Biology subjects

Mysorekar, I. U.

Publications and source records attributed to Mysorekar, I. U..

14 recordsLinked to original sources

bioRxiv2026

Human placental trophoblasts support sustained Treponema pallidum replication and reveal new candidate host pathways implicated in congenital syphilis

Congenital syphilis is a leading cause of preventable stillbirth, yet the mechanisms that enable Treponema pallidum subsp. pallidum, the etiological agent of syphilis, to traverse and persist within the human placenta are virtually unknown. This knowledge gap reflects, in part, the fastidious nature of T. pallidum, which has historically only been propagated in rabbit epithelial cells. Here, we demonstrate that T. pallidum replicates and can be propagated long-term in human placental trophoblast models. We identified three human trophoblast cell lines, JEG-3, BeWo, and HTR-8, that sustained robust T. pallidum replication for over 55 days. Confocal imaging with GFP-expressing T. pallidum demonstrated bacterial adherence to trophoblast cultures. To identify human placental pathways that may support T. pallidum replication and persistence, we performed bulk transcriptomic profiling of the three trophoblast lines co-cultured with T. pallidum for 3 or 7 days. Our findings revealed conserved host responses involving increased cholesterol synthesis, suppressed type I interferon signaling, and disrupted extracellular matrix organization. These data implicate host metabolic rewiring, innate immune attenuation, and extracellular matrix remodeling as candidate pathways that may promote T. pallidum invasion, replication and persistence within the placenta. Together, these models represent the first human placental systems capable of supporting efficient, long-term T. pallidum growth and provide a foundation for mechanistic studies of congenital syphilis pathogenesis.

microbiology↗

bioRxiv2026

Trophoblast ferroptosis restricts SARS-CoV-2 spread in the placenta

Prenatal SARS-CoV-2 infection is associated with adverse pregnancy outcomes, but placental mechanisms that restrict viral spread remain unclear. Here we show that SARS-CoV-2 exposure induces ferroptosis-linked iron dysregulation in the placenta as a host defense. Human placentas from early gestation SARS-CoV-2-exposed pregnancies exhibited persistent viral protein expression at term, iron accumulation, disrupted localization of iron transport proteins, and reduced expression of the ferroptosis inhibitor, GPX4. In trophoblast cells and newly generated stem cell-derived trophoblast organoids (SC-TOs) with physiological apical-out polarity, infection with live SARS-CoV-2 Delta variant suppressed expression of iron efflux transporter, ferroportin and ferroptosis inhibitors, GPX4 and PLA2G6, promoting lipid peroxidation and ferroptotic signaling. Sub-lethal pharmacological activation of ferroptosis reduced viral titers in trophoblasts, indicating an antiviral function. Together, these results uncover a new mechanism through which the placenta attempts to restrict SARS-CoV-2 replication. However, this protective response is accompanied by placental iron sequestration, which may compromise maternal-fetal iron transfer and help explain iron deficiency and anemia reported in infants born after prenatal SARS-CoV-2 exposure, highlighting a delicate balance between iron and ferroptosis-mediated protection and damage with implications for pregnancy outcomes.

cell biology↗

bioRxiv2025

Bacteriophage-mediated reduction of uropathogenic E. coli from the urogenital epithelium

