Biology subjects

Shiloh, M. U.

Publications and source records attributed to Shiloh, M. U..

16 recordsLinked to original sources

bioRxiv2026

CMPK2 restricts Mycobacterium tuberculosis replication and regulates macrophage gene expression

Host cell metabolic pathways influence innate immune responses to intracellular pathogens, but the contribution of nucleotide metabolism to antimicrobial defense remains incompletely defined. Here, we identify the mitochondrial nucleoside monophosphate kinase CMPK2 as a regulator of macrophage responses to Mycobacterium tuberculosis (Mtb). Using a targeted genetic screen of candidate host factors, we found that depletion of CMPK2 enhances intracellular Mtb replication in human macrophages. This phenotype was confirmed using both shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout approaches. CMPK2 expression increased following macrophage activation and Mtb infection. Transcriptomic profiling revealed that loss of CMPK2 is associated with broad alterations in gene expression, including reduced expression of genes linked to innate immune and inflammatory responses early after infection. In contrast, myeloid-specific deletion of Cmpk2 in mice did not significantly alter bacterial burden or survival following aerosol Mtb infection, indicating that the contribution of CMPK2 to host defense is context dependent. Together, these findings identify CMPK2 as a host factor that limits Mtb replication in human macrophages and shapes innate immune gene expression programs.

immunology↗

bioRxiv2026

Sorting nexin 5 mediates antigen presentation and immunity against Mycobacterium tuberculosis

Tuberculosis (TB) remains one of the leading causes of death from a single infectious agent worldwide, yet the host pathways that regulate antigen presentation and lung inflammation during Mycobacterium tuberculosis (Mtb) infection are incompletely defined. Sorting nexin 5 (SNX5) is a protein best known for roles in endosomal trafficking, antigen processing, and antiviral host defense, but its contribution to immunity during Mtb infection is unknown. Here, we show that SNX5-deficient mice exhibit markedly increased mortality following low-dose aerosol infection despite unchanged pulmonary bacterial burden compared to wild-type mice. Snx5-/- mice developed exacerbated lung inflammation without major alterations in immune cell recruitment. In macrophages, Snx5 did not affect phagocytosis, vacuolar maturation, intracellular bacterial control, or global transcriptional responses to Mtb, but was required for efficient MHC class II antigen presentation. Snx5 deficiency reduced antigen degradation, limited peptide loading onto MHC-II and impaired activation of antigen-specific CD4+ T cells without altering surface MHC-II abundance or expression of costimulatory molecules. Together, these findings identify SNX5 as a previously unrecognized regulator of MHC-II peptide loading that shapes inflammatory outcomes during pulmonary Mtb infection, highlighting a role for the endosomal sorting machinery in immunity to intracellular pathogens.

immunology↗

bioRxiv2025

Pglyrp1-Cre Marks Distinct Epithelial and Immune Lineages Across Mucosal Sites

Mucosa-associated lymphoid tissue (MALT) initiates immune responses at mucosal entry sites. Within MALT, microfold (M) cells sample luminal antigens and deliver them to underlying immune cells. Despite their functional importance, few tools enable selective manipulation of M cells in vivo. Here we report the generation and characterization of a peptidoglycan recognition protein 1 (Pglyrp1)-Cre knock-in mouse designed to allow conditional genetic access to M cells. Using Rosa26-tdTomato reporter mice, we found strong Pglyrp1 promoter activity in gut epithelial cells, including goblet and M cells, whereas activity in nasal-associated lymphoid tissue (NALT) was more heterogeneous and skewed towards immune cells, particularly neutrophils. To functionally interrogate Pglyrp1-expressing cells, we performed Cre-mediated ablation using three DTA-based models. The Rosa26GFP-DTA line caused marked perinatal lethality in double-positive pups, suggesting essential roles for Pglyrp1-positive cells early in life. In contrast, Rosa26DTA and Rosa26iDTR crosses produced minimal depletion of mucosal populations, including M cells, even at the highest non-lethal diphtheria toxin dose. These findings demonstrate tissue-specific Pglyrp1 promoter activity and highlight challenges in achieving M cell-specific targeting. Although not M cell-restricted, the Pglyrp1-Cre mouse provides a useful tool for manipulating Pglyrp1-expressing lineages and probing their roles in mucosal homeostasis and immunity.

immunology↗

bioRxiv2025

Airflow Determines Natural Airborne Transmission of Tuberculosis in a Guinea Pig Model

