Uncovering Ribosomal Gene Disruptions in Human Tuberculosis: A Spatial Transcriptomics Study of Submucosal Glands

Abstract Rationale: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease-related mortality, responsible for 1.3 million deaths in 2023. Damage to host tissues occurs during and after active infection, impairing host physiology to variable degrees, though, the mechanisms by which mycobacterium tuberculosis evades host immune defenses are not completely elucidated. Spatial variation in transcript expression has been demonstrated in Mtb granulomas across species. We previously observed histopathologic submucosal gland (SMG) abnormalities in latent and active pulmonary Mtb, thus we leveraged spatial transcriptomics to further elucidate important biological mediators. Methods: Human lung tissue from active and latent Mtb cases and controls specimens were obtained from autopsy and resection from King DinuZulu Hospital Complex, KwaZulu Natal Province, South Africa and Birmingham, AL, USA. Latent specimens were identified via micro-CT scanning and confirmed of Mtb- origin. Tissue sections were stained with H&E & immunohistochemistry for SMGs. Tissue was prepared according to GeoMx DSP antigen retrieval protocol, stained with immunofluorescent morphometry markers (i.e. MUC5B, Ag85) (Fig. 1), and analyzed using the Nanostring GeoMx Whole Transcriptome Atlas. Groups (control, latent, active) were compared by analyzing technical replicates. Counts were normalized (log2), differential gene expression (p<0.05, log2 fold change >0.4) was determined using a mixed linear model. Results: Twelve specimens (5 active, 5 latent, 2 control) underwent GeoMX analysis. Active and latent Mtb showed 840 and 618 differentially expressed genes versus controls, respectively (p<0.05) (Fig. 1). No significant differential gene expression was found between Mtb SMG subgroups based on Ag85 immunofluorescence. Principal component analysis identified 50 highly differentially expressed genes; 33 downregulated genes have gene ontology (GO) associations with ribosomal assembly and protein synthesis, (including RPL5, RPL9, RPS29). STRING analysis revealed enriched interaction between all ribosomal proteins with functional enrichment connections to several other downregulated proteins involved in antigen binding and protein binding, folding, and translocation (i.e. AZGP1, HNRNPA2B1, PFDN5, SSR4, respectively). Conclusions: Our data suggest ribosome biogenesis, essential for protein synthesis and cell growth, may be impaired within SMG in active and latent Mtb infection. Prior RNAseq data and Mtb host-pathogen interaction analysis correlate with our findings. Impairments in ribosome biogenesis are associated with impaired innate immunity. SMG proteins include mucin glycoproteins, an essential first line immune defense; Ribosome dysfunction may impair mucin production and function. Future research is needed to assess mucous function in Mtb. Differential gene expression in latent Mtb raises concerns of ongoing pathology and warrants further research.