Biology subjects

Johnson, J. R.

Publications and source records attributed to Johnson, J. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Integrative proteomic analysis and molecular dynamics simulations of ANKLE2 reveal mechanisms of microcephaly

ANKLE2 is a scaffolding protein with crucial roles in neuroprogenitor cell division and embryonic brain development. Pathogenic variants in ANKLE2 cause primary microcephaly, a congenital disorder characterized by impaired neurodevelopment. Nonetheless, the underlying dysfunction of ANKLE2 is not understood. Here, we define the ANKLE2 protein interaction landscape, which includes cell division proteins and novel microcephaly candidates in the APC (anaphase-promoting complex). Analysis of six pathogenic variants reveals changes in this interactome that likely result in microcephaly. Using molecular simulations, we identify the structural consequences of five pathogenic substitutions, including disruption of important helical structures. Combined, these techniques suggest loss of interaction with the PP2A (protein phosphatase 2A) complex is a common mechanism for ANKLE2 pathogenesis. Phosphoproteomics reveals widespread alterations in ANKLE2- and PP2A-dependent phosphorylation of cell division proteins. Together, our integrative proteomics and simulation approach improves the molecular understanding of ANKLE2 function and its pathogenic changes in primary microcephaly.

systems biology↗

bioRxiv2026

The Hao-Fountain syndrome gene USP7 restricts neurotropic orthoflavivirus entry through cell intrinsic control of endosomal dynamics

Neurodevelopmental disorders are increasingly associated with immune phenotypes, including autoinflammation, immunodeficiency, and increased susceptibility to severe infection. To determine whether neurodevelopmental disorders-associated genes exert immune functions, we performed an arrayed siRNA screen targeting 28 genes with nonredundant cellular roles and assessed their effects on Zika virus (ZIKV) infection and innate immune pathways. We identified hits that intrinsically restrict ZIKV infection and modulate inflammatory pathways following infection. We further characterized the antiviral activity of the Hao-Fountain syndrome gene USP7, which potently restricts selected neurotropic orthoflaviviruses. USP7 inhibits ZIKV internalization before viral membrane fusion and genome release into the cytoplasm. Because USP7 plays a role in endosomal tubulation and recycling, we investigated whether endosomal recycling pathways restrict ZIKV infection. We identified the USP7-regulated E3 ubiquitin ligase TRIM27, as well as the recycling-associated Rab GTPases RAB11 and RAB35, as potent regulators of ZIKV infection. Infection assays using cell lines expressing pathogenic USP7 variants and primary fibroblasts from individuals with Hao-Fountain syndrome demonstrated that disease-associated USP7 mutations impair its antiviral activity and increase permissivity to ZIKV infection. These findings are consistent with recent case reports of unusually severe viral infection during early life in individuals with Hao-Fountain syndrome. Collectively, our study identifies endosomal recycling pathways as important intrinsic restriction mechanisms against neurotropic orthoflaviviruses and nominates pathogenic USP7 variation as a candidate inborn error of immunity.

microbiology↗

bioRxiv2026

Powassan Virus NS5 Antagonizes TYK2-Mediated Immune Signaling Pathways

Powassan virus (POWV) is an emerging neurotropic tick-borne flavivirus, yet the mechanisms by which POWV evades host antiviral immunity remain poorly defined. Here, we identify multiple mechanisms of POWV innate immune antagonism with the viral polymerase NS5 protein as a central inhibitor of cytokine signaling. Both POWV lineages potently inhibited type I interferon (IFN) signaling, and NS5 expression suppressed signaling and downstream interferon-stimulated gene expression. Affinity purification-mass spectrometry identified the host kinase TYK2 as a conserved NS5 interactor. POWV NS5 binds the TYK2 kinase domain through a discrete interface within the RNA-dependent RNA polymerase (RdRp) region between catalytic motifs B and C and inhibits TYK2 phosphorylation. Disruption of this interface abrogated TYK2 binding and reduced NS5-mediated IFN antagonism, while revealing additional TYK2-independent mechanisms of immune suppression. POWV NS5 also inhibited TYK2-dependent IFN-{lambda} and IL-12 signaling, demonstrating that its immune antagonism extends beyond type I IFN. Together, these findings identify TYK2 as a central target of POWV immune evasion and implicate the variable RdRp B-C region as an interface for flavivirus-host interactions. More broadly, our results reveal how POWV can coordinately suppress multiple antiviral cytokine pathways and provide insight into mechanisms that may shape tick-borne flavivirus host adaptation and pathogenesis.

