Biology subjects

Patel, A. M.

Publications and source records attributed to Patel, A. M..

8 recordsLinked to original sources

bioRxiv2025

Characterization of a Novel Cell Wall-Associated Nucleotidase of Enterococcus faecalis that Degrades Extracellular c-di-AMP

Enterococcus faecalis is a prolific opportunistic pathogen responsible for a range of life-threatening infections for which treatment options are increasingly limited due to the high prevalence of multidrug-resistant isolates. Cyclic di-AMP has emerged as an essential bacterial signaling molecule due to its impact on physiological processes, including osmotic adaptation, cell wall homeostasis, antibiotic tolerance, and virulence. In addition, c-di-AMP is a potent pathogen-associated molecular pattern (PAMP) molecule recognized by the host immune system to trigger protective responses. In previous work, we identified and characterized the enzymes responsible for the synthesis and degradation of intracellular c-di-AMP in E. faecalis, demonstrating that maintaining c-di-AMP homeostasis is vital for bacterial fitness and virulence. In addition to the intracellular enzymes that regulate c-di-AMP levels, a limited number of bacteria encode surface-associated nucleotidases capable of cleaving extracellular c-di-AMP, potentially facilitating immune evasion. Here, we characterize a novel and unique cell wall-anchored phosphodiesterase, termed EecP (E. faecalis extracellular c-di-AMP phosphodiesterase), which features duplicated catalytic domains and specifically degrades extracellular c-di-AMP. Deletion of eecP ({Delta}eecP) resulted in a marked accumulation of extracellular c-di-AMP. Although the {Delta}eecP strain exhibited comparable growth and behavior to the parent strain in vitro, it displayed increased susceptibility to killing by phagocytic cells. Using two murine infection models, we show that the impact of eecP deletion and the consequent buildup of extracellular c-di-AMP on E. faecalis pathogenesis may be site-specific. Notably, disseminated infection was more severe in mice infected with the {Delta}eecP strain, suggesting that extracellular c-di-AMP influences infection outcomes, likely through modulation of host immune responses. Author SummaryEnterococcus faecalis is a major opportunistic pathogen and a leading cause of several life-threatening hospital-associated infections. Cyclic di-AMP is a bacterial second messenger nucleotide that regulates essential cellular processes and plays key roles in bacterial pathogenesis and host immune activation. We previously characterized the enzymes responsible for the synthesis and degradation of c-di-AMP in E. faecalis, demonstrating that this signaling molecule is crucial for bacterial fitness and virulence. In this study, we describe the characterization of EecP, a novel cell wall-associated enzyme that degrades c-di-AMP extracellularly. Our findings identify EecP as a new virulence factor in E. faecalis, capable of modulating infection outcomes.

microbiology↗

bioRxiv2025

Recombination and the Species Structure of the Genus Bacteroides

Bacteroides play prominent roles in maintaining gut homeostasis through the fermentation of dietary carbohydrates, the production of metabolites, and the regulation of host immune response. The genus Bacteroides comprises several dozen named species, several of which serve as a nexus of gene exchange in gut microbiomes, especially in humans. Because gene exchange will blur the historically defined species boundaries, we assessed the patterns and extent of recombination for nearly 2,000 fully sequenced Bacteroides genomes representing 36 named species, and redefined species boundaries based on their capacity to exchange genes. We recognized numerous incongruences between traditional classification schemes, those based on nucleotide sequence identity thresholds, and species defined by gene flow. Almost all named species of sufficient sample size for analysis included genomes that failed to recombine with other genomes that were historically assigned to the same species. Such misclassifications occurred even for cases in which sequence identities surpassed the thresholds regularly used to define species. Conversely, we detected instances in which species previously considered distinct were found to recombine extensively--the most striking of which supported unification of B. kribbi, B. koreensis, and B. ovatus (which freely recombines with B. xylanisolvens). Among the broadly distributed populations of B. fragilis, we detected elevated levels of recombination among human-derived strains, although this result is confounded by geographic and sampling biases. Our findings emphasize the utility of a universal species concept based on gene flow, not only for refining bacterial classifications but also for understanding the variation within and across gut microbiomes. IMPORTANCE STATEMENTBacteroides is a diverse genus comprising some of the most pervasive taxa in the mammalian gut microbiome, with several species playing key roles in the maintenance of gut ecosystem stability. The propensity for Bacteroides to exchange genes, both within and among species, has eluded efforts to understand its diversity and to delineate species boundaries, both by traditional classification schemes as well as methods that rely on genomic sequence identity. To circumvent these obstacles and to circumscribe species in a manner that can be uniformly applied across lifeforms, we analyzed all complete genomes assigned to Bacteroides and reclassified them based on their capacity for gene flow, uncovering numerous inconsistencies with both recent and historical classifications. Our approach offers a biologically grounded alternative to the use of sequence identity thresholds, offering a better understanding of the diversity and relationships among genomes within the genus Bacteroides at large.

