Microbial Pathogenesis in the Era of Spatial Omics

Abstract

The biology of a cell, whether it is a unicellular organism or part of a multicellular network, is influenced by cell type, temporal changes in cell state, and the cell's environment. Spatial cues play a critical role in the regulation of microbial pathogenesis strategies. Information about where the pathogen is-in a tissue or in proximity to a host cell-regulates gene expression and the compartmentalization of gene products in the microbe and the host. Our understanding of host and pathogen identity has bloomed with the accessibility of transcriptomics and proteomics techniques. A missing piece of the puzzle has been our ability to evaluate global transcript and protein expression in the context of the subcellular niche, primary cell, or native tissue environment during infection. This barrier is now lower with the advent of new spatial omics techniques to understand how location regulates cellular functions. This review will discuss how recent advances in spatial proteomics and transcriptomics approaches can address outstanding questions in microbial pathogenesis.

Keywords: host-pathogen interactions; microbial pathogenesis; spatial proteomics; spatial transcriptomics.

FIG 1

Questions in microbial pathogenesis to approach with spatial omics tools. (A) Pathogens that lack model organisms or tools for experimental manipulation (e.g., a virus recently transferred to a zoonotic host); (B) studying the biology of infected primary cell types or tissues with complex architecture that are poorly modeled in vitro (e.g., infected neurons or bacteria encroaching on intestinal mucus [green]); (C) understanding the constituents of novel structures that form during infection like a bacterial pedestal or granuloma (green core); (D) evaluating subpopulations of microorganisms (e.g., bacteria deep within versus peripheral to a biofilm [green], a constituent of the gut commensal population, and tissue-resident versus circulating parasites). Red indicates a population of interest, and purple indicates nontargeted. The figure was generated by BioRender.

FIG 2

Predicting molecular interactions and subcellular localization by enzyme-mediated proximity ligation with the APEX, BioID, and TSA-BAR approaches. (A) The labeling enzyme (teal Pac-Man) is conjugated to a bait protein (gray) or signal sequence that is abundant and robustly localized to a target organelle to label resident proteins or nucleic acids (light aqua region), here depicting a microbial (red) vacuole. (B) A bait protein is enzyme conjugated to label any nearby molecules (i), such as a target protein with an engineered acceptor peptide (ii), using split GFP (green) where the larger N terminus of GFP is conjugated to the enzyme under inducible control and the smaller C terminus is conjugated to the bait protein (iii). (C) In protein mapping approaches, cell lines are generated where each expresses a label-targeting protein (described in panel A) and comparative proteomics is used to map host and/or microbial protein abundance in each compartment. (D) In TSA-BAR and nanobody-APEX, a label-targeting enzyme is targeted via an antibody (blue) raised against a specific bait antigen. The figure was generated by BioRender.

FIG 3

Microscopy-mediated omics tools. (A) In LCM, structures of interest are imaged and stabilized or non-target structures are ablated in a photopolymer matrix. Cells are processed for RNA-seq or MS. (B) In DVP, an artificial intelligence platform identifies structures of interest and ablative LCM is used to isolate them for MS analysis. (C) In autoSTOMP, photo-biotin tags are conjugated to proteins in target structures using confocal microscopy and then purified for LC-MS. (D) In FISSEQ, cDNA synthesis and rolling circle amplification are used to identify transcripts in situ. (E and F) Slides containing a barcoded capture oligonucleotide array (10×/Visium [E]) or a bead array (SlideSeq [F]) are overlaid with tissue. Upon permeabilization, RNAs are captured for library prep in vitro and next-generation sequencing (NGS). (G) In HDST, a hexagonal well array containing capture beads is overlaid with tissue. RNAs are captured for in vitro library prep and NGS; however, the resolution is enhanced by binning reads from neighboring hexagonal wells. (H) In Light-seq, barcoded oligonucleotides are hybridized to RNAs for a cross-junctional synthesis reaction that facilitates cDNA amplification, library preparation and NGS. The figure was generated by BioRender.