Biology subjects

Russell, D. G.

Publications and source records attributed to Russell, D. G..

9 recordsLinked to original sources

bioRxiv2026

Screening of epigenetic modifiers identifies novel host-directed agents that suppress HIV-1 replication in primary human macrophages

Epigenetic modifications play a critical role in diverse biological processes, including HIV-1 replication in lymphoid and myeloid cells, particularly in the establishment and maintenance of latency. As such, epigenetic targets represent potential candidates for novel host directed therapy (HDT). In this study, we developed a novel in vitro screening platform using human primary macrophages, including both monocyte-derived macrophages and alveolar macrophages, infected with a replication-competent luminescent virus. In contrast to previous screens, this platform enables the identification of compounds and epigenetic targets that modulate viral replication during a spreading infection, capturing not only transcriptional regulation but other vulnerabilities across the complete HIV-1 life cycle. Our data reveal multiple novel compounds targeting distinct epigenetic regulators, effectively inhibiting viral replication at various stages in primary macrophages, which may serve as potential candidates/targets for HDT.

immunology↗

bioRxiv2026

Ubiquitylation by the GID/CTLH complex regulates the metabolic and innate immune response of macrophages to infection by Mycobacterium tuberculosis

The GID/CTLH E3 ligase complex is implicated in several biological processes, yet its full substrate repertoire remains poorly defined. We recently identified the complex as a broad modulator of macrophage responses to Mycobacterium tuberculosis (Mtb) infection. Here, we use label-free proteomics and diGly capture analysis of Mtb-infected macrophages to define the GID/CTLH-dependent ubiquitylome. We identify thousands of dynamically altered ubiquitylation sites, with strong enrichment among proteins involved in cellular metabolism and innate immune signaling. Concurrent proteome analysis revealed extensive rewiring in GID/CTLH-deficient macrophages, with >90% of enriched pathways among increased proteins consisting of metabolic targets. Notably, inhibitory phosphatases (PTEN, INPP5D) also emerged as candidate substrates. Functional studies revealed proteasome-dependent stabilization of PTEN and INPP5D in GID/CTLH-deficient macrophages with each phosphatase individually exerting an influence on Mtb intracellular survival. Together, our study defines a GID/CTLH-dependent ubiquitylome in macrophages and identifies the complex as a central regulator of metabolism and antimicrobial immunity. Author summaryMycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), survives and replicates within macrophages, key immune cells that normally eliminate pathogens. How macrophages control their internal cellular environment in response to infection remains incompletely understood. One such important cellular control system is ubiquitylation, in which proteins are tagged with ubiquitin to determine their functional fate or target them for degradation. We recently identified the GID/CTLH E3 ligase ubiquitylation complex as a critical modulator of macrophage antimicrobial responses to Mtb. Here, we used proteomics approaches to define the proteins controlled by the GID/CTLH complex in Mtb-infected macrophages. We found that this complex ubiquitylates a broad network of proteins involved in cellular metabolism and immune signaling. When the complex is disrupted, macrophages undergo extensive metabolic reprogramming, particularly increased mitochondrial energy production, while showing reduced inflammatory signaling. Despite this dampened immune response, these cells are better able to restrict Mtb growth. We also identified the phosphatases PTEN and INPP5D as targets controlled by the GID/CTLH complex that independently influence intracellular bacterial survival. Our findings demonstrate that the GID/CTLH complex is a critical regulator of metabolism and immune function, shaping the outcomes of Mtb infection.

immunology↗

bioRxiv2025

Versatile HIV Rev-dependent reporter cell system for stringent and sensitive quantification of viral reservoirs, neutralizing antibodies, and restriction factors

Detecting and measuring HIV reservoirs, neutralizing antibodies, and restriction factors are important for HIV cure research and the development of new therapeutics and vaccines. Here we describe the development and validation of several HIV Rev-dependent indicator cell lines for these purposes. These reporter cells come from different T-lymphoblast cell lines, including MOLT-4-R5, SupT1-R5, CEM-SS, A3R5, and from the adherent TZM cell platform based on HeLa clone JC53. These cells express CD4, CXCR4, and various levels of CCR5. We compared these cell lines for responsiveness to both X4 and R5-tropic viruses, and confirmed that reporter expression in these cells is not affected by stimulation from mitogens but is responsive to HIV Tat and Rev, reducing non-specific reporter induction from the leaky LTR promoter. To validate the sensitivity of the Rev-dependent reporter cell systems, we conducted a viral dilution assay with three primary HIV-1 clade C swarms from an adult in Malawi. We also validated the systems for quantifying antibody neutralization and screening restriction factors; these systems are also sensitive for viral outgrowth assays for quantifying viral reservoirs in clinical and basic research settings. Given that the systems can measure HIV accurately in complex environments with mitogens or other substances, they can be used for versatile applications, such as quantifying latent reservoirs, testing inhibitory compounds, conducting neutralizing antibody assays, and identifying new restriction factors.