microbiology↗

bioRxiv2024

The enzyme glutamate-cysteine ligase (GCL) is a target for ferroptosis induction in cancer

Despite glutathiones long-recognized role as a major cellular antioxidant and its central role in ferroptosis defense, inhibition of glutathione biosynthetic enzymes has received little attention as a target for the therapeutic induction of ferroptosis. Here, we report that small-molecule inhibition of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of glutathione biosynthesis, selectively and potently kills cancer cells by ferroptosis. We further describe novel GCL inhibitors including KOJ-1 and KOJ-2, compounds with excellent cellular potency and pharmacological properties, representing valuable tools to study the biology of ferroptosis and glutathione.

cancer biology↗

bioRxiv2021

The Antiviral Enzyme, Viperin, Activates Protein Ubiquitination by the E3 Ubiquitin Ligase, TRAF6

Viperin is a broadly conserved radical SAM enzyme that synthesizes the antiviral nucleotide ddhCTP. In higher animals viperin expression also accelerates the degradation of various cellular and viral proteins necessary for viral replication; however, the details of this process remain largely unknown. Here we show that viperin activates a component of the protein ubiquitination machinery, which plays an important role in both protein degradation and immune signaling pathways. We demonstrate that viperin binds the E3 ubiquitin ligase, TRAF6, which catalyzes K63-linked ubiquitination associated with immune signaling pathways. Viperin activates ubiquitin transfer by TRAF6 ~2.5-fold and causes a significant increase in polyubiquitinated forms of TRAF6 that are important for mediating signal transduction. Our observations both imply a role for viperin as an agonist of immune signaling and suggest that viperin may activate other K48-linked E3-ligases involved in targeting proteins for proteasomal degradation.

biochemistry↗

bioRxiv2021

Viperin inhibits cholesterol biosynthesis and interacts with enzymes in the cholesterol biosynthetic pathway

Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (virus inhibitory protein endoplasmic reticulum-associated, interferon-induced) is an ER membrane-associated enzyme that when expressed in response to viral infections exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. Here we have investigated the effect of viperin expression on cholesterol biosynthesis. We found that viperin expression reduces cholesterol levels by 20 - 30 % in HEK293T cells. A proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits. The two most highly enriched proteins were lanosterol synthase and squalene monooxygenase, enzymes that catalyze key steps establishing the sterol carbon skeleton. Co-immunoprecipitation experiments established that viperin, lanosterol synthase and squalene monooxygenase form a complex at the ER membrane. Co-expression of viperin was found to significantly inhibit the specific activity of lanosterol synthase in HEK293T cell lysates. Co-expression of viperin had no effect on the specific activity of squalene monooxygenase, but reduced its expression levels in the cells by approximately 30 %. Despite these inhibitory effects, co-expression of either LS or SM failed to reverse the viperin-induced depletion of cellular cholesterol levels in HEK293T cells. Our results establish a clear link between the down-regulation of cholesterol biosynthesis and viperin, although at this point the effect cannot be unambiguously attributed interactions between viperin and a specific biosynthetic enzyme.

biochemistry↗

bioRxiv2020

The human liver microenvironment shapes the homing and function of CD4+ T-cell populations