microbiology↗

bioRxiv2026

An Inverse Transwell Assay for Airway Mucus Barrier Function Reveals both Virus- and Mucin-Specific Impacts on Infection

Respiratory viruses are a significant cause of morbidity and mortality world-wide and an important trigger of acute exacerbation in chronic lung disease. Secreted airway mucus - a front-line defense system against respiratory virus infection - is largely composed of glycosylated mucins that promote virus trapping via steric and adhesive interactions. Still, the degree to which mucus can trap specific viruses is unclear. Further, mucin expression is altered in chronic lung disease with undefined impacts on host susceptibility to infection. Here, we devised an inverse Transwell assay (ITA) to specifically probe the barrier function of mucus towards infection without the confounding effects of ongoing mucus secretion and transport on viral dynamics. Using the ITA, we assessed the barrier function of human airway epithelial (HAE) culture-derived mucus towards influenza (IAV), rhinovirus (RV), adenovirus, and parainfluenza virus. Results showed differences in mucus penetration efficiency between viruses, with IAV being the most inhibited relative to no mucus controls. Notably, IAV and RV penetration efficiency was similar between normal mucus and mucus sourced from an in-vitro model of asthmatic epithelium. To further explore the role of specific mucins during infection, we employed CRISPR/Cas9-modified HAE cultures lacking either MUC5B or MUC5AC expression. Direct infection in these cultures with IAV yielded higher viral titers compared to control HAE, suggesting both MUC5B and MUC5AC contribute to antiviral defense. Application of mucus harvested from specific mucin-knockout or control HAE in the ITA revealed that while RV penetration was similar across conditions, IAV was more efficient in breaching MUC5AC-depleted gels. Subsequent biophysical analysis of these mucus gels revealed a larger pore structure in the absence of MUC5AC. Together, these data indicate mucus-mediated restriction is virus dependent and highlight the contribution of MUC5AC to mucus structure and antiviral capabilities. Further, they establish the ITA as a tunable platform enabling investigation into mucus penetration by diverse viruses and the effects of altered mucus composition on barrier function. Clinical RelevanceMucus composition is altered in chronic lung disease states and during inflammation with unknown consequences on its barrier function towards respiratory virus infection. Using an inverse Transwell assay, we describe virus-specific kinetics through mucus representative of health and disease and identify a critical role for MUC5AC in defense towards influenza A virus. This work can inform future strategies to improve infection prevention or mucus targeted therapies, and may help explain differences in susceptibility to viral infections across the population.

microbiology↗

bioRxiv2025

Nuclear activities and interactome of the NS5 protein of Tick-Borne Encephalitis Virus

Orthoflaviviruses are RNA viruses responsible for significant diseases in humans, domesticated animals and wildlife. Their NS5 protein is central in viral replication, functioning both as an RNA-dependent RNA polymerase and a methyltransferase, while also modulating cellular processes, including the interferon response. Although viral replication is cytoplasmic, the NS5 protein of several mosquito-borne orthoflaviviruses cycles between the cytoplasm and the nucleus of infected human cells. However, the nuclear localization and function of NS5 of tick-borne orthoflaviviruses, such as tick-borne encephalitis virus (TBEV), remained poorly understood. Microscopy analysis and cell fractionation revealed that the NS5 protein of TBEV localized to both the cytoplasm and nucleoplasm of infected cells. Mutagenesis studies identified critical residues required for its nuclear targeting. Mutating these residues in a TBEV replicon abolished viral replication. Immunoprecipitation-mass spectrometry analyses performed in two human cell lines infected with TBEV recovered 352 NS5 partners. Among them, 187 were nuclear or partially nuclear. By integrating our interactome data with that of Powassan virus (POWV), another tick-borne orthoflavivirus, we refined a list of 20 high-confidence NS5 partners, including splicing factors and chromatin modulators. Functional analysis revealed that seven of these nuclear partners significantly modulated viral replication, further underscoring the importance of nuclear NS5 in the viral life cycle. Our work advances our understanding of the nuclear function of the NS5 proteins of tick-borne orthoflaviviruses. ImportanceTick-borne orthoflaviviruses are emerging globally, spreading across Europe, Asia, and North America, where they infect humans, domesticated animals, and wildlife. These viruses produce a protein called NS5, which drives viral replication and helps evade the innate immune response. We observed that the NS5 protein of tick-borne encephalitis virus (TBEV) localized both in the cytoplasm and nucleoplasm of infected human cells. We identified the specific residues responsible for its nuclear addressing and showed that it interacts with numerous nuclear proteins, including some involved in regulating gene expression. Seven of these nuclear partners significantly influenced viral replication, highlighting the importance of NS5s nuclear activity. This work sheds light on how tick-borne orthoflaviviruses manipulate host cells, deepening our understanding of their replication strategies.

