Biology subjects

St. Leger, A. J.

Publications and source records attributed to St. Leger, A. J..

7 recordsLinked to original sources

bioRxiv2026

Defining the Functional Consequence of the Human Ocular Microbiome

The ocular surface is one of the body's most environmentally exposed mucosal barriers, yet it is considered low biomass and harbors orders of magnitude fewer bacteria than microbe-rich sites like the gut. This has cast doubt on whether a functional core ocular microbiome exists at all. Having previously defined a protective gd T cell and IL-17 axis engaged by the murine commensal Corynebacterium mastitidis, we asked whether human-derived bacteria could do the same. Pairing sequencing and culturomics of healthy human conjunctiva from two geographically distinct cohorts, we define a stable core microbiome and identify Corynebacterium bovis as a consistent eye-resident commensal rather than a transient contaminant. In humans, C. bovis elicits a non-pathogenic response marked by IL-17 secretion by {gamma}{delta} T cells and circulating C. bovis-specific IgG, which suggests commensalism rather than pathology. Colonizing mice with human C. bovis strains from Pittsburgh or Miami reproduced this program and conferred protection against ocular infection with Staphylococcus aureus. Together, these findings identify C. bovis as a human-derived ocular commensal that can drive protective {gamma}{delta} T cell immunity, translating a mechanism previously defined only in mice into human relevance and a rational target against sight-threatening infection.

immunology↗

bioRxiv2026

Extracellular matrix proteoglycans lumican and biglycan promote innate immune signals and viral clearance in HSV-1 infections of mouse corneas

The extracellular matrix (ECM) regulates innate immunity, but its role in HSV-1 keratitis is unclear. Here, we identify the ECM proteoglycans lumican (Lum) and biglycan (Bgn) as key regulators of early antiviral defense in the cornea. Lum-/- and BgnKO mice showed impaired viral clearance, increased corneal opacity, and worse keratitis than wild-type controls. These defects were associated with reduced early recruitment of neutrophils, monocytes, and plasmacytoid dendritic cells, and blunted induction of pro-inflammatory cytokines, chemokines, and type I interferons. In infected wild-type corneas, Lum and Bgn were upregulated, including in the basal epithelium, where Ccl2 emerged as an early epithelial response that was diminished in knockout mice. In human corneal epithelial cells, recombinant LUM or BGN enhanced HSV-1-induced CCL2 secretion, and human corneal organoids recapitulated epithelial viral tropism and CCL2 induction. Together, these findings identify Lum and Bgn as regulators of epithelial-innate immune crosstalk that promote early antiviral immunity during corneal HSV-1 infection.

immunology↗

bioRxiv2026

The miR-183/96/182 Cluster Regulates Trigeminal Ganglion Sensory Neurons Response to Pseudomonas aeruginosa Infection

PurposeTo uncover the molecular mechanisms of corneal sensory nerves (CSN)s involvement in the initiation of Pseudomonas aeruginosa (PA) keratitis and the roles of the miR-183/96/182 cluster (miR-183C) in this process. MethodsmiR-183C conventional knockout (KO) or sensory neuron-specific (SNS) conditional (C)KO mice and their age- and sex-matched wild type (WT) controls were used. TG SN were isolated. Neurite growth and branching were analyzed by neurite tracing. Custom-made microfluidic chambers (MFC) were used to separate the neuronal cell bodies in the soma chamber and their neurites/nerve endings in the axon chamber. TG SNs response to lipopolysaccharide (LPS) or PA infection of the neurites/nerve endings was studied by ELISA assays of CX3CL1 and substance P (sP) in the axon chamber. Target luciferase reporter assays were performed to validate key downstream target genes of miR-183C. ResultsThe total neurite length and number of branches per TG SN were decreased in the CKO vs WT mice, and in the male vs female WT mice. PA infection, but not LPS alone, induced the production and secretion of CX3CL1 and sP in WT mice; while TG SN of miR-183C KO mice responded to both LPS and PA and were significantly enhanced when compared to WT mice. Antagonists to TLR4 and/or FPR1 inhibited PA-induced responses. Target luciferase reporter assays confirmed that genes encoding NRP1, TAC1-the precursor gene of sP, CX3CL1 and ADAM10, a metalloproteinase involved in the production of soluble CX3CL1, were direct targets of miR-183C. ConclusionsPA directly activates TG SN and induces chemokine and neuropeptide production/secretion through TLR4 and FPR1 receptors, which may contribute to the initiation of PA keratitis. miR-183C regulates TG SN neurite growth, chemokine and neuropeptide production/secretion and the response to PA infection by targeting a collection of key genes involved in axon guidance/projection-, chemokine and neuropeptide biogenesis- and receptors mediating PA-induced activation.

immunology↗

bioRxiv2026

C. mastitidis requires the protein Sortase F to colonize the eye

The ocular surface is a mucosal tissue that is constantly exposed to environmental antigens and potential pathogens. Human microbiomes play a critical role in the balance of surveillance and inflammation at sites of colonization. Historically, the investigation of the ocular microbiome has been difficult due to its paucibacterial nature and the inhospitable environment of the ocular surface. Despite this, Corynebacterium mastitidis (C. mast) developed a unique ability to colonize the eye and elicit a protective immune response characterized by induction of IL-17 from {gamma}{delta} T cells and protection from corneal infection. Therefore, we sought to understand the unique bacterial machinery that C. mast utilizes to colonize the eye and how it affects the induction of an eye-specific immune signature. Using a C. mast transposon mutant library, we identified a mutant that completely lacked an ability to form biofilm, colonize the eye, and induce in vivo immunity. Whole genome sequencing revealed a disruption in the sortase F gene, which anchors proteins to the cell wall of C. mast, governing biofilm formation and tethering of adhesins to the cell surface. Additionally, we show that mutation in individual C. mast adhesins does not affect ocular colonization or immune induction. By understanding the molecular mechanism of ocular microbial colonization, this work advances our understanding of how bacteria colonize and induce immune responses on the eye, providing a foundation for developing novel therapeutic strategies against ocular infections.