Urinary tract infections (UTIs), primarily caused by uropathogenic Escherichia coli (UPEC), affect millions annually. UPEC gains access to the urinary tract through mucosal reservoirs including the vaginal tract. With rising antibiotic resistance and frequent recurrence, alternative non-antibiotic strategies like bacteriophage (phage) therapy are gaining attention. We explored the potential of a lytic phage, {Phi}HP3, as well as a phage cocktail to decolonize UPEC from the urogenital tract using in vitro and in vivo models. Phage significantly inhibited UPEC growth in both bacteriologic medium and simulated vaginal fluid. Pretreatment of human vaginal epithelial cells (VK2/E6E7) and bladder carcinoma cells (HTB-9) with phage reduced adhesion and invasion of UPEC compared with controls. Phage treatment was further able to reduce intracellular UPEC in VK2 cells. Notably, phage pretreatment did not impact phage resistant UPEC strains, indicating that phage lysis was the primary driver of phenotypes. Live confocal microscopy confirmed interaction of phage particles with UPEC and with both epithelial cell lines. In vivo, daily intravaginal {Phi}HP3 administration in humanized microbiota mice significantly reduced vaginal UPEC burden after 4 days. Treatment with a phage cocktail also reduced vaginal and cervical tissue burdens by day 7 post-treatment. UPEC dissemination was observed to uterine and kidney tissues, but burdens were not different between phage and mock-treated groups. In conclusion, we demonstrate that phage and phage cocktails can modestly reduce UPEC urogenital colonization, highlighting the potential of phage therapy as a viable treatment option for UTI prevention. IMPORTANCEUrinary tract infections (UTIs) are among the most common infections worldwide, with millions of cases each year. Due to frequent recurrence and increasing antibiotic resistance, UTIs are becoming more difficult to treat, and non-antibiotic prevention options remain limited. The bacteria responsible for UTIs, such as uropathogenic E. coli (UPEC), often colonize other body sites, such as the intestines or vagina, before causing infection. In this study, we investigated whether bacteriophage (phage), viruses that infect bacteria, could reduce UPEC colonization. We found that phage treatment decreased UPEC adherence to vaginal and bladder cell lines, but only modestly reduced UPEC vaginal colonization in a mouse model. These findings suggest that phages may offer a potential strategy for UTI prevention, though further research is needed to optimize their therapeutic use.

microbiology↗

bioRxiv2025

A scalable organoid model of urothelial aging for metabolic interrogation, infection modeling, and reversal of age-associated changes

Aging leads to a progressive decline in overall bladder function resulting in lower urinary tract symptoms and increased susceptibility to infections. However, tissue-specific mechanisms of aging, specifically the contributions of the aged urothelium remain elusive. Here, we introduce mouse bladder epithelium-derived organoids (mBEDOs) as a scalable platform to model urothelial aging. mBEDOs from aged mice recapitulate key features of age-associated cellular reprogramming, including oxidative stress, senescence, and DNA damage. We demonstrate the utility of mBEDOs for modeling Uropathogenic Escherichia coli (UPEC) infection, generating assembloids between mBEDOs and macrophages to model epithelial-immune interactions, and genetic perturbation. Using the mBEDO platform, we also identify urothelium-specific changes in purine, amino acid, and glycerophospholipid metabolism which may contribute to age-associated cellular perturbations. Lastly, supplementation with depleted metabolites, nicotinamide (NAM) and D-mannose, reduce DNA damage and oxidative stress and restore mitochondrial integrity in aged mBEDOs. These findings establish mBEDOs as an effective platform for investigating molecular and cellular underpinnings of urothelial aging and exploring metabolism-based interventions for age-associated bladder dysfunction.

bioengineering↗

bioRxiv2025

SARS-CoV-2 infection disrupts syncytial and endothelial integrity and alters PLGF levels in the placenta

IntroductionSARS-CoV-2 infection during pregnancy has been associated with an increased risk for several pregnancy-related disorders, particularly preeclampsia (PE). However, there are limited studies determining the impact of SARS-CoV-2 on placental physiology and function. MethodsPlacental samples were acquired from two large prospective cohorts: STOP-COVID19 and REBRACO studies. Placental villous tissues (VTs) were collected from pregnant women who tested positive for SARS-CoV-2 without PE during pregnancy. Immunohistochemistry and immunofluorescence were used to assess pathological features known to be altered in PE, including 1) syncytial knot formation; 2) alterations in renin-angiotensin system components; 3) and endothelial integrity. Maternal serum was collected to examine AT1 autoantibodies levels using an immunoassay. ResultsSARS-CoV-2 viral proteins spike, nucleocapsid, and ORF3a were observed in the syncytiotrophoblast layer and stroma of placental VT. SARS-CoV-2-infected placentas exhibited increased numbers of syncytial knots, which were positive for Flt-1 and SARS-CoV-2 viral proteins. In addition, the presence of placental infarctions and excessive fibrin deposits was also observed in infected placentas. Infection was associated with decreased placental expression of PlGF and an increase in the placental Flt-1/PlGF expression ratio, mostly driven by PlGF. No significant changes in maternal serum AT1AA levels were observed. Finally, SARS-CoV-2-infected placentas exhibited a significant decrease in vimentin expression. DiscussionSARS-CoV-2 infection negatively impacts placental integrity in the form of increased syncytial knots, dysregulated RAS components, and endothelial damage. Since all these features are similarly disrupted in PE, this could be a mechanism through which SARS-CoV-2 infection during pregnancy increases the risk of a PE-like syndrome.

