBB function during neonatal meningitis remains poorly understood. To investigate the mechanisms by which GBS disrupts and penetrates the BBB, we utilized induced pluripotent stem cell (iPSC)-derived brain-like endothelial cells. EZ-Sphere-derived neurons generated from the same iPSC source were incorporated into an isogenic BBB model to determine whether neuronal-endothelial communication influences GBS interaction with BECs. Neuronal co-culture significantly reduced GBS adherence to and invasion of BECs while preserving tight junction integrity during infection. Application of neuron-conditioned medium similarly decreased bacterial adherence and invasion, suggesting that neuron-derived soluble factors enhance barrier integrity during GBS infection. Together, these findings demonstrate that neuronal signaling enhances BBB resistance to GBS and highlight a previously underappreciated role for neurovascular crosstalk in limiting bacterial pathogenesis. ImportanceGroup B Streptococcus (GBS) is the leading cause of bacterial meningitis in newborns. GBS interacts with and crosses the blood-brain barrier (BBB) contributing to a potentially fatal infection without treatment. Understanding how the BBB interacts with bacterial pathogens is critical for developing new strategies to protect vulnerable infants. In this study, we used human stem cell-derived models to recapitulate the BBB and examine how communication between brain endothelial cells and neurons influence host response to bacterial infection. These findings identify neuronal-endothelial communication as a potential contributor to BBB protection and provide a foundation for future studies aimed at preventing GBS invasion of the central nervous system.

microbiology↗

bioRxiv2026

Global Lipidomic Analysis of Lytic KSHV Infection: The Lipid Chaperone FABP4 is Required for Maximal Infectious Virion Production

Kaposis Sarcoma Herpesvirus (KSHV), an enveloped double-stranded DNA virus, is the etiological agent of Kaposis sarcoma (KS), an endothelial cell-based tumor. KSHV is a leading cause of infection-related cancers in sub-Saharan Africa and immunocompromised individuals worldwide. Therefore, it is vital to identify the underlying mechanisms of viral infection and transmission to effectively identify specific therapeutic strategies and combat the disease. Here, we demonstrate that KSHV rewires the host cell lipidome during lytic infection. Bulk lipidomic analysis shows significant changes in the abundance of neutral lipids and phospholipids during lytic infection. We further investigated fatty acid-binding proteins (FABPs) to understand the underlying mechanisms that support KSHV pathogenesis. Using the doxycyclin-inducible iSLK.BAC16 cell line, we find that FABP genes are differentially regulated by lytic KSHV infection compared to latent infection. We report that FABP4 is significantly upregulated during lytic infection. Loss of FABP4 during lytic infection does not impact viral gene transcription however, lytic protein translation is reduced. Moreover, our intracellular and extracellular viral titers indicate that FABP4 affects maximal infectious virion production. This study highlights the role of FABP4 and its therapeutic potential as a target that facilitates KSHV infection and pathogenesis.

