Biology subjects

Candor, K.

Publications and source records attributed to Candor, K..

5 recordsLinked to original sources

bioRxiv2025

HIV-1 infection of human microglia activates inflammatory pathways associated with HIV-associated neurocognitive disorders (HAND)

HIV-associated neurocognitive disorders (HAND) cause significant dysfunction among people living with HIV. Microglia are the primary immune cells of the central nervous system and are readily infected by HIV. Microglia are thought to contribute to neuroinflammation and cognitive dysfunction in neurodegenerative diseases such as Alzheimers Disease and are likely to play an important role in the pathogenesis of HAND. In order to identify pathways that may contribute to neuropathogenesis in HAND, we infected induced pluripotent stem cell-derived microglia (iMG) with HIV and defined gene expression changes over an 8-day period. Monocyte-derived macrophages (MDMs) were studied in parallel to identify common pathways stimulated in myeloid cells versus the unique aspects of microglia infection. Infection of iMG led to the induction of a robust early inflammatory response triggered within hours of infection, a pattern that differed significantly from that seen in MDMs. Remarkably, gene expression changes in iMG reproduced many of the characteristic genetic signatures previously identified in brain tissues obtained from individuals clinically diagnosed with HAND. Inflammatory activation representing interferon-mediated signaling, TNF/NF-{kappa}B, and IL-6/JAK/STAT pathways were particularly prominent over the time course of infection. Interferon-mediated signaling led to enhanced expression of multiple HIV restriction factors, yet viral replication in iMG remained robust. These findings suggest that HIV infection of microglia is the key cellular driver of neuroinflammation in the CNS of HIV-infected individuals. Further studies of infected microglia are likely to aid in understanding the pathogenesis of HAND and in evaluating therapeutic strategies to limit or eliminate HIV-induced neuropathogenesis. Author SummaryPersons living with HIV frequently develop debilitating problems with brain function that are collectively known as HIV-associated neurocognitive disorders (HAND). Treatment with antiretroviral drugs to control HIV has largely eliminated the most severe form of HIV-related brain dysfunction, HIV encephalitis, but a significant proportion of HIV-infected individuals receiving antiviral therapy still develop significant problems with cognitive function. Microglia are the major macrophage-like cells of the brain and are highly susceptible to HIV infection. Inflammation in the brain following infection contributes to damage to neurons and is the likely source of decline of brain function. This study used microglia that were derived from induced pluripotent stem cells, termed iMG, to study the kinetics of gene expression changes in HIV-infected microglia and compared those changes to those seen in monocyte-derived macrophages (MDMs). iMG were found to be easily infected with a macrophage-tropic HIV strain and exhibited a rapid increase in gene expression associated with inflammatory signaling through interferon- and tumor necrosis factor/NF-kappa B-mediated pathways. Remarkably, the patterns of gene expression in microglia strongly overlapped with gene expression signatures derived from brain tissue samples of HIV patients with HAND. The rapid onset and magnitude of inflammation as well as induction of specific HIV restriction factors differed from that seen in infected macrophages/MDMs. These studies reinforce the central role of microglia in the pathogenesis of HAND and provide insights into HIV-microglia interactions that can help direct interventions to reduce inflammation and preserve brain function.

microbiology↗

bioRxiv2024

The CLIC/GEEC pathway regulates particle endocytosis and formation of the virus-containing compartment (VCC) in HIV-1-infected macrophages

HIV-1 particles are captured by the immunoglobulin superfamily member Siglec-1 on the surface of macrophages and dendritic cells, leading to particle internalization and facilitating trans-infection of CD4+ T cells. HIV-1-infected macrophages develop a unique intracellular compartment termed the virus-containing compartment (VCC) that exhibits characteristic markers of the late endosome and is enriched in components of the plasma membrane (PM). The VCC has been proposed as the major site of particle assembly in macrophages. Depleting Siglec-1 from macrophages significantly reduces VCC formation, implying a link between the capture and endocytosis of external HIV-1 particles and the development of VCCs within HIV-infected cells. We found that endocytosis of particles to the VCC was independent of clathrin, but required dynamin-2. CD98 and CD44, classical markers of the CLIC/GEEC pathway, colocalized with Siglec-1 and HIV-1 particles within the VCC. Inhibition of CLIC/GEEC-mediated endocytosis by chemical or genetic means resulted in the arrest of captured HIV-1 particles on the macrophage cell surface and prevented VCC formation. Virus-like particles (VLPs) were taken up within CD98 and Siglec-1-enriched tubular membranes that migrated centripetally over time to form VCC-like structures. These findings indicate that following capture of virus by Siglec-1, particles follow an endocytic route to the VCC that requires both the CLIC/GEEC pathway and dynamin-2. We propose a model in which internalization of HIV-1 particles together with CLIC/GEEC membranes leads to the formation of the VCC in HIV-infected macrophages, creating an intracellular platform that facilitates further particle assembly and budding. Author SummaryThe major cell types infected by HIV are CD4+ T cells and macrophages. Infection of macrophages is of great interest because this cell type can contribute to transmission of virus within tissues, and because infected macrophages contribute to HIV-related complications that include neurologic disorders, endocrine disorders, and cardiovascular disease. HIV infection of macrophages may also create a latent reservoir that persists in infected individuals despite administration of antiretroviral therapy. Here we focused on a compartment that forms in HIV-infected macrophages termed the virus-containing compartment or VCC. The VCC is an intracellular compartment that has been described as a site of assembly and as a holding compartment for viruses, and contains components of both intracellular organelles and the plasma membrane. We identified an endocytosis pathway that helps to explain the origins of the VCC, the CLIC/GEEC pathway. Inhibition of the CLIC/GEEC pathway prevented virus-like particle delivery to the VCC and prevented the VCC from forming in HIV-infected macrophages. The GTPase dynamin-2 was also required for delivery of HIV particles to the VCC. This study identifies a new facet of how HIV interacts with macrophages, suggesting that disruption of this pathway could be a therapeutic strategy with implications for HIV cure efforts.