microbiology↗

bioRxiv2025

The role of amygdala calcitonin gene-related peptide receptors on the development of persistent bladder pain in mice.

Bladder pain significantly impacts millions worldwide, severely affecting their quality of life and posing a major clinical challenge. Understanding the mechanisms underlying persistent bladder pain is critical for developing better therapeutic strategies. In this study, we investigate the effects of cyclophosphamide (CYP)-induced persistent bladder sensitization to explore the lateralized contribution of amygdala calcitonin gene-related peptide receptors (CGRP-Rs) on pain-like changes in mice. We demonstrate that CYP induces hypersensitivity lasting up to 14 days post-injury (DPI) in the urinary bladder distention assay and up to 21 DPI when assessing abdominal mechanical sensitivity. Despite persistent pain-like changes, no significant bladder histological changes were observed. Based on previous findings that CGRP signaling from the parabrachial nucleus contributes to central amygdala (CeA) lateralization, we hypothesized that CGRP-Rs play a key role in driving visceral bladder pain-related hemispherical differences. We show that inhibiting CGRP-R activity with the antagonist CGRP8-37, in the right CeA attenuates bladder pain-like behavior, whereas left CeA inhibition sustains CYP-induced hypersensitivity. Electrophysiological recordings revealed increased firing frequency in CGRP-R positive cells in the right CeA 7 DPI. In vivo single photon calcium imaging demonstrated increased Ca transients in CGRP-R-positive cells in the right CeA, upon the presentation of a stimulus at 0 DPI, and overall at 2DPI, further confirming the pronociceptive role of CGRP-Rs in the right CeA. Taken together, these findings provide a crucial foundation for understanding pain-induced CeA lateralization and for further studies identifying how targeting CGRP signaling could provide bladder pain relief.

neuroscience↗

bioRxiv2024

SARS-CoV-2 ORF3a Protein Impairs Syncytiotrophoblast Maturation, Alters ZO-1 Localization, and Shifts Autophagic Pathways in Trophoblast Cells and 3D Organoids

SARS-CoV-2 infection poses a significant risk to placental physiology, but its impact on placental homeostasis is not well understood. We and others have previously shown that SARS-CoV-2 can colonize maternal and fetal placental cells, yet the specific mechanisms remain unclear. In this study, we investigate ORF3a, a key accessory protein of SARS-CoV-2 that exhibits continuous mutations. Our findings reveal that ORF3a is present in placental tissue from pregnant women infected with SARS-CoV-2 and disrupts autophagic flux in placental cell lines and 3D stem-cell-derived trophoblast organoids (SC-TOs), impairing syncytiotrophoblast differentiation and trophoblast invasion. This disruption leads to protein aggregation in cytotrophoblasts (CTB) and activates secretory autophagy, increasing CD63+ extracellular vesicle secretion, along with ORF3a itself. ORF3a also compromises CTB barrier integrity by disrupting tight junctions via interaction with ZO-1, mediated by its PDZ-binding motif, SVPL. Colocalization of ORF3a and ZO-1 in SARS-CoV-2-infected human placental tissue supports our in vitro findings. Deleting the PDZ binding motif in the ORF3a protein (ORF3a-noPBM mutant) restored proper ZO-1 localization at the cell junctions in an autophagy-independent manner. Lastly, we demonstrate that constitutive ORF3a expression induces SC-TOs to transition towards a secretory autophagy pathway likely via the PBM motif, as the ORF3a-NoPBM mutants showed a significant lack of CD63 expression. This study demonstrates the functional impact of ORF3a on placental autophagy and reveals a new mechanism for the activation of secretory autophagy, which may lead to increased extracellular vesicle secretion. These findings provide a foundation for exploring therapeutic approaches targeting ORF3a, specifically focusing on its PBM region to block its interactions with host cellular proteins and limiting placental impact.