microbiology↗

bioRxiv2025

HIV-1 latency reversing agents converge on phosphoregulation of nuclear protein complexes

Despite the success of antiretroviral therapy (ART), HIV-1 persists in latently infected cells, posing a central barrier to a cure. "Shock-and-kill" strategies using latency-reversing agents (LRAs) have shown some promise in reactivating viral gene expression ex vivo, but have yielded little clinical efficacy, underscoring the need for deeper insight into the molecular mechanisms that govern reactivation. Here, we performed deep quantitative phosphoproteomics of J-Lat 10.6 cells treated with diverse LRAs: SAHA, PMA, or prostratin. We identified 48,476 confidently localized phosphorylation sites mapping to 6,672 proteins, with SAHA inducing the most extensive changes. Regulated phosphoproteins were enriched in chromatin organization, transcription, RNA processing, nuclear transport, and cytoskeletal remodeling. Although LRAs regulated overlapping pathways, they elicited divergent kinase activities and site-specific phosphorylation patterns. A reproducible core of 3,502 phosphorylation sites on 1,432 proteins mapped to 39 nuclear protein complexes, including the spliceosome, Mediator, NF-{kappa}B, and RNA polymerase II. Remarkably, 20 protein complexes were phosphoregulated by all three LRAs, but at distinct sites, revealing convergence on shared nuclear machinery through distinct mechanisms. This study provides a comprehensive map of protein complex phosphorylation remodeling during HIV-1 reactivation and highlights signaling mechanisms that could guide the rational design of next-generation LRAs with improved efficacy and reduced toxicity.

microbiology↗

bioRxiv2025

Modelling cigarette smoke-induced lung vascular dysfunction using an alveolus-on-chip

The alveolus is central to gas exchange in the lung, and alveolar damage is a characteristics of a variety of lung diseases. Understanding alveolar microvascular dynamics and epithelial-endothelial interactions is essential for accurately modeling alveolar physiology and its dysfunction in lung diseases such as Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis and acute respiratory distress syndrome (ARDS). In this study, we present an open-top, membrane-free alveolus-on-chip platform incorporating self-assembled, perfusable 3D vascular networks by primary human lung endothelial cells and pericytes, co-cultured with alveolar epithelial type 2 (AEC2) cells. These vascular networks were developed within 6 days under continuous flow and remained stable for at least 12 days. The inclusion of pericytes supported capillary-like vessel formation and increased gene expression of EDNRB1, a gene enriched in alveolar microvascular endothelial cells. Furthermore, CD31 gene expression was higher in 3D endothelial networks compared to 2D endothelial monolayers, suggesting increased cell-cell adhesion. Monocytes could be successfully perfused through the networks, expanding the platforms potential for studying immune interactions in lung disorders. Culturing AEC2 monolayers directly on the vascularized hydrogel enabled physiologically relevant cell-cell interactions without artificial membranes, while maintaining air-liquid interface conditions. Importantly, exposure of the AEC2 layer to whole cigarette smoke (WCS) led to complete disintegration of the underlying vascular network, an effect not observed in the absence of AEC2. This chip model provides a human-relevant system for investigating vascular-epithelial crosstalk in the alveolus, smoke-induced lung injury, and immune recruitment, offering a valuable platform for future disease modeling and drug testing applications.

bioengineering↗

bioRxiv2025

Glypican-1 upregulation elicited in response to a cell-impermeable kinase inhibitor and its overexpression enhance HIV-1 infection

