Biology subjects

Chauhan, M.

Publications and source records attributed to Chauhan, M..

6 recordsLinked to original sources

bioRxiv2024

Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus.

The Toll pathway plays a pivotal role in innate immune responses against pathogens. The evolutionary conserved pathogen recognition receptors (PRRs), including Toll like receptors (TLRs), play a crucial role in recognition of pathogen associated molecular patterns (PAMPs). The Drosophila genome encodes nine Toll receptors that are orthologous to mammalian TLRs. While mammalian TLRs directly recognize PAMPs, most Drosophila Tolls recognize the proteolytically cleaved ligand Spatzle to activate downstream signaling cascades. In this study, we demonstrated that Toll-9 is crucial for antiviral immunity against Drosophila C virus (DCV), a natural pathogen of Drosophila. A transposable element insertion in the Toll-9 gene renders the flies more susceptible to DCV. The stable expression of Toll-9 in S2 cells confers resistance against DCV infection by upregulation of the RNAi pathway. Toll-9 promotes the dephosphorylation of AKT, resulting in the induction of antiviral RNAi genes to inhibit DCV replication. Toll-9 localizes to the endosome where it binds dsRNA, suggesting its role to detect viral dsRNA. Toll-9 also induces apoptosis during DCV infection, contributing to its antiviral role. Together, this work identifies the role of Toll-9 in antiviral immunity against DCV infection through its ability to bind dsRNA and induce AKT-mediated RNAi antiviral immunity. IMPORTANCEInsects rely on innate immunity and RNA interference (RNAi) to combat viral infections. Our study underscores the pivotal role of Drosophila Toll-9 in antiviral immunity, aligning with findings in Bombyx mori, where Toll-9 activation upregulates the RNAi component Dicer2. We demonstrate that Drosophila Toll-9 functions as a pattern recognition receptor (PRR) for double-stranded RNA (dsRNA) during Drosophila C virus (DCV) infection, akin to mammalian TLRs. Toll-9 activation leads to the upregulation of key RNAi components, Dicer2 and Argonaute2, and dephosphorylation of AKT triggers apoptosis via induction of proapoptotic genes Hid and Reaper. This study also reveals that Toll-9 localizes in endosomal compartments where it interacts with dsRNA. These insights enhance our understanding of Drosophila innate immune mechanisms, reflecting the evolutionary conservation of immune responses across diverse species and providing impetus for further research into the conserved roles of TLRs across the animal kingdom.

immunology↗

bioRxiv2024

AAV-DJ is Superior to AAV9 for Targeting Brain and Spinal Cord, and De-Targeting Liver Across Multiple Delivery Routes in Mice

Highly efficient adeno associated viruses (AAVs) targeting the central nervous system (CNS) are needed to deliver safe and effective therapies for inherited neurological disorders. The goal of this study was to compare the organ-specific transduction efficiencies of two AAV capsids across three different delivery routes. We compared AAV9-CBA-fLucYFP to AAV-DJ-CBA-fLucYFP using the following delivery routes in mice: intracerebroventricular (ICV) 1x1012 vg/kg, intrathecal (IT) 1x1012 vg/kg, and intravenous (IV) 1x1013 vg/kg body weight. Our evaluations revealed that following ICV and IT administrations, AAV-DJ demonstrated significantly increased vector genome (vg) uptake throughout the CNS as compared to AAV9. Through the IV route, AAV9 demonstrated significantly increased vg uptake in the CNS. However, significantly fewer vgs were detected in the off-target organs (kidney and liver) following administration of AAV-DJ using the IT and IV delivery routes as compared to AAV9. Distributions of vgs correlate well with transgene transcript levels, luciferase enzyme activities, and immunofluorescence detection of YFP. Overall, between the two vectors, AAV-DJ resulted in better targeting and expression in CNS tissues paired with de-targeting and reduced expression in liver and kidneys. Our findings support further examination of AAV-DJ as a gene therapy capsid for the treatment of neurological disorders.

molecular biology↗

bioRxiv2024

The Candida auris Hog1 MAP kinase is essential for the colonization of murine skin and intradermal persistence

Candida auris, a multidrug-resistant human fungal pathogen, was first identified in 2009 in Japan. Since then, systemic C. auris infections have now been reported in more than 50 countries, with mortality rates of 30-60%. A major contributing factor to its high inter- and intrahospital clonal transmission is that C. auris, unlike most Candida species, displays unique skin tropism and can stay on human skin for a prolonged period. However, the molecular mechanisms responsible for C. auris skin colonization, intradermal persistence, and systemic virulence are poorly understood. Here, we report that C. auris Hog1 mitogen-activated protein kinase (MAPK) is essential for efficient skin colonization, intradermal persistence, as well as systemic virulence. RNA-seq analysis of wildtype parental and hog1{Delta} mutant strains revealed marked down-regulation of genes involved in processes such as cell adhesion, cell-wall rearrangement, and pathogenesis in hog1{Delta} mutant compared to the wildtype parent. Consistent with these data, we found a prominent role for Hog1 in maintaining cell-wall architecture, as the hog1{Delta} mutant demonstrated a significant increase in cell-surface {beta}-glucan exposure and a concomitant reduction in chitin content. Additionally, we observed that Hog1 was required for biofilm formation in vitro and fungal survival when challenged with primary murine macrophages and neutrophils ex vivo. Collectively, these findings have important implications for understanding the C. auris skin adherence mechanisms and penetration of skin epithelial layers preceding bloodstream infections. ImportanceCandida auris is a World Health Organization (WHO) fungal priority pathogen and an urgent public health threat recognized by the Centers for Disease Control and Prevention (CDC). C. auris has a unique ability to colonize human skin. It also persists on abiotic surfaces in healthcare environments for an extended period of time. These attributes facilitate the inter- and intrahospital clonal transmission of C. auris. Therefore, understanding C. auris skin colonization mechanisms are critical for infection control, especially in hospitals and nursing homes. However, despite its profound clinical relevance, the molecular and genetic basis of C. auris skin colonization mechanisms are poorly understood. Herein, we present data on the identification of the Hog1 MAP kinase as a key regulator of C. auris skin colonization. These findings lay foundation for further characterization of unique mechanisms that promote fungal persistence on human skin.