microbiology↗

bioRxiv2024

Placentas from SARS-CoV-2 infection during pregnancy exhibit foci of oxidative stress and DNA damage

ProblemCOVID-19 during pregnancy is linked to increased maternal morbidity and a higher incidence of preterm births, yet the underlying mechanisms remain unclear. Cellular senescence, characterized by the irreversible cessation of cell division, is a critical process in placental function and its dysregulation has been implicated in pregnancy complications like preterm birth. Senescence can be induced by various stressors, including oxidative stress, DNA damage, and viral infections. Method of StudyIn this study, we determined whether COVID-19 had an impact on placental senescence. We examined placentas from women infected with SARS-CoV-2 (n=10 term, 4 preterm) compared to uninfected controls (n=10 term, 3 preterm). The placentas were analyzed for SARS-CoV-2 infection/replication (Spike and Nucleocapsid viral proteins), markers of DNA damage ({gamma}H2AX) and oxidative stress (ROS), and senescence (telomere length; cell cycle regulators, SASP). ResultsWhile no overall differences in cellular senescence markers were observed between the COVID-19 positive and negative groups, we found increased secreted SASP markers and confocal microscopy revealed localized areas of oxidative stress and DNA damage in the placentas from COVID-19 positive cases. ConclusionsThese findings indicate that SARS-CoV-2 infection induces localized placental damage, warranting further investigation into its impact on maternal and perinatal outcomes.

microbiology↗

bioRxiv2024

A new role for IFRD1 in regulation of ER stress in bladder epithelial homeostasis

A healthy bladder requires the homeostatic maintenance of and rapid regeneration of urothelium upon stress/injury/infection. Several factors have been identified to play important roles in urothelial development, injury and disease response, however, little is known about urothelial regulation at homeostasis. Here, we identify a new role for IFRD1, a stress-induced gene that has recently been demonstrated to play a critical role in adult tissue proliferation and regeneration, in maintenance of urothelial function/ homeostasis in a mouse model. We show that the mouse bladder expresses IFRD1 at homeostasis and its loss alters the global transcriptome of the bladder with significant accumulation of cellular organelles including multivesicular bodies with undigested cargo, lysosomes and mitochondria. We demonstrate that IFRD1 interacts with several mRNA-translation-regulating factors in human urothelial cells and that the urothelium of Ifrd1-/- mice reveal decreased global translation and enhanced endoplasmic reticulum (ER) stress response. Ifrd1-/- bladders have activation of the unfolded protein response (UPR) pathway, specifically the PERK arm, with a concomitant increase in oxidative stress and spontaneous exfoliation of urothelial cells. Further, we show that such increase in cell shedding is associated with a compensatory proliferation of the basal cells but impaired regeneration of superficial cells. Finally, we show that upon loss of IFRD1, mice display aberrant voiding behavior. Thus, we propose that IFRD1 is at the center of many crucial cellular pathways that work together to maintain urothelial homeostasis, highlighting its importance as a target for diagnosis and/or therapy in bladder conditions.

cell biology↗

bioRxiv2022

Discovery And Characterization of Small Molecule Inhibitors of Zika Virus Replication