Studies of herpes simplex virus (HSV) entry uncovered a previously unappreciated "outside-in" signaling pathway whereby activation of the calcium (Ca2+) responsive enzyme phospholipid scramblase 1 (PLSCR1), which is known to trigger the bidirectional movement of phosphatidylserines (PS) between the inner and outer leaflet of the plasma membrane, also induces the translocation and subsequent extracellular activation of intracellular proteins, including Akt. We hypothesized that HIV-1, which has been shown to elicit scramblase TMEM16F-mediated PS externalization, may trigger a similar "outside-in" signaling cascade involving exofacial kinase activity to promote its entry into CD4+ T cells. To study this process, we utilized a cell impermeable staurosporine analogue, alkyl-CIMSS, which is a broadly active kinase inhibitor that blocks HSV-induced exofacial Akt phosphorylation and HSV infection. Using multiple cell types including TZM-bl, Jurkat T cells, and human peripheral blood mononuclear cells (PBMCs), we show that, in contrast to the effects on HSV, treatment of cells with alkyl-CIMSS enhances HIV-1 infection post-entry that is not dependent on TMEM16F. To identify potential biological processes that are responsive to alkyl-CIMSS, we performed bulk RNA-sequencing and whole cell proteomics and found that alkyl-CIMSS treatment of cells robustly upregulates the cell surface density of the proteoglycan glypican-1 (GPC1). Lentiviral delivery of GPC1 overexpression and shRNA knockdown constructs reveal that the presence and absence of GPC1 independently of alkyl-CIMSS treatment significantly impact HIV-1 infection, with the effect on infection corresponding to GPC1 expression. Further, we demonstrate that the influence of GPC1 on HIV-1 infection is in part mediated by TGF-{beta} signaling. Collectively, these findings implicate a cell surface protein susceptible to alkyl-CIMSS in restricting HIV-1 infection and identify GPC1 as a novel modulator of HIV-1 infection. Author SummaryWe utilized a cell-impermeable pan-kinase tool compound, alkyl-CIMSS, to identify cell surface molecules that might be involved in viral infection. Treatment of CD4+ T cells with alkyl-CIMSS increased the expression of the cell-surface protein glypican-1, which led to an increase in early HIV reverse transcriptase products and promoted viral infection. Conversely, alkyl-CIMSS inhibited herpes simplex virus entry and infection. These findings illustrate that viruses interact with exofacial cell membrane molecules differently to promote or impede infection.

microbiology↗

bioRxiv2025

A proteome-wide, MS-based screen identifies SUMOylation of host RNA splicing factors induced by HIV-1 infection

HIV-1 exploits host cell post-translation modifications (PTMs) to facilitate production of infectious particles. These modifications include SUMOylation, a dynamically regulated PTM involving covalent attachment of small ubiquitin-like modifiers (SUMOs) to lysine (K) residues of target proteins. SUMOylation modulates the activity of thousands of proteins and multiple fundamental host cellular processes, including pathways hijacked by HIV-1 to promote infection and spread. The SUMOylation of several proteins during HIV-1 infection has been characterized. However, the broad effects of HIV-1 infection on the SUMOylation of the host cell proteome is largely unknown. To date, SUMOylation has not been explored by large-scale proteomics in the context of HIV infection, where many SUMO-regulated host dependency factors remain to be identified. In this study, we performed a proteome-wide, mass spectrometry (MS)-based screen to identify proteins that are SUMOylated during HIV-1 infection. Here, and in biochemical assays, infection with HIV-1 led to the widespread increased SUMOylation of the heterogeneous nuclear ribonucleoprotein (HNRNP) A/B proteins, a protein family central to the regulation of alternative splicing. Intriguingly, this phenotype was found to be driven by expression of the HIV-1 Viral Infectivity Factor (Vif), suggesting a novel function for this protein aside from APOBEC3G degradation. We selected HNRNPA2B1 (A2/B1) and HNRNPA3 for further study, where depletion of these proteins led to the altered splicing of HIV-1 viral RNAs and dramatically reduced HIV-1 infectivity. Considering the enrichment of SUMOylation sites within the RNA-binding domains of the HNRNPA/B family, our data suggest a novel mechanism involving HIV-1-induced, Vif-mediated SUMOylation of host RNA splicing factors as a means to regulate HIV-1 alternative splicing. Broadly, our findings suggest that infection with HIV-1 alters the SUMOylation of many unexplored host cellular proteins, and provides a significant proteomic resource for their future mechanistic study.