Tuberculosis (TB) is transmitted through the air, yet the determinants of natural airborne transmission remain poorly defined. Early twentieth-century guinea pig studies demonstrated efficient airborne transmission of Mycobacterium tuberculosis (Mtb), but this paradigm has not been reestablished in contemporary containment facilities. Here, we show that ventilation can impose airflow constraints that suppress transmission under otherwise permissive conditions. Using a guinea pig model of animal-to-animal exposure, we combined transmission experiments with quantitative airflow measurements and particle transport modeling to explain why some housing configurations fail to support effective exposure. Static environments and excessive unidirectional airflow prevented transmission, whereas controlled low-velocity airflow restored evidence of exposure, including tuberculin skin test conversion, antigen-specific immune responses, and pulmonary inflammation consistent with early infection. These findings identify airflow as a critical constraint on airborne TB transmission and provide a reproducible framework to dissect host, microbial, and environmental determinants of spread.

physiology↗

bioRxiv2025

SMURF2 inhibits autophagic control of Mycobacterium tuberculosis in macrophages

Autophagy is a critical host defense mechanism that restricts intracellular pathogens such as Mycobacterium tuberculosis (Mtb). A key step in this process is the ubiquitination of Mtb or Mtb-associated structures. The E3 ligase SMURF1 catalyzes K48-linked ubiquitination, promoting bacterial clearance. However, the function of its homolog, SMURF2, in host defense remains undefined. Here, we demonstrate that Smurf2 deletion in murine macrophages increases SMURF1 levels, enhances LC3B lipidation, augments K48 ubiquitination of Mtb-associated structures, and reduces intracellular Mtb replication. These effects are reversed by Smurf1 deletion, indicating that SMURF2 restricts autophagy in a SMURF1-dependent manner. Mice with myeloid-specific Smurf2 deletion exhibit modestly prolonged survival following aerosol Mtb infection. In human macrophages, SMURF2 knockdown or its pharmacological inhibition with the HECT ligase inhibitor Heclin reduces Mtb replication. Together, our findings identify SMURF2 as a negative regulator of selective autophagy and host immunity to Mtb and suggest that targeting SMURF2 may represent a novel host-directed therapeutic strategy for tuberculosis.

immunology↗

bioRxiv2025

Cysteine reactivity profiling identifies host regulators of Mycobacterium tuberculosis replication in human macrophages

Innate immune cells such as monocytes and macrophages provide the earliest defense against infection by intracellular pathogens by initiating signaling pathways and restricting pathogen replication. However, the full complement of proteins that mediate cell-autonomous immunity remains incompletely defined. Here, we applied cysteine-directed activity-based protein profiling (ABPP) to map proteome-wide cysteine reactivity changes in THP-1 monocytes and primary human monocyte-derived macrophages during Mycobacterium tuberculosis (Mtb) infection. Across both cell types, we quantified 148 cysteine residues with altered reactivity. Genetic perturbation of a subset of proteins harboring these changes significantly impacted Mtb replication, revealing functional links between site-specific cysteine reactivity and antimicrobial defense. These data define previously unrecognized host protein changes during Mtb infection and provide a resource for investigating post-translational events that regulate innate immune responses to intracellular bacteria.

immunology↗

bioRxiv2025

Deubiquitinase USP15 restricts autophagy and macrophage immunity to Mycobacterium tuberculosis

Autophagy enables macrophages to degrade intracellular Mycobacterium tuberculosis (Mtb), and this defense depends on E3 ubiquitin ligases such as PARKIN and SMURF1, which tag Mtb-associated structures for lysosomal clearance. Deubiquitinases (DUBs) counter ubiquitin ligases by removing ubiquitin from molecular targets. We hypothesized that DUBs might offset ubiquitin ligase activity and negatively regulate host immunity to Mtb. Here, we identify ubiquitin-specific protease 15 (USP15) as a negative regulator of autophagy-mediated macrophage immunity to Mtb. Using a targeted knockdown screen in mouse macrophages, we found that Usp15 loss increased K63-linked ubiquitination and LC3 recruitment to Mtb-associated structures, leading to reduced bacterial replication. These effects required USP15s catalytic activity and were reversed by knockdown of PARKIN (Park2) or inhibition of autophagy initiation. In primary human macrophages, USP15 knockdown similarly enhanced LC3 targeting and restricted Mtb growth. Importantly, pharmacologic inhibition of USP15 with a selective small molecule decreased Mtb burden in human macrophages. Our findings identify USP15 as a suppressor of macrophage immunity and suggest that targeting deubiquitinases may represent a promising host-directed therapeutic strategy against tuberculosis.

immunology↗

bioRxiv2025

Mycobacterium tuberculosis sulfolipid-1 (Sl-1) increases the excitability of mouse and human TRPV1-positive sensory neurons in a YM254890-reversible fashion