microbiology↗

bioRxiv2025

Spatially-distinct programming of macrophage diversity within the granulomas of Mycobacterium tuberculosis infected nonhuman primates

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is defined by granulomas-- immune aggregates that either contain or support bacterial replication. Macrophages, fundamental components of these lesions, are crucial to TB pathogenesis, yet their phenotypic and functional diversity is incompletely understood. Here, we used single-cell RNA sequencing and immunofluorescence to profile macrophages in lung tissue and granulomas from a nonhuman primate model of early TB. We identified distinct subsets, including embryonic-origin tissue-resident alveolar macrophages and monocyte-derived alveolar and interstitial macrophages, with distinct spatial localization in granulomas. Tissue-resident alveolar macrophages and a subset undergoing epithelial-to-mesenchymal transition accounted for the highest frequency of Mtb-infected cells. Infected cells exhibited differential expression of immune- and migration-associated genes compared to uninfected counterparts, suggesting Mtb either induces or exploits these pathways as a survival strategy. These findings highlight macrophage heterogeneity as a major driver of differential susceptibility to Mtb and provide insights relevant to future immunomodulatory strategies.

immunology↗

bioRxiv2025

Incorporation of macrophage immune stresses into an assay for drug tolerance in intracellular Mycobacterium tuberculosis

Development of new and improved tuberculosis (TB) chemotherapies is hampered by antibiotic resistance and drug tolerance by Mycobacterium tuberculosis (Mtb). Phenotypic drug tolerance, a phenomenon where Mtb populations can temporarily survive therapeutic antibiotic concentrations, represents a significant hurdle to TB treatment and is indeed one of the factors responsible for prolonged TB therapy. Assays that can identify compounds with improved efficacy against drug tolerant Mtb are urgently required to improve TB treatment regimens. Here, we report the development of a 96-well plate assay capable of identifying anti-Mtb drugs with activity against drug tolerant Mtb in physiologically relevant intracellular environments within macrophages. Primary murine macrophages modified either by immunological activation or specific CRISPR/Cas9 gene knockouts to generate tolerance-inducing environments were infected with an Mtb strain constitutively expressing luciferase. Following drug exposure, differences in bacterial survival were measured by bacterial outgrowth after lysis of the host macrophages. By monitoring Mtb luciferase in infected macrophages before, during and after drug treatment, we confirmed earlier observations that host immune stresses trigger induction of drug tolerance. However, while host stresses induced tolerance against some anti-TB compounds, the same host stresses were synergistic with other anti-TB drugs. Our assay provides the ability to profile the activities of anti-TB drugs on bacteria in intracellular host environments which is critical to the rational design of drug combinations that provide optimal coverage of the Mtb sub-populations in the infected host. Author summaryTB treatment is a lengthy process which at minimum takes 6-9 months in cases of drug sensitive Mtb. Long TB treatments are in part, due to drug tolerance phenotypes in the bacterial population which can be a result of immune related stresses on the bacteria in the infected host cells. We used this knowledge to develop a luciferase-based assay which can be used to screen, optimize and discriminate anti-TB drugs with enhanced activity against drug tolerant Mtb inside the infected host cells. Primary murine macrophages were modified either by cytokine activation or genetic knockout of certain host genes to mimic immune related stresses experienced by the bacteria in vivo. By infecting the modified macrophages with an Mtb strain expressing luciferase followed by drug addition, our assay was able to confirm that immune related stress induce drug tolerance to Mtb, and that the drug tolerance phenotypes are unique to certain drug classes consistent with their known mode of action. The assay provides an important addition to anti-TB drug discovery by providing a means to readily screen new drugs and drug combinations that display improved activity in tolerance inducing environments in a physiologically relevant context.

microbiology↗

bioRxiv2024

Accessing anti-HIV activity through the attenuation of USP18 activity: novel insights from molecular dynamic simulations, free-energy profiling, and multi-cellular inhibition assays