microbiology↗

bioRxiv2026

vGPCR-mediated ANO1 Modulation of Apoptosis Regulates Lytic KSHV Infection

Kaposis Sarcoma herpesvirus (KSHV) is an oncogenic, enveloped double-stranded DNA virus that causes Kaposis Sarcoma (KS), an endothelial-derived tumor that primarily affects immunocompromised individuals. KSHV persists in a biphasic lifecycle of latency and lytic reactivation, with the lytic phase driving viral dissemination and disease progression through expression of viral proteins that reprogram host survival pathways. One such protein is the viral G protein-coupled receptor (vGPCR), a constitutively active lytic gene product that promotes angiogenic and pro-survival signaling, although the host mediators of these effects remain incompletely defined. Here, we identify Anoctamin-1 (ANO1), a calcium-activated chloride channel with ant-apoptotic functions, as a downstream target of vGPCR in endothelial cells. RNA sequencing and RT-qPCR in vGPCR-expressing HMEC-1 cells revealed broad transcriptional reprogramming, including strong ANO1 induction. Functional studies showed that ANO1 depletion had little effect on viability in full-serum conditions but selectively sensitized vGPCR-expressing cells to apoptosis during serum starvation. This loss of viability was rescued by the pan-caspase inhibitor Z-VADFMK, indicating caspase-dependent apoptosis. To examine relevance during infection, we analyzed iSLK.BAC16 cells undergoing lytic reactivation. ANO1 was similarly upregulated during reactivation. Genetic knockdown or pharmacological inhibition of ANO1 with DES or Ani9 increased late-stage apoptosis while minimally affecting early apoptosis. Notably, ANO1 loss enhanced early and late lytic gene expression and increased production of infectious virions. These findings identify ANO1 as a vGPCR-induced host survival factor that suppresses apoptosis while modulating KSHV lytic replication, revealing a host ion-channel-dependent checkpoint linking cell survival to viral replication and KSHV pathogenesis. IMPORTANCEKSHV drives tumorigenesis by manipulating host cell survival pathways, yet how apoptosis is regulated during infection remains incompletely understood. We identify ANO1 as a host factor induced by the vGPCR that suppresses caspase-dependent apoptosis in endothelial cells. ANO1 is upregulated in both vGPCR-expressing endothelial cells and during KSHV lytic reactivation in iSLK.BAC16 cells. Disruption of ANO1 increases apoptotic cell death and is accompanied by elevated lytic gene expression and greater production of infectious virions, suggesting that apoptosis can coincide with enhanced lytic replication. These findings implicate host ion-channel signaling in the control of apoptosis during KSHV infection and indicate that the balance between cell survival and apoptosis influences viral gene expression and replication. Understanding how KSHV manipulates host survival pathways provides insight into mechanisms governing viral replication and may inform approaches to limit KSHV-associated disease.

microbiology↗

bioRxiv2026

Global Metabolomic Analysis of Lytic KSHV Infection: Induced Host Nucleotide Metabolism is Required for Infectious Virus Production

Kaposis Sarcoma Herpes Virus (KSHV) is the etiological agent of Kaposis Sarcoma (KS) which is known to cause metabolic stress in infected host cells. KSHV reprograms host metabolic pathways for efficient viral replication and infectious virion production. Here, we report a time-course global metabolomics study conducted in the doxycycline-inducible iSLK.BAC16 cells to compare latent and lytic KSHV infection. Our data show that amino acid, central carbon, and nucleotide metabolic pathways are highly dysregulated upon reactivation to lytic replication. During lytic KSHV infection, pathway enrichment analysis shows that the top two most significantly impacted and dysregulated pathways are purine and pyrimidine metabolism. Further experiments have shown that nucleotide metabolism is required during lytic KSHV infection to produce maximal infectious virus. Treatment with the FDA-approved drug, methotrexate (MTX), a folate antagonist that inhibits cellular DHFR and decreases nucleotide metabolism by reducing tetrahydrofolate cofactors, significantly reduced KSHV late lytic viral gene expression upon reactivation compared to control. Additionally, titering cell-free supernatants from MTX-treated lytic KSHV-infected cells showed a significant reduction in infectious virion production. Furthermore, by adding folinic acid (FA), a downstream metabolite of the MTX-DHFR inhibition step, in the presence of MTX, late lytic gene expression and infectious virion production were significantly rescued. Furthermore, we observed a significant decrease in viral titer of murine herpesvirus 68 (MHV-68), a model virus to study gammaherpesvirus, after MTX treatment. Overall, our study demonstrates that metabolic inhibition during lytic gammaherpesvirus infection decreases productive infection and hence, serves as a potential therapeutic antiviral target.

microbiology↗

bioRxiv2026

LANA-dependent Interaction of Host Factors DAXX and BRD4 Impact KSHV Lytic Replication