molecular biology↗

bioRxiv2024

The EZH2 inhibitor tazemetostat mitigates HIV immune evasion, reduces reservoir formation, and promotes durable CD8+ T-cell revitalization

Persistent HIV reservoirs in CD4 T-cells pose a barrier to curing HIV infection. We identified overexpression of enhancer of zeste homolog 2 (EZH2) in HIV-infected CD4 T- cells that survive cytotoxic T lymphocyte (CTL) exposure, suggesting a mechanism of CTL resistance. Inhibition of EZH2 with the FDA-approved drug tazemetostat increased surface expression of major histocompatibility complex class I (MHC-I) on CD4 T-cells, counterbalancing HIV Nef-mediated MHC-I downregulation. This improved CTL-mediated elimination of HIV-infected cells and suppressed viral replication in vitro. In a participant-derived xenograft mouse model, tazemetostat elevated MHC-I and the pro-apoptotic protein BIM in CD4 T-cells, facilitating CD8 T-cell-mediated reductions of HIV reservoir seeding. Additionally, tazemetostat promoted sustained skewing of CD8 T-cells toward less differentiated and exhausted phenotypes. Our findings reveal EZH2 overexpression as a novel mechanism of CTL resistance and support the clinical evaluation of tazemetostat to enhance clearance of HIV reservoirs and improve CD8+ T-cell function.

immunology↗

bioRxiv2024

Broad-spectrum RNA antiviral inspired by ISG15-/- deficiency

Type I interferons (IFN-I) are cytokines with potent antiviral and inflammatory capacities. IFN-I signaling drives the expression of hundreds of IFN-I stimulated genes (ISGs), whose aggregate function results in the control of viral infection. A few of these ISGs are tasked with negatively regulating the IFN-I response to prevent overt inflammation. ISG15 is a negative regulator whose absence leads to persistent, low-grade elevation of ISG expression and concurrent, self-resolving mild autoinflammation. The limited breadth and low-grade persistence of ISGs expressed in ISG15 deficiency are sufficient to confer broad-spectrum antiviral resistance. Inspired by ISG15 deficiency, we have identified a nominal collection of 10 ISGs that recapitulate the broad antiviral potential of the IFN-I system. The expression of the 10 ISG collection in an IFN-I non-responsive cell line increased cellular resistance to Zika, Vesicular Stomatitis, Influenza A (IAV), and SARS-CoV-2 viruses. A deliverable prophylactic formulation of this syndicate of 10 ISGs significantly inhibited IAV PR8 replication in vivo in mice and protected hamsters against a lethal SARS-CoV-2 challenge, suggesting its potential as a broad-spectrum antiviral against many current and future emerging viral pathogens. One-Sentence SummaryHuman inborn error of immunity-guided discovery and development of a broad-spectrum RNA antiviral therapy

immunology↗

bioRxiv2024

Global mapping of the Chlamydia trachomatis conventional secreted effector-host interactome reveals CebN interacts with nucleoporins and Rae1 to impede STAT1 nuclear translocation.

To usurp host defenses and establish a replicative niche, obligate intracellular pathogens are tasked with remodeling the host cell using a comparatively small repertoire of effector proteins. For Chlamydia trachomatis (C.t), discovery of secreted proteins and their host targets has been particularly challenging due to the bacteriums historical genetic intractability. Using affinity purification-mass spectrometry, we defined host interaction partners for 21 secreted effector proteins, providing the first comprehensive type III secretion system (T3SS) effector- host interactome generated during infection. Among these, we show that the C-terminus of CebN (CT584) binds multiple nucleoporins and Rae1, host factors previously associated only with viral immune evasion. Remarkably, we shown that CebN localizes to the nuclear envelope not only in infected cells but also in uninfected bystander cells. Functionally, CebN is both necessary and sufficient to perturb STAT1 nuclear import following IFN-{gamma} stimulation and its expression is critical for C.t. survival, as evidenced by reduced bacterial replication and smaller inclusions in cells infected with a CebN mutant. Together, these finds expand our understanding of chlamydia effector biology and highlight novel bacterial strategies for manipulating host defenses at the nuclear pore. SIGNIFICANCEChlamydia trachomatis (C.t.) is a leading cause of sexually transmitted infections and blindness, yet the molecular mechanisms it uses to manipulate host defenses remain poorly defined. Unlike many pathogens, C.t. relies on a limited set of effectors to remodel the host cell and establish its niche. We identified host targets for 21 C.t. effector proteins. Focusing on CebN, we show that it binds nucleoporins and Rae1, host factors previously linked only to viral immune antagonism. CebN localizes to the nuclear envelope of both infected and bystander cells, and is critical for replication, inclusion development, and perturbation of STAT1 nuclear import following IFN-{gamma} stimulation. These findings uncover a novel strategy by which C.t. manipulates nuclear pore function to evade host defenses and establish infection.