Zika virus (ZIKV) is a flavivirus, and ZIKV infections in the past 15 years have been linked to Guillain-Barre syndrome and severe complications during pregnancy associated with congenital Zika syndrome. There are no approved therapies or vaccines for ZIKV. In recent years, advances in structure-based drug design methodologies have accelerated drug development pipelines for identifying promising inhibitory compounds against viral diseases. Among ZIKV proteins, NS2B-NS3 protease is an attractive target for antiviral drug development due to its vital role in the proteolytic processing of the single polyprotein. To find potential inhibitors against ZIKV, we used molecular docking at the NS2B-NS3 protease active site as a virtual screening approach with small molecules diverse scaffold-based library with rigorous druglikeness filters. The top-hit compounds with stable molecular dynamics trajectories were then subjected to in-vitro efficacy testing against ZIKV. In docking and molecular dynamics simulation studies, compound F1289-0194 showed stable binding to the NS2B-NS3 protease active site. Furthermore, viral load assays, immunofluorescence, and plaque reduction assays demonstrated that compound F1289-0194 significantly reduced ZIKV load and replication in Vero cells while maintaining cellular integrity. Thus, the compound F1289-0194 merits further investigation as a novel inhibitor against ZIKV replication.

cell biology↗

bioRxiv2022

D-mannose ameliorates age-associated cellular senescence in the bladder urothelium and NLRP3/Gasdermin/IL-1β -driven pyroptotic epithelial cell shedding

Aging is a risk factor for disease via increased susceptibility to infection, decreased ability to maintain homeostasis, inefficiency in combatting stress, and decreased regenerative capacity. Multiple diseases including urinary tract infection (UTI), are more prevalent with age; however, the mechanisms underlying how aging affects the urinary tract mucosa and the reason why aging correlates with disease are poorly understood. Here, we show that, relative to young (8-12 weeks) mice, the urothelium of aged (18-24 months) female mice accumulates large lysosomes with decreased acid phosphatase activity and shows overall decreased autophagic flux. Aged bladders exhibit basally high accumulation of reactive oxygen species (ROS) and dampened redox response. Furthermore, the aged urothelium exhibits a canonical senescence-associated secretory phenotype (SASP) at baseline with continuous NLRP3-inflammasome- and Gasdermin D (GSDMD)-dependent pyroptotic cell death. Accordingly, we find that aged mice chronically exfoliate epithelial cells. When infected with uropathogenic E. coli, infected aged mice harbor more bacterial reservoirs post-infection and are prone to spontaneous recurrent UTI. Finally, treatment of aged mice with D-Mannose, a natural bioactive monosaccharide, rescues autophagy flux, reverses SASP, and limits pyroptotic epithelial shedding. Thus, normal aging dramatically affects bladder physiology with aging alone increasing baseline cellular stress and susceptibility to infection. Additionally, our results suggest that mannose supplementation could serve as a senotherapeutic to limit age-associated urothelial dysfunction.

cell biology↗

bioRxiv2020

Coxsackievirus B3 Infection Early in Pregnancy Induces Congenital Heart DefectsThrough Suppression of Fetal Cardiomyocyte Proliferation

AimsCoxsackievirus B (CVB), the most common cause of viral myocarditis, targets cardiomyocytes through Coxsackie and Adenovirus Receptor, which is highly expressed in the fetal heart. We hypothesized CVB3, a well-recognized culprit for viral myocarditis, can precipitate congenital heart defects (CHD), when fetal infection occurs during critical window of gestation. Methods & ResultsWe infected C57Bl/6 pregnant mice with CVB3 during serial time points in early gestation (E5, E7, E9 and E11). We used different viral titers to examine possible dose- response relationship and assessed viral loads in various fetal organs as well as kinetics of virus passage into the fetus during gestation. Provided viral exposure occurred between E7-E9, we observed characteristic features of ventricular septal defect (33.6%), abnormal myocardial architecture resembling non-compaction (23.5%), and double outlet right ventricle (4.4%) among 209 viable fetuses examined. We observed a direct relationship between viral titers, severity and incidence of CHD, with apparent predominance among female fetuses. Infected dams remained healthy; we did not observe any maternal heart or placental injury suggestive of direct viral effects on developing heart as likely cause of CHD. We examined signaling pathways in CVB3-exposed hearts using RNAseq, KEGG enrichment analysis and immunohistochemistry. Signaling proteins of the Hippo, tight junction, transforming growth factor {beta}1 and extracellular matrix proteins were the most highly enriched in CVB3-infected fetuses with VSD (log fold change >1.9, P<0.02). Moreover, cardiomyocyte proliferation was 50% lower in fetuses with VSD compared with uninfected controls. ConclusionPrenatal CVB3 infection can induce CHD, provided the infection occurs during a critical window. Alterations in myocardial proliferate capacity and consequent changes in cardiac architecture and trabeculation appear to account for the majority of observed phenotypes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/104844v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@79d320org.highwire.dtl.DTLVardef@1419b8org.highwire.dtl.DTLVardef@1a49082org.highwire.dtl.DTLVardef@10b72bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