Kaposis Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic gammaherpesvirus that causes Kaposis Sarcoma (KS). KSHV alternates between latent and lytic phases. Latency is marked by limited viral gene expression and absence of virion production, whereas the lytic phase is characterized by the expression of all viral genes in a temporally and sequentially regulated cascade of immediate-early, early, and late genes, culminating in viral genome replication and infectious virion production. Both KSHV latent and lytic viral phases contribute to its pathogenesis and the development of KS. KSHV proteins hijack host proteins, rewiring host cellular processes and signaling pathways, and modulating host gene expression. KSHV establishes latency through stable interactions between viral proteins and host factors, with the Latency Associated Nuclear Antigen (LANA) serving as a key regulator that interacts with host machinery to maintain the viral episome and regulate viral replication. However, the consequences for host proteins binding viral proteins, including changes in their interaction networks and their impact on viral replication, remains poorly understood. Here, using Immunoprecipitation Mass Spectrometry (IP-MS), we identified and validated the host death domain-associated protein (DAXX) and DNA ligase 3 (LIG3) as major LANA-associated factors and discovered a LANA-dependent recruitment of Bromodomain-containing protein 4 (BRD4) to DAXX. This remodeling of the DAXX interactome guided functional analyses demonstrating roles for DAXX and BRD4 in supporting KSHV infection. Functional studies show that siRNA knockdown of DAXX or BRD4 in iSLK.BAC16 cells, followed by lytic reactivation, significantly induced viral gene transcription and protein expression of KSHV lytic genes ORF45, ORF59, ORF26, and K8.1. Conversely, siRNA knockdown of LIG3 reduced the transcription and protein expression of KSHV lytic genes. Viral genome replication and infectious virion production were elevated upon knockdown of DAXX or BRD4 and reduced after the knockdown of LIG3. Additionally, chemical inhibition of BRD4 activity by the drug JQ1 in iSLK.BAC16 cells, followed by lytic reactivation, resulted in elevated KSHV lytic gene expression, genome replication, and infectious virus production. Together, these data suggest that both DAXX and BRD4 host genes contribute to KSHV latency maintenance, while LIG3 is required for lytic reactivation. Understanding the functional significance of these LANA-interacting host factors in regulating KSHV infection is critical for identifying therapeutic targets and developing potential treatment strategies.

microbiology↗

bioRxiv2025

TLR4+ Dermal fibroblasts induce acute and transitional pain states

The prominence of non-neuronal cells driving pain states has gained attention in recent years. Fibroblasts, a major stromal cell, perform essential functions during inflammation, tissue remodeling, and wound healing; however, recent studies suggest that fibroblasts may play a role in pain. Toll-like receptor 4 (TLR4) is an essential component of the innate immune system and activation of the receptor promotes pain. This study utilized a novel mouse model with dermal fibroblast specific expression of TLR4 on a TLR4-null background, which allows us to understand the sufficiency of skin fibroblast activation in pain development. Here we demonstrate that dermal fibroblast activation induces both acute inflammatory pain and hyperalgesic priming in both male and female mice. In vivo, activated dermal fibroblasts change cellular morphology in mice and humans. In vitro we observed pro-inflammatory cytokine production and activation of calcium signaling pathways. These data demonstrate that dermal fibroblast activation can cause acute pain and drive mechanisms involved in the transition to chronic pain.

neuroscience↗

bioRxiv2021

Glucagon Receptor-mediated Regulation of Gluconeogenic Gene Transcription is Endocytosis-dependent in Primary Hepatocytes

A number of G protein-coupled receptors (GPCRs) are now thought to use endocytosis to promote cellular cAMP signaling that drives downstream transcription of cAMP-dependent genes. We tested if this is true for the Glucagon Receptor (GCGR), which mediates physiological regulation of hepatic glucose metabolism via cAMP signaling. We show that epitope-tagged GCGRs undergo clathrin and dynamin-dependent endocytosis in HEK293 cells after activation by glucagon, and transit via EEA1-marked endosomes shown previously to be sites of GPCR/Gs-stimulated production of cAMP. We further show that endocytosis potentiates cytoplasmic cAMP elevation produced by GCGR activation and promotes transcription of PCK1, the gene which encodes the enzyme catalyzing the rate-limiting step in gluconeogenesis. We verify endocytosis-dependent induction of PCK1 expression by endogenous GCGRs in primary hepatocytes, and show similar control of two other gluconeogenic genes (PGC1 and G6PC). Together, these results implicate the endosomal signaling paradigm in metabolic regulation by glucagon.

cell biology↗