microbiology↗

bioRxiv2024

Insects visit Fusarium xyrophilum pseudoflowers on the host Xyris surinamensis (Xyridaceae) and carry fungal DNA on their bodies

The fungus Fusarium xyrophilum produces flower-like structures (i.e., pseudoflowers) that were recently discovered on yellow-eyed grasses (Xyris spp.) in Guyana. It is unknown whether these pseudoflowers, which are composed entirely of fungal tissue, are true mimics that attract insects as a means of fungal dispersal. We evaluated the potential of F. xyrophilum to affect insect visitation patterns to flowers and pseudoflowers by 1) documenting insect visitation to X. surinamensis in Guyana, 2) measuring the presence of F. xyrophilum DNA on insects, and 3) evaluating fluorescence and volatile production on flowers and pseudoflowers. We report for the first time Vespidae, Formicidae, Salticidae, Acrididae, and Tetrigidae visiting Xyris. Diverse insects, including Conocephalini spp. (meadow katydids; Tettigoniidae), Camponotus spp. (carpenter ants; Formicidae), and a Geometridae sp. (geometer moths) were found to visit flowers and pseudoflowers. Fusarium xyrophilum DNA was detected on 3/12 (25%) of captured insect bodies using conventional and quantitative PCR. Volatiles produced in the field by pseudoflowers and flowers were similar, except for the presence of a sesquiterpene, putatively identified here as -gurjunene, which was detected both in F. xyrophilum pure cultures and field-collected pseudoflower samples, but not from flowers. The production of this sesquiterpene by F. xyrophilum and the fluorescence of X. surinamensis peduncles represent potential signals involved in insect attraction for this system. These observations, along with the overlap in insect visitors of flowers and pseudoflowers and the detection of F. xyrophilum DNA on insect bodies, are consistent with insect visitors being vectors of Xyris pollen and F. xyrophilum propagules between host plants.

ecology↗

bioRxiv2024

Spatial control of sensory adaptation modulates mechanosensing in Pseudomonas aeruginosa

Sensory signaling pathways use adaptation to dynamically respond to changes in their environment. Here, we report the mechanism of sensory adaptation in the Pil-Chp mechanosensory system, which the important human pathogen Pseudomonas aeruginosa uses to sense mechanical stimuli during surface exploration. Using biochemistry, genetics, and cell biology, we discovered that the enzymes responsible for adaptation, a methyltransferase and a methylesterase, are segregated to opposing cell poles as P. aeruginosa explore surfaces. By coordinating the localization of both enzymes, we found that the Pil-Chp response regulators influence local receptor methylation, the molecular basis of bacterial sensory adaptation. We propose a model in which adaptation during mechanosensing spatially resets local receptor methylation, and thus Pil-Chp signaling, to modulate the pathway outputs, which are involved in P. aeruginosa virulence. Despite decades of bacterial sensory adaptation studies, our work has uncovered an unrecognized mechanism that bacteria use to achieve adaptation to sensory stimuli.

microbiology↗

bioRxiv2022

Deficiency in Galectin-3, -8, and -9 impairs immunity to chronic Mycobacterium tuberculosis infection but not acute infection with multiple intracellular pathogens

Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity in vivo, we generated Gal-3, -8, and - 9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective lysosomal trafficking but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection. Author SummaryIntracellular bacterial pathogens cause many of the worlds most deadly infectious diseases. A common requirement for nearly all intracellular pathogens is the ability to damage the endomembrane compartments in which they reside, which allows pathogens access to the nutrient-rich cytosol of the host. However, membrane damage also creates a "pattern of pathogenesis" that triggers antimicrobial immune responses. Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) act as a surveillance system for membrane damage and Gal-3 and Gal-8 inhibit bacterial growth by activating autophagy, a cellular pathway that can capture cytosolic bacteria and degrade them in lysosomes. Membrane damage-sensing galectins were hypothesized to promote bacterial killing during acute infection yet their role in the immune response of an infected animal remains unclear. Here, we show that mice deficient for Gal-3, -8, and -9 had no defects in resistance to acute infection with the pathogens Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Mycobacterium tuberculosis (Mtb), and were only modestly susceptible to chronic Mtb infection. Our data suggest that Gal-3, -8 and -9 are not critical for innate immune responses during acute infection and may play a more prominent role in the adaptive immune response. These results broaden our understanding of the role of membrane damage-sensing pathways in host defense against bacterial infection.

immunology↗

bioRxiv2022

Hyperglycemia increases the severity of Staphylococcus aureus osteomyelitis and influences bacterial genes required for survival in bone.

Hyperglycemia, or elevated blood glucose, renders individuals more prone to developing severe Staphylococcus aureus infections. S. aureus is the most common etiological agent of musculoskeletal infection, which is a common manifestation of disease in hyperglycemic patients. However, the mechanisms by which S. aureus causes severe musculoskeletal infection during hyperglycemia are incompletely characterized. To examine the influence of hyperglycemia on S. aureus virulence during invasive infection, we used a murine model of osteomyelitis and induced hyperglycemia with streptozotocin. We discovered that hyperglycemic mice exhibited increased bacterial burdens in bone and enhanced dissemination compared to control mice. Furthermore, infected hyperglycemic mice sustained increased bone destruction relative to euglycemic controls, suggesting that hyperglycemia exacerbates infection-associated bone loss. To identify genes contributing to S. aureus pathogenesis during osteomyelitis in hyperglycemic animals relative to euglycemic controls, we used transposon sequencing (TnSeq). We identified 71 genes uniquely essential for S. aureus survival in osteomyelitis in hyperglycemic mice and another 61 mutants with compromised fitness. Among the genes essential for S. aureus survival in hyperglycemic mice was superoxide dismutase A (sodA), one of two S. aureus superoxide dismutases involved in detoxifying reactive oxygen species (ROS). We determined that a sodA mutant exhibits attenuated growth in vitro in high glucose and in vivo during osteomyelitis in hyperglycemic mice. SodA therefore serves an important role during growth in high glucose and promotes S. aureus survival in bone. Collectively, these studies demonstrate that hyperglycemia increases the severity of osteomyelitis and identify genes contributing to S. aureus survival during hyperglycemic infection.

microbiology↗

bioRxiv2022

Immunological landscape of human lymph nodes during ex vivo measles virus infection

In humans, lymph nodes are the primary site of measles virus (MeV) replication. To understand the immunological events that occur at this site, we infected human lymphoid tissue explants using a pathogenic strain of MeV that expresses GFP. We found that MeV infected between 5-15% of cells across donors. Using single cell RNA-Seq (scRNA-Seq) and flow cytometry, we found that while most of the 29 cell populations identified in the lymphoid culture were susceptible to MeV, there was a broad preferential infection of B cells and reduced infection of T cells. Further subsetting of T cells revealed that this reduction may be driven by the decreased infection of naive T cells. Transcriptional changes in infected B cells were dominated by an interferon stimulated gene (ISG) signature. To determine which of these ISGs were most substantial, we evaluated the proteome of MeV-infected Raji cells by mass spectrometry. We found that IFIT1, IFIT2, IFIT3, ISG15, CXCL10, MX2, and XAF1 proteins were the most highly induced, and positively correlated with their expression in the transcriptome. These data provide insight into the immunological events that occur in lymph nodes during infection and may lead to the development of therapeutic interventions.

immunology↗

bioRxiv2022

Quantitative proteomic analysis reveals apoE4-dependent phosphorylation of the actin-regulating protein VASP

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimers Disease (AD). While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in VASP S235 phosphorylation in a PKA-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.

neuroscience↗