Cough is a hallmark sign of tuberculosis and key driver of transmission. While traditionally attributed to host-driven inflammation, we previously demonstrated that Mycobacterium tuberculosis lipid extract (Mtb extract) and its component sulfolipid-1 (SL-1) directly activate nociceptive neurons to induce cough in guinea pigs. However, the cellular mechanisms by which Mtb extract and SL-1 modulate nociceptive sensory neurons remain incompletely understood. Here, we show that Mtb extract enhances action potential (AP) generation in mouse nodose nociceptors via an SL-1-dependent mechanism. Using calcium imaging, we found that Mtb extract and SL-1 increased intracellular Ca{superscript 2} signals in TRPV1 neurons from both mouse nodose and human dorsal root ganglia (hDRG). These Ca{superscript 2} signals were attenuated by the Gq/11 pathway inhibitor YM254890, even in the absence of extracellular Ca{superscript 2}, suggesting involvement of intracellular Ca{superscript 2} stores. Together, these findings indicate that SL-1 engages Gq/11-coupled pathways to sensitize nociceptors via intracellular Ca2+ release, providing mechanistic insight into tuberculosis-associated cough and potential targets for therapeutic intervention.

neuroscience↗

bioRxiv2025

Mycobacterial Phenolic Glycolipid Triggers ATP-Mediated Neuronal P2X3 Signaling and Cough

Cough drives respiratory pathogen transmission, yet how microbes directly engage host sensory neurons to trigger cough is largely unknown. We previously demonstrated that the Mycobacterium tuberculosis (Mtb) glycolipid sulfolipid-1 (SL-1) activates neurons and induces cough. Here, we reveal that phenolic glycolipid (PGL) produced by the hypertransmissible HN878 Mtb strain activates both mouse and human nociceptive neurons in vitro using calcium imaging and electrophysiology and is sufficient to induce cough using plethysmography. Combined with SL-1, PGL potently triggers neuronal activation. By synthesizing various PGL analogs, we show that neuroactivity is proportional to saccharide chain length and structure. Mechanistically, PGL stimulates rapid extracellular ATP release, which engages neuronal P2X3 purinergic receptors--an effect blocked by a P2X3 antagonist. These findings uncover a neuronal activation pathway co-opted by certain Mtb strains to enhance transmission via cough and suggest inhibition of purinergic signaling as a potential strategy to block airborne spread of Mtb.

neuroscience↗

bioRxiv2024

Single cell transcriptional analysis of human adenoids identifies molecular features of airway microfold cells

The nasal, oropharyngeal, and bronchial mucosa are primary contact points for airborne pathogens like Mycobacterium tuberculosis (Mtb), SARS-CoV-2, and influenza virus. While mucosal surfaces can function as both entry points and barriers to infection, mucosa-associated lymphoid tissues (MALT) facilitate early immune responses to mucosal antigens. MALT contains a variety of specialized epithelial cells, including a rare cell type called a microfold cell (M cell) that functions to transport apical antigens to basolateral antigen-presenting cells, a crucial step in the initiation of mucosal immunity. M cells have been extensively characterized in the gastrointestinal (GI) tract in murine and human models. However, the precise development and functions of human airway M cells is unknown. Here, using single-nucleus RNA sequencing (snRNA-seq), we generated an atlas of cells from the human adenoid and identified 16 unique cell types representing basal, club, hillock, and hematopoietic lineages, defined their developmental trajectories, and determined cell-cell relationships. Using trajectory analysis, we found that human airway M cells develop from progenitor club cells and express a gene signature distinct from intestinal M cells. Surprisingly, we also identified a heretofore unknown epithelial cell type demonstrating a robust interferon-stimulated gene signature. Our analysis of human adenoid cells enhances our understanding of mucosal immune responses and the role of M cells in airway immunity. This work also provides a resource for understanding early interactions of pathogens with airway mucosa and a platform for development of mucosal vaccines.

immunology↗

bioRxiv2024

An alveolus lung-on-a-chip model of Mycobacterium fortuitum lung infection

Lung disease due to non-tuberculous mycobacteria (NTM) is rising in incidence. While both two dimensional cell culture and animal models exist for NTM infections, a major knowledge gap is the early responses of human alveolar and innate immune cells to NTM within the human alveolar microenvironment. Here we describe development of a humanized, three-dimensional, alveolus lung-on-a-chip (ALoC) model of Mycobacterium fortuitum lung infection that incorporates only primary human cells such as pulmonary vascular endothelial cells in a vascular channel, and type I and II alveolar cells and monocyte-derived macrophages in an alveolar channel along an air-liquid interface. M. fortuitum introduced into the alveolar channel primarily infected macrophages, with rare bacteria inside alveolar cells. Bulk-RNA sequencing of infected chips revealed marked upregulation of transcripts for cytokines, chemokines and secreted protease inhibitors (SERPINs). Our results demonstrate how a humanized ALoC system can identify critical early immune and epithelial responses to M. fortuitum infection. We envision potential application of the ALoC to other NTM and for studies of new antibiotics.

