Biology subjects

Silverman, N.

Publications and source records attributed to Silverman, N..

5 recordsLinked to original sources

bioRxiv2025

B. pertussis tracheal cytotoxin biases NOD signaling to suppress IL-1 mediated inflammation and evade adaptive immunity

Bordetella pertussis releases the monomeric peptidoglycan (PGN) fragment tracheal cytotoxin (TCT) due to inefficient recycling by the permease AmpG. Releasing this PGN is metabolically costly and potentially immune alarming and the benefits to B. pertussis are unclear. While TCT has been characterized as a potent NOD1 agonist capable of causing the extrusion of ciliated cells, in vitro, the consequences of its release have yet to be studied in vivo. Here we show that selective PGN release by B. pertussis biases host PGN sensing toward NOD1 and away from NOD2, suppressing IL-1{beta}-driven inflammation and blunting adaptive immune recruitment. Mice infected with a TCT over-releasing strain (TCT(+)) exhibit reduced pulmonary immunopathology relative to wild type (WT) and a TCT-under-releasing strain (TCT(-)), despite similar bacterial burdens. NOD reporter assays demonstrate that TCT release enhances NOD1 activation and inversely correlates with NOD2 activation. Bulk transcriptomic analysis of infected lungs shows that B. pertussis PGN release dampens pro-inflammatory transcriptional programs. Single-cell transcriptomic determined Nod2 expression is limited to inflammatory myeloid subsets. IL-1 family genes were highly enriched in Nod2- but not Nod1 expressing alveolar macrophages. Upstream regulator analysis predicted IL-1{beta} as a major driver of B. pertussis inflammation, which was enhanced by the absence of PGN release. Flow cytometry shows that PGN release skews macrophages polarization toward M2 and away from M1 in a NOD1 dependent manner. Finally, extracellular release of PGN and subsequent reduced IL-1 production facilitated the suppression fibroblast chemokine programs (e.g., CXCL13, CCL19), diminished recruitment of B and T cells, reduced iBALT formation, and limited immune memory development. Conversely, IL-1R1 deficiency impairs adaptive recruitment and bacterial clearance despite similar innate infiltration. Together, these data suggest PGN release by B. pertussis is an immune-evasion strategy, favoring NOD1 activation over NOD2, reducing IL-1-dependent fibroblast reprogramming, and curtailing chemokine-driven adaptive responses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/669383v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@ada335org.highwire.dtl.DTLVardef@1b61a0eorg.highwire.dtl.DTLVardef@68c66eorg.highwire.dtl.DTLVardef@bb6e54_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 8.C_FLOATNO Graphic Abstract B. pertussis can produce both NOD1 and NOD2 activating PGNs. Release of TCT promotes NOD1 activation and diminishes NOD2 activation. NOD2 activation in myeloid cells drives M1 polarization of macrophages and IL-1 family cytokine production. IL-1 family cytokines skew fibroblasts towards an inflammatory phenotype, leading to chemokine release, extracellular remodeling, and recruitment of lymphocytes. Therefore, TCT release tempers long-term immunity to B. pertussis. C_FIG

microbiology↗

bioRxiv2024

CRISPR editing of candidate host factors that impact influenza A virus infection

Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that can cause epidemics and pandemics. The host factors required for the complete IAV infectious cycle have not been fully identified. Here, we examined select host factors that were identified by independent CRISPR screens as candidate contributors to IAV infectivity. We performed CRISPR-mediated knockout of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) as well as CRISPR-mediated overexpression of beta-1,4 N-acetylgalactosaminyltransferase 2 (B4GALNT2) and adenosine deaminase acting on RNA 1 (ADAR1) in the human bronchial epithelial A549 cell line and evaluated IAV infectivity. We confirmed that the knockout of CMAS or overexpression of B4GALNT2 restricts IAV infection by diminishing binding to the cell surface but has no effect on vesicular stomatitis virus infection. While ADAR1 overexpression does not significantly inhibit IAV replication, it has a pro-viral effect with coxsackie B virus (CVB) infection. This pro-viral effect is not likely secondary to reduced type I interferon (IFN) production, as the induction of the IFN-stimulated genes ISG15 and CXCL10 is negligible in both parent and ADAR1-overexpressing A549 cells following CVB challenge. In contrast, ISG15 and CXCL10 production is robust and equal for parent and ADAR1-overexpressing A549 cells challenged with IAV. Taken together, these data provide insight into how host factors identified in CRISPR screens can be further explored to understand the dynamics of pro- and anti-viral factors. ImportanceInfluenza A virus (IAV) remains a global threat due to its ability to cause pandemics, making the identification of host factors essential for developing new antiviral strategies. In this study, we utilized CRISPR-based techniques to investigate host factors identified in screens as reducing IAV infectivity. Knockout of CMAS, a key enzyme in sialic acid biosynthesis, significantly reduced IAV binding and infection by disrupting sialic acid production on the cell surface. Overexpression of B4GALNT2 had similar effects, conferring resistance to IAV infection through diminished cell-surface binding. While overexpression of ADAR1, known for its role in RNA editing and immune regulation, slightly reduced IAV replication, it increased coxsackie B virus replication. Such findings reveal the diverse roles of host factors in viral infection, offering insights for targeted therapeutic development against IAV and other pathogens.

immunology↗

bioRxiv2023

Synthesis and validation of click-modified of NOD1/2 agonists

NOD1 and NOD2 sense small bacterial peptidoglycan fragments often called muropeptides. These muropeptides include iE-DAP and MDP, the minimal agonists for NOD1 and NOD2, respectively. Here, we synthesized and validated alkyne-modified muropeptides, iE-DAP-Alk and MDP-Alk, for use in click-chemistry reactions. While it has long been known that many cell types respond to extracellular exposure to muropeptides, it is unclear how these innate immune activators access their cytosolic innate immune receptors, NOD1 and NOD2. The subcellular trafficking and transport mechanisms by which muropeptides access these cytosolic innate immune receptors are a major gap in our understanding of these critical host responses. The clickchemistry-enabled agonists developed here will be particularly powerful to decipher the underlying cell biology and biochemistry of NOD1 and NOD2 innate immune sensing.

immunology↗

bioRxiv2022

Methotrexate inhibition of muropeptide transporter SLC46A2 controls psoriatic skin inflammation

Cytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here we focus on Slc46a2, which is highly expressed in mammalian epidermal keratinocytes, and show that it is critical for delivery of DAP-muropeptides and activation of NOD1 in keratinocytes, while the related transporter Slc46a3 is critical for responding to MDP, the NOD2 ligand. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, while methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively these studies define SLC46A2 as a transporter of NOD1 activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention.

immunology↗

bioRxiv2021

Leishmania amazonensis sabotages host cell SUMOylation for intracellular survival

Leishmania parasites use elaborate virulence mechanisms to invade and thrive in macrophages. These virulence mechanisms inhibit host cell defense responses and generate a specialized replicative niche, the parasitophorous vacuole. In this work, we performed a genome-wide RNAi screen in Drosophila macrophage-like cells to identify host factors necessary for Leishmania amazonensis infection. This screen identified 52 conserved genes required specifically for parasite entry, including several components of the SUMOylation machinery. Further studies in mammalian macrophages found that L. amazonensis infection inhibited SUMOylation within infected macrophages and this inhibition enhanced parasitophorous vacuole growth and parasite proliferation through modulation of multiple genes especially ATP6V0D2, which in turn effects CD36 expression and cholesterol levels. Together, these data suggest that parasites actively sabotage host SUMOylation and alter host transcription to improve their intracellular niche and enhance their replication.

cell biology↗