Tertiary lymphoid tissue develops during normal aging in mice and humans

Aging has multifaceted effects on the immune system in the context of systemic responses to specific vaccines and pathogens, but how aging affects tissue-specific immunity is not well-defined. Chronic bladder inflammation is highly prevalent in older women, but mechanisms by which aging promotes these pathologies remain unknown. Here we report distinct, age-associated changes to the immune compartment in the otherwise normal female (but not in male) mouse urinary bladder and parallel changes in older women with chronic bladder inflammation. In aged mice, the bladder epithelium became more permeable, and the homeostatic immune landscape shifted from a limited, innate immune-predominant surveillance to an inflammatory, adaptive immune-predominant environment. Strikingly, lymphoid cells were organized into tertiary lymphoid tissues, hereafter named bladder tertiary lymphoid tissue (bTLT). Analogous bTLTs were found in older women, many of whom had a history of recurrent urinary tract infection (UTI). Aged mice responded poorly to experimental UTI, experiencing spontaneous recurrences at higher rates than young mice. However, bTLT formation was dependent on aging and independent of infection. Furthermore, bTLTs in aged mice played a role in de novo antibody responses and urinary IgA production by recruitment of naive B cells that form germinal centers and mature into IgA-secreting plasma cells. Finally, TNF was a key driver of bTLT formation, as aged TNF-/- mice lacked bTLTs. Both aged TNF-/- and wild type mice exhibited increased bladder permeability, suggesting that epithelial dysfunction may be an upstream mediator of chronic, age-associated bladder inflammation. Thus, bTLTs arise as a function of age and may underlie chronic, age-associated bladder inflammation. Our model establishes a platform for further investigation of age-association tissue inflammation and translation to new treatment strategies.\n\nOne Sentence SummaryMice develop bladder tertiary lymphoid tissue (bTLT) during aging that is dependent on TNF and independent of urinary tract infection.

immunology↗

bioRxiv2019

Functional characterization of Ralstonia insidiosa, a bona fide resident at the maternal-fetal interface

Controversy about whether there are microbes in the placenta and if they have any functional importance during pregnancy and for neonatal health is ongoing. Previous work has demonstrated that the basal plate (BP), comprising maternal and fetal derived cells harbors intracellular bacteria. 16S sequencing and bacterial species-specific analysis of term placentas revealed that the gram-negative bacillus Ralstonia insidiosa, native to aqueous environments and an effective biofilm promoter, comprises the most abundant species in the BP. Here, we demonstrate whether R. insidiosa cells home to a particular niche in the BP, how they may arrive there, and whether they are associated with adverse outcomes. We developed methods to detect and study cell-specific localization of R. insidiosa using ex vivo and in vitro models. Additionally, we studied potential routes of R. insidiosa entry into the placenta. We show that R. insidiosa is a bona fide resident in human placental BP. It can access trophoblast cells in culture and within basal plate tissues where it localizes to intracellular single-membrane vacuoles and can replicate. However, the presence of R. insidiosa does not cause cell death and does not induce a pro-inflammatory immune response suggesting that it is not harmful in and of itself. Finally, we show that in a pregnant mouse model, R. insidiosa traffics to the placenta via the intrauterine route but does not induce preterm labor or preterm birth. Together, our findings provide a foundation for understanding non-pathogenic placental cell-microbe interactions and the functional importance of R. insidiosa in placental health and physiology.

microbiology↗