microbiology↗

bioRxiv2023

Bioorthogonal metabolic labeling of the virulence factor phenolic glycolipid in mycobacteria

Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical Mycobacterium tuberculosis strains. PGL is also found in Mycobacterium marinum where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host-pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor p-hydroxybenzoic acid (pHB) with an azide group (3-azido pHB). When fed to mycobacteria, 3-azido pHB was incorporated into the cell surface, which could then be visualized via bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido pHB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing Mycobacterium marinum and Mycobacterium tuberculosis strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL which may facilitate studies of mycobacterial pathogenesis.

biochemistry↗

bioRxiv2022

CDKL5 regulates p62-mediated selective autophagy and host antiviral defense

Virophagy, the selective autophagosomal engulfment and degradation of viral components, is crucial for antiviral immunity. However, the mechanisms leading to viral antigen recognition and autophagy induction remain poorly understood. Here, we identify a novel kinase, Cyclin-dependent kinase-like 5 (CDKL5), as an essential regulator of virophagy. Deletion of CDKL5 or abrogation of its kinase activity reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid proteins and cellular cytotoxicity. Mechanistically, through direct phosphorylation of the selective autophagy receptor p62, CDKL5 promoted formation of p62 inclusion bodies that bound capsid. Loss of CDKL5 disrupted the capsid-p62 interaction, and a p62 phosphomimetic mutant rescued the interaction. CDKL5 knockout mice demonstrated increased neuronal cell death after SINV infection and enhanced lethality after infection with several human viruses. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear toxic viral capsid aggregates during infection.

microbiology↗

bioRxiv2022

BECN1 F121A mutation increases autophagic flux in aged mice and improves aging phenotypes in an organ-dependent manner

Autophagy is necessary for lifespan extension in multiple model organisms and autophagy dysfunction impacts age-related phenotypes and diseases. Introduction of an F121A mutation into the essential autophagy protein BECN1 constitutively increases basal autophagy in young mice and reduces cardiac and renal age-related changes in longer-lived Becn1F121A mutant mice. However, both autophagic and lysosomal activity have been described to decline with age. Thus, whether autophagic flux is maintained during aging and whether it is enhanced in Becn1F121A mice is unknown. Here we demonstrate that old wild type mice maintained functional autophagic flux in heart, kidney and skeletal muscle but not liver, and old Becn1F121A mice had increased autophagic flux in those same organs compared to wild type. In parallel, Becn1F121A mice were not protected against age-associated hepatic phenotypes but demonstrated reduced skeletal muscle fiber atrophy. These findings identify an organ-specific role for the ability of autophagy to impact organ aging phenotypes.

physiology↗

bioRxiv2019

Identification of scavenger receptor B1 as the airway microfold cell receptor for Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) can enter the body through multiple routes, including via specialized transcytotic cells called microfold cells (M cell). However, the mechanistic basis for M cell entry remains undefined. Here, we show that M cell transcytosis depends on the Mtb Type VII secretion machine and its major virulence factor EsxA. We identify scavenger receptor B1 (SR-B1) as an EsxA receptor on airway M cells. SR-B1 is required for Mtb binding to and translocation across M cells in mouse and human tissue. Together, our data demonstrate a previously undescribed role for Mtb EsxA in mucosal invasion and identify SR-B1 as the airway M cell receptor for Mtb.

immunology↗

bioRxiv2018

Screening Mycobacterium tuberculosis secreted proteins identifies Mpt64 as eukaryotic membrane-binding virulence factor

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secret virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb exported proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 exported proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb exported proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and was necessary for Mtb survival in primary human macrophages. These data highlight the importance of exported proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.\n\nImportanceAdvances have been made to identify exported proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis exported proteins for eukaryotic membrane binding, host subcellular localization and interactions with host vesicular trafficking. In addition, we observed the localization of one exported protein, Mpt64, during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the Type VII Secretion System. Finally, we observed that Mpt64 contributes to the virulence of M. tuberculosis during infection of primary human macrophages.

microbiology↗