Poster #68: Shasmeen Azhar

Spatially Resolving Bacterial Burden, Iron Restriction, and Immune Recruitment in Gingival Tissue

Jason LaPierre, BS¹; Mallory Cunningham, BS²; Shasmeen Azhar, BS¹; Chris Oldfield, PhD²; Megan Williams, PhD²; Myrna Serrano, PhD²˒³; Gregory Buck, PhD²˒³˒⁴; Katarzyna M. Tyc, PhD⁵˒⁶. ¹School of Life Sciences and Sustainability, Virginia Commonwealth University; ²Department of Microbiology and Immunology, School of Medicine, Virginia Commonwealth University; ³Center for Microbiome Engineering and Data Analysis, Virginia Commonwealth University; ⁴VCU Genomics Core, Virginia Commonwealth University; ⁵Department of Biostatistics, School of Public Health, Virginia Commonwealth University; ⁶Massey Bioinformatics Shared Resource, Virginia Commonwealth University.

Spatial transcriptomics enables high-resolution characterization of tissue organization but remains limited in its ability to quantitatively integrate microbial burden with localized host immune and iron-regulatory responses. We are developing a computational spatial transcriptomics framework to identify bacterial infection foci and determine how host-mediated iron restriction is associated with immune recruitment in gingival tissue. Bacterial burden will be quantified from Visium HD spatial transcriptomic data using bacterial 16S rRNA transcripts together with a targeted panel of approximately 30 highly expressed Prevotella intermedia OMA14 genes, including genes involved in iron acquisition and metabolism. These microbial signals will be integrated to identify infected regions and generate quantitative estimates of bacterial burden at the spot or cell level. Host transcriptional responses will subsequently be evaluated at single-cell resolution to characterize spatial gradients surrounding infection foci. To quantify local iron restriction, expression of HAMP, SLC40A1 (FPN), FTH1/FTL, TFRC, and SLC11A1 (NRAMP1) will be integrated into an iron-restriction score for each spatial location. Regions with elevated scores will be evaluated for associations with bacterial burden and recruitment of activated macrophages, neutrophils, and T cells. Spatial analyses will quantify immune-cell density, proximity to HAMP-expressing regions, neighborhood enrichment, and clustering around iron-retaining macrophages. We will additionally assess spatial relationships between iron-regulation genes and chemokine expression and characterize immune infiltration gradients surrounding infection foci. We hypothesize that bacterial regions with elevated HAMP activity will exhibit increased immune recruitment and macrophage iron-retention phenotypes characterized by increased HAMP, reduced FPN, and elevated ferritin expression. Together, this framework will provide a quantitative approach for linking localized bacterial burden with host iron regulation and immune-cell organization, enabling spatial characterization of inflammatory microenvironments associated with P. intermedia infection.