microbiology↗

bioRxiv2023

Incorporation of the HIV-1 envelope glycoprotein into viral particles is regulated by the tubular recycling endosome in a cell type-specific manner

The HIV-1 envelope glycoprotein (Env) is incorporated into particles during assembly on the plasma membrane (PM). Env initially reaches the PM through the secretory pathway, after which it is rapidly endocytosed via an AP-2- and clathrin-dependent mechanism. Here we show that endocytosed cell surface Env enters the tubular recycling endosome compartment (TRE). Trafficking to the TRE was dependent upon motifs within the CT previously implicated in Env recycling and particle incorporation. Depletion of TRE components MICAL-L1 or EHD1 led to defects in Env incorporation, particle infectivity, and viral replication. Remarkably, defects were limited to cell types defined as nonpermissive for incorporation of CT-deleted Env, including monocyte-derived macrophages, and not observed in 293T, HeLa, or MT-4 cells. This work identifies the TRE as an essential component of Env trafficking and particle incorporation, and provides evidence that the cell type-dependent incorporation of Env is defined by interactions with components of the TRE.

microbiology↗

bioRxiv2023

Utilization of a Histoplasma capsulatum zinc reporter reveals the complexities of fungal sensing of metal deprivation

Histoplasma capsulatum is a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into yeasts that reside primarily within phagocytes. Macrophages (M{Phi}) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. M{Phi} activated by granulocyte-M{Phi} colony stimulating factor (GM-CSF) induce metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the high affinity zinc importer, ZRT2, was important for fungal survival in vivo. Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under the control of this importer. This reporter accurately responds to medium devoid of Zn. ZRT2 expression increased ([~]5-fold) in GM-CSF, but not interferon-{gamma}, stimulated M{Phi}. To examine the in vivo response, we infected mice with reporter yeasts and assessed ZRT2 expression at 0-, 3-, 7-, and 14-days post-infection (dpi). ZRT2 expression minimally increased at 3-dpi and peaked on 7-dpi, corresponding with onset of adaptive immunity. We discovered that the major phagocyte populations that restrict Zn to the fungus are interstitial M{Phi} and exudate M{Phi}. Neutralizing GM-CSF blunted control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates M{Phi} to control H. capsulatum replication, M-CSF. These findings illustrate the reporters ability to sense Zn in vitro and in vivo and correlate ZRT2 activity with GM-CSF and M-CSF activation of M{Phi}. ImportancePhagocytes use an arsenal of defenses to control replication of Histoplasma yeasts, one of which is limitation of trace metals. On the other hand, H. capsulatum combats metal restriction by upregulating metal importers such as the Zn importer ZRT2. This transporter contributes to H. capsulatum pathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 reporter to probe H. capsulatum Zn sensing during infection and exposed a role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation in vivo and reveal the potential of metal-sensing reporters. The work adds a new dimension to studying how intracellular pathogens sense and respond to the changing environments of the host.

microbiology↗

bioRxiv2020

Dual-purpose isocyanides produced by Aspergillus fumigatus contribute to cellular copper sufficiency and exhibit antimicrobial activity

The maintenance of sufficient but non-toxic pools of metal micronutrients is accomplished through diverse homeostasis mechanisms in fungi. Siderophores play a well-established role for iron homeostasis; however, no copper-binding analogs have been found in fungi. Here we demonstrate that in Aspergillus fumigatus isocyanides derived from the xan biosynthetic gene cluster (BGC) bind copper, impact cellular copper content, and have significant metal-dependent antimicrobial properties. xan BGC-derived isocyanides are secreted and bind copper as visualized by a chrome azurol S (CAS) assay and inductively coupled plasma-mass spectrometry (ICP-MS) analysis of A. fumigatus intracellular copper pools demonstrated a role for xan cluster metabolites in the accumulation of copper. A. fumigatus coculture with A. nidulans, Candida albicans and a variety of pathogenic bacteria establish copper-dependent antimicrobial properties of xan BGC metabolites including inhibition of laccase activity. Similarly, inhibition of Pseudomonas aeruginosa by low concentrations of the xan isocyanide xanthocillin was copper-dependent. Other metals also reduced xanthocillins antimicrobial properties, but less efficiently than copper. As variations of the xan BGC exist in other filamentous fungi, we suggest that xanthocillin-like natural products represent a first example for fungal small molecules that serve to maintain copper sufficiency and mediate interactions with competing microbes. SignificanceMetal homeostasis is an integral part of metabolism for any organism. A vast array of small molecules are already known to mediate metal homeostasis in fungi and bacteria; however, unlike their bacterial counterparts, to-date there are no known fungal small molecules that function to maintain copper homeostasis. Discovery of copper binding small molecules produced by A. fumigatus gives insight into mechanisms other than the extensively studied copper transporters or metalloproteins for how fungi can regulate copper. This has important ecological implications as securing scarce nutrients is central for fitness and survival. Additionally, studying this mechanism in A. fumigatus provides a basis for investigation of copper regulation pathways in other fungi.

microbiology↗