The feasibility of achieving anti-HIV activity from the attenuation of USP18 activity was explored for the first time. A cheminformatic survey demonstrated that the current known USP18 isopeptidase inhibitors are derivatives of a bis-aryl pyranone scaffold that possesses undesirable toxicity profiles. Molecular modelling approaches applied to these active bis-aryl pyranones isolated the likely mechanism that perturbs the isopeptidase activity of USP18. Molecular dynamic simulations and free-energy profiling showed that induced-fit effects on the catalytic triad and the IBB-1 domain residues of USP18 drive a reversible non-competitive isopeptidase inhibition mechanism. Proof-of-concept multi-cellular HIV inhibition assays demonstrate the utility of achieving anti-HIV-1 activity from attenuating the activity of USP18 using small molecules. This study motivates for the pursuit of scaffolds that target the allosteric site of USP18, fine-tuning the IFN response as a strategy to enhance the natural control mechanisms that lead to an antiviral state potentially curing viral infection.

immunology↗

bioRxiv2024

Genome-wide screen of Mycobacterium tuberculosis-infected macrophages identified the GID/CTLH complex as a determinant of intracellular bacterial growth.

SummaryThe eukaryotic GID/CTLH complex is a highly conserved E3 ubiquitin ligase involved in a broad range of biological processes. However, a role of this complex in host antimicrobial defenses has not been described. We exploited Mycobacterium tuberculosis (Mtb) induced cytotoxicity in macrophages in a FACS based CRISPR genetic screen to identify host determinants of intracellular Mtb growth restriction. Our screen identified 5 (GID8, YPEL5, WDR26, UBE2H, MAEA) of the 10 predicted members of the GID/CTLH complex as determinants of intracellular growth of both Mtb and Salmonella serovar Typhimurium. We show that the antimicrobial properties of the GID/CTLH complex knockdown macrophages are mediated by enhanced GABAergic signaling, activated AMPK, increased autophagic flux and resistance to cell death. Meanwhile, Mtb isolated from GID/CTLH knockdown macrophages are nutritionally starved and oxidatively stressed. Our study identifies the GID/CTLH complex activity as broadly suppressive of host antimicrobial responses against intracellular bacterial infections. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/592714v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1fc273aorg.highwire.dtl.DTLVardef@173927borg.highwire.dtl.DTLVardef@95cb02org.highwire.dtl.DTLVardef@42fcea_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

HIV-1 latent infection triggers broader epigenomic and transcriptional changes in protein-coding and long non-coding RNAs than active infection of SupT1 cells

Latent HIV-1 infection poses a major challenge in complete viral remission and cure. HIV-1 latency is a multi-dimensional, dynamic process and many aspects of how the viral latency is established and maintained still remains incompletely characterized. Here, we have investigated the host chromatin organization and transcriptomic changes in active- and latently-infected SupT1 cells. We employed an in vitro model of HIV-1 latency in SupT1 cells using a dual-reporter virus, HIVGKO, which enables high purity sorting and characterization of active- and latently-infected cells. We found a significant divergence in chromatin organization and gene expression pattern between active and latent infection compared to uninfected cells. Latent infection results in a repressive reorganization of the host chromatin, while active infection leads to an overall increase in chromatin accessibility. A stronger correlation was also observed between chromatin accessibility and gene expression in latent infection, which was manifested in a greater alteration of the cellular transcriptome in latent than active infection, for both protein-coding and long-non-coding RNAs (lncRNAs). We identified a number of novel lncRNAs associated with either active and latent infection. A reversal in expression pattern of latency-associated lncRNAs following PMA-induced reactivation indicated their infection-state-specific expression and potential roles in HIV-1 latency. Taken together, this integrated, comparative study revealed that latent HIV-1 infection requires a substantially greater alteration in cellular epigenome and transcriptome. Understanding of the distinct cellular states conducive to active and latent infection may support devising strategies for specific modulation of host cellular functions as a curative intervention for HIV-1.

microbiology↗

bioRxiv2017

Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis

Pathogenic bacteria have evolved highly specialized systems to extract essential nutrients from their hosts and Mycobacterium tuberculosis (Mtb) scavenges lipids (cholesterol and fatty acids) to maintain infection in mammals. While the uptake of cholesterol by Mtb is mediated by the Mce4 transporter, the route(s) of uptake of fatty acids remain unknown. Here, we demonstrate that an uncharacterized protein LucA, integrates the assimilation of both cholesterol and fatty acids in Mtb. LucA interacts with subunits of the Mce1 and Mce4 complexes to coordinate the activities of these nutrient transporters. We also demonstrate that Mce1 functions as an important fatty acid transporter in Mtb and we determine that the integration of cholesterol and fatty acid transport by LucA is required for full bacterial virulence in vivo. These data establish that fatty acid and cholesterol assimilation are inexorably linked in Mtb and reveals a key role for LucA in coordinating both transport activities.

microbiology↗