microbiology↗

bioRxiv2025

Commensal-derived Trehalose Monocorynomycolate Triggers γδ T Cell-driven Protective Ocular Barrier Immunity

Commensals shape host physiology through molecular crosstalk with host receptors. Identifying specific microbial factors that causally influence host immunity is key to understanding homeostasis at the host-microbe interface and advancing microbial-based therapeutics. Here, we identify trehalose monocorynomycolate (TMCM) from Corynebacterium mastitidis (C. mast) as a potent stimulator of IL-17 production by {gamma}{delta} T cells at the ocular surface. Mechanistically, TMCM-driven IL-17 responses require both IL-1 signals and {gamma}{delta} TCR signaling, which also supports endogenous {gamma}{delta} T cell IL-1R1 expression. Notably, synthetic TMCM alone is sufficient to mimic the effect of C. mast in inducing {gamma}{delta} T cell immunity and protect against pathogenic corneal infection. Our findings establish TMCM as a key mediator of commensal-driven immune defense, highlighting its potential as a {gamma}{delta} T cell adjuvant and a microbiome-informed therapeutic to enhance IL-17-driven protection at barrier sites such as the ocular surface. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/643820v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@63139forg.highwire.dtl.DTLVardef@9d6978org.highwire.dtl.DTLVardef@775e37org.highwire.dtl.DTLVardef@ca84b6_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LICorynomycolates enable ocular C. mast colonization and protective IL-17 immunity C_LIO_LITMCM drives IL-17 from {gamma}{delta} T cells through TCR and IL-1R signaling C_LIO_LI{gamma}{delta} TCR signaling maintains the expression of endogenous IL-1R1 C_LIO_LISynthetic TMCM mimics the ability of C. mast to induce {gamma}{delta} T cell immunity in the eye C_LIO_LITMCM protects against P. aeruginosa keratitis, highlighting its therapeutic potential C_LI

immunology↗

bioRxiv2024

TLR2 Supports γδ T cell IL-17A Response to ocular surface commensals by Metabolic Reprogramming

The ocular surface is a mucosal barrier tissue colonized by commensal microbes, which tune local immunity by eliciting IL-17 from conjunctival {gamma}{delta} T cells to prevent pathogenic infection. The commensal Corynebacterium mastitidis (C. mast) elicits protective IL-17 responses from conjunctival V{gamma}4 T cells through a combination of {gamma}{delta} TCR ligation and IL-1 signaling. Here, we identify V{gamma}6 T cells as a major C. mast-responsive subset in the conjunctiva and uncover its unique activation requirements. We demonstrate that V{gamma}6 cells require not only extrinsic (via dendritic cells) but also intrinsic TLR2 stimulation for optimal IL-17A response. Mechanistically, intrinsic TLR2 signaling was associated with epigenetic changes and enhanced expression of genes responsible for metabolic shift to fatty acid oxidation to support Il17a transcription. We identify one key transcription factor, I{kappa}B{zeta}, which is upregulated by TLR2 stimulation and is essential for this program. Our study highlights the importance of intrinsic TLR2 signaling in driving metabolic reprogramming and production of IL-17A in microbiome-specific mucosal {gamma}{delta} T cells. SummaryOcular commensal Corynebacterium mastitidis (C. mast) induces IL-17 responses from {gamma}{delta} T cells by activating TLR2 signaling. {gamma}{delta} T cell-intrinsic TLR2 stimulation can increase Il17a transcription and promote fatty acid oxidation, favoring {gamma}{delta} T cell IL-17A responses. Highlights(1) V{gamma}6 T cells are a major C. mast-responsive subset in the conjunctiva (2) TLR2-deficient mice exhibit reduced {gamma}{delta} T cell responses to ocular commensal bacteria. (3) {gamma}{delta} T cell-intrinsic TLR2 deficiency causes defects of fatty acid oxidation and IL-17A production in a {gamma}{delta} subset-specific manner. (4) The transcription factor, I{kappa}B{zeta} is upregulated by TLR2 stimulation and supports {gamma}{delta} IL-17A production through fatty acid oxidation. O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/587519v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1d1ba87org.highwire.dtl.DTLVardef@2d17d9org.highwire.dtl.DTLVardef@78e846org.highwire.dtl.DTLVardef@1d96dc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

Generation of Xylose-inducible promoter tools for Pseudomonas species and their use in implicating a role for the Type II secretion system protein XcpQ in inhibition of corneal epithelial wound closure

Tunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and P. fluorescens. The Pxut promoter derived from the P. flurorescens xut operon was incorporated into a broad host-range pBBR1-based plasmid and compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. GFP-fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate cloning of toxic genes using E. coli to generate plasmids. The Pxut promoter was expressed at a lower inducer concentration than PBAD in P. fluorescens and higher gfp levels were achieved using Pxut. Flow cytometry analysis indicated that Pxut was more leaky than PBAD in the tested Pseudomonas species, but was expressed in a higher proportion of cells when induced. D-xylose did not support growth of P. aeruginosa or P. fluorescens as a sole carbon source and is less expensive than many other commonly used inducers which could facilitate large scale applications. The efficacy of this system aided in demonstrating a role for the P. aeruginosa type II secretion system gene from xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species. ImportancePseudomonas species are enormously important in human infections, biotechnology, and as a model system for interrogating basic science questions. In this study we have developed a xylose-inducible promoter system and evaluated it in P. aeruginosa and P. fluorescens and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type 2 secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

microbiology↗