Background & AimsTissue-resident memory T cells (TRM) are important immune sentinels that provide efficient in situ immunity. Liver-resident CD8+ TRM have been previously described, and contribute to viral control in persistent hepatotropic infections. However, little is known regarding liver CD4+ TRM cells. Here we profiled resident and non-resident intrahepatic CD4+ T cell subsets, assessing their phenotype, function, differential generation requirements and roles in hepatotropic infection. MethodsLiver tissue was obtained from 173 subjects with (n=109) or without (n=64) hepatic pathology. Multiparametric flow cytometry and immunofluorescence imaging examined T cell phenotype, functionality and location. Liver T cell function was determined after stimulation with anti-CD3/CD28 and PMA/Ionomycin. Co-cultures of blood-derived lymphocytes with hepatocyte cell lines, primary biliary epithelial cells, and precision-cut autologous liver slices were used to investigate the acquisition of liver-resident phenotypes. ResultsCD69 expression delineated two distinct subsets in the human liver. CD69HI cells were identified as CD4+ TRM due to exclusion from the circulation, a residency-associated phenotype (CXCR6+CD49a+S1PR1-PD-1+), restriction to specific liver niches, and ability to produce robust type-1 multifunctional cytokine responses. Conversely, CD69INT were an activated T cell population also found in the peripheral circulation, with a distinct homing profile (CX3CR1+CXCR3+CXCR1+), and a bias towards IL-4 production. Frequencies of CD69INT cells correlated with the degree of fibrosis in chronic hepatitis B virus infection. Interaction with hepatic epithelia was sufficient to generate CD69INT cells, while additional signals from the liver microenvironment were required to generate liver-resident CD69HI cells. ConclusionsIntermediate and high CD69 expression demarcates two discrete intrahepatic CD4+ T cell subsets with distinct developmental and functional profiles. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY HighlightsO_LICD69HI (CXCR6+CD49a+S1PR1-PD-1+) are the CD4+ TRM of the human liver C_LIO_LIHepatic CD69INTCD4+ T-cells are distinct, activated, and recirculation-competent C_LIO_LIStimulation evokes respective IFN-{gamma} and IL-4 responses in CD69HI and CD69INT cells C_LIO_LICD69INT cell frequencies correlate with worsening fibrosis in chronic HBV patients C_LIO_LILiver slice cultures allow differentiation of CD69INT and CD69HI cells from blood C_LI Lay summaryTissue-resident memory T cells (TRM) orchestrate regional immune responses, but much of the biology of liver-resident CD4+ TRM remains unknown. We found high expression of cell-surface protein CD69 defined hepatic CD4+ TRM, while simultaneously uncovering a distinct novel recirculatory CD69INT CD4+ T cell subset. Both subsets displayed unique immune receptor profiles, were functionally skewed towards type-1 and type-2 responses respectively, and had distinct generation requirements, highlighting the potential for differential roles in the immunopathology of chronic liver diseases.

immunology↗

bioRxiv2019

The Interaction of Viperin with Hepatitis C Virus Non-Structural Protein 5A Inhibits the Catalytic Activity of Viperin

The radical SAM enzyme viperin exerts a wide range of antiviral effects through both the synthesis of the antiviral nucleotide 3-deoxy-3, 4-didehydro-CTP (ddhCTP) and through its interactions with various cellular and viral proteins. Here we investigate the interaction of viperin with hepatitis C virus non-structural protein 5A (NS5A) and the host sterol regulatory protein, vesicle-associated membrane protein A (VAP-33). NS5A and VAP-33 form part of the viral replication complex that is essential for copying the RNA genome of the virus. Using transfected enzymes in HEK293T cells, we show that viperin binds to both NS5A and VAP-33 independently and that this interaction is dependent on all three proteins being localized to the ER membrane. Co-expression of viperin with VAP-33 and NS5A was found to reduce NS5A levels, most likely by increasing the rate of proteasomal degradation. However, co-expression of viperin with VAP-33 and NS5A also reduces the specific activity of viperin by ~ 3-fold. This observation suggests that NS5A may have evolved to bind viperin as a strategy to reduce ddhCTP synthesis, thereby reducing possibility of the replication complex introducing this chain-terminating nucleotide during genome synthesis.

biochemistry↗

bioRxiv2018

Regulation of IRAK1 Ubiquitination by the Antiviral Radical SAM Enzyme Viperin

Viperin is a radical S-adenosylmethionine (SAM) enzyme that plays a multifaceted role in the cellular antiviral response. Viperin was recently shown to catalyze the SAM-dependent formation of 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), which inhibits some viral RNA polymerases. Viperin is also implicated in regulating K63-linked poly-ubiquitination of interleukin-1 receptor-associated kinase-1 (IRAK1) by the E3 ubiquitin ligase TNF Receptor-Associated Factor 6 (TRAF6) as part of the Toll-like receptor-7 and 9 (TLR7/9) innate immune signaling pathways. We show that IRAK1 and TRAF6 activate viperin to efficiently catalyze the radical-mediated dehydration of CTP to ddhCTP. Furthermore, poly-ubiquitination of IRAK1 requires the association of viperin with IRAK1 and TRAF6. Poly-ubiquitination appears dependent on structural changes induced by SAM binding to viperin but does not require catalytically active viperin. The synergistic activation of viperin and IRAK1 provides a mechanism that couples innate immune signaling with the production of the antiviral nucleotide ddhCTP.

biochemistry↗