microbiology↗

bioRxiv2023

A possible mechanistic insight on how Compromised Hydrolysis of Triacylglycerol 7 (CHT7) restrains the involvement of it's DNA binding CXC domain from quiescence repression

CHT7 is a regulator of quiescence repression and TAG degradation between the nitrogen deprived and the nitrogen replenished states in Chlamydomonas reinhardtii. Initially it was thought that the CHT7s repression activity is managed by its DNA binding CXC domain which is a tandem repeat of two cysteine rich subdomains. Later, it was found that the CXC (CHT7_CXC) domain is effectively dispensable for CHT7s activities. Rather, CHT7s predicted protein binding domains are proposed to be involved in gene regulation activities by binding through other repressors in the cell. Yet, it remains unclear why and how CHT7 manages to refrain its own CXC domain from participating in any transcriptional activities. The question becomes more intriguing, because CXC binding regions are available in promoter regions of some of the misregulated genes in the CHT7 mutant (cht7). Through the combination of biophysical experiments and molecular dynamics approaches, we have studied the DNA recognition behavior of CHT7_CXC. The results show that CHT7_CXC domain is highly selective towards DNA sequences and this selectivity is imparted due to the differential binding abilities of the CXC subdomains. Further, to understand if the case is - that CXC looses its DNA binding capabilities in the vicinity of other repressor molecules, we carried out CHT7_CXCs DNA binding stability test by simulating the spatial constraint conditions using the AsLOV2- CXC fusion. Our test results show limited ability of CHT7_CXC to withstand steric forces and provide insights to why and how algal cells may hold back CHT7_CXCs indulgence in quiescence repression. SignificanceMicroalgae, under nutrient rich conditions, provide biomass. Whereas, nutrient deprivation leads to accumulation of biofuel feedstock, but cells enter quiescence. Net enhancement in feedstock, therefore relies on the precision of the quiescence regulator. In Chlamydomonas reinhardtii, CHT7 is a central regulator of quiescence. Surprisingly, rather than using its own DNA binding domain (DBD) for the regulatory activities, CHT7 recruits external transcriptional regulators using its non DBDs. To ensure smooth functioning, CHT7s DBD must rapidly switch to inactive form. Modifications in DNA binding profiles of DBDs due to non DBDs are seen in transcription factors of many organisms. The switching mechanism discussed could therefore be a generic approach of timely regulation of individual components of the complex transcriptional machineries.

biophysics↗

bioRxiv2022

Novel insights into the fundamentals of palatal shelf elevation dynamics in normal mouse embryos

Embryonic palate development involves bilateral vertical growth of palatal shelves - extensions from the maxillary processes - next to the tongue until embryonic day (E) 13.5. Following vertical growth, palatal shelves elevate and adhere above the tongue by E14.5. Current models indicate that this process of elevation involves a complex vertical to horizontal reorienting of the palatal shelves. While earlier studies have implied that this is a rapid process, the precise timing has not been resolved. To understand the dynamics of palatal shelf elevation, we employed time-restricted pregnancies with a one-hour resolution and magnetic resonance imaging of intermediate stages. Our data showed that in almost all C57BL/6J embryos, palatal shelves have not yet elevated by E14.0. However, six hours later at E14.25, palatal shelves have completed elevation in 80% of embryos. Interestingly, all E14.25 embryos with unelevated palatal shelves (20%) were female, suggesting a delay in female embryos. In FVB/NJ embryos, the elevation window started earlier (E13.875-E14.25) without any noticeable sex differences. We frequently captured an intermediate stage with unilateral elevation of either right or left palatal shelf. Magnetic resonance imaging of various stages showed that palatal shelf elevation began with the formation of bilateral bulges in the posterior. These bulges progressed laterally and anteriorly over time. During elevation, we observed increased cell proliferation in the lingual region of the palatal shelf. Within the bulge, cell orientation was acutely tilted towards the tongue and actomyosin activity was increased, which together may participate in the projection of the bulge in the horizontal direction. Thus, our data reveal novel insights into the rapid dynamic changes in palatal shelf elevation that lay the foundation for future studies of normal and abnormal palatogenesis.

developmental biology↗

bioRxiv2021

The mitochondrial gene CMPK2 functions as a rheostat for macrophage homeostasis in inflammation

In addition to their role in cellular energy production, mitochondria are increasingly recognized as regulators of the innate immune response of phagocytes. Here, we demonstrate that altering expression levels of the mitochondria associated enzyme, cytidine monophosphate kinase 2 (CMPK2), disrupts mitochondrial physiology and significantly de-regulates the resting immune homeostasis of macrophages. Both CMPK2 silenced as well as constitutively over expressing macrophage lines portray mitochondrial stress with marked depolarization of their membrane potential, enhanced ROS and disturbed architecture culminating in the enhanced expression of the pro-inflammatory genes-IL1{beta}, TNF and IL8. Interestingly, the long-term modulation of CMPK2 expression resulted in an increased glycolytic flux of macrophages akin to the altered physiological state of activated M1 macrophages. While infection induced inflammation for restricting pathogens is regulated, our observation of a total dysregulation of basal inflammation by bidirectional alteration of CMPK2 expression, only highlights a critical role of this gene in mitochondria mediated control of inflammation.

immunology↗