Sunday, June 1, 2025
6/1/2025
Interrogating complex mycobacterial population structures using single-cell RNA sequencing - Project Summary Tuberculosis (TB) is a leading killer among infectious diseases, and global progress against this pandemic has been set back by COVID-19. Major difficulties in combating TB are the need for months of multidrug therapy to ensure a high probability of relapse-free cure, and its ability to survive in the air as infectious aerosols and create a large pool of infected people. The requirement for prolonged drug treatment is likely due to the minority of bacteria that are able to survive antibiotics for a long duration, even in the absence of genetically-encoded resistance. Such bacteria are termed persisters. Similarly, transmission events rely on the subset of bacteria that are able to survive aerosolization stress until it reaches a new host. While recent strides have been made in understanding these subpopulations of cells, a way to interrogate their transcriptional states at a single-cell resolution in a high-throughput, unbiased manner does not exist. This limits understanding of the biology of these cells over time, and restricts identification of vulnerabilities that could be exploited to shorten TB treatment duration or reduce transmission. The aim of this proposal is to apply a method of single-cell RNA sequencing (sc-RNAseq) that allows for simultaneous comparison of the transcriptomes of thousands of cells of Mycobacterium tuberculosis (Mtb), the causative agent of TB. Preliminary data demonstrate that the protocol, which relies on in situ reverse transcription and barcoding with combinatorial indexing, is feasible in Mtb. In this application, the first aim is to test the limitations and characteristics of the technique in pre-defined mixtures of Mtb containing different plasmids. Over the first year of the grant, we will evaluate (1) the number of genes detected per cell, (2) the detection threshold of the smallest predefined subpopulation, and (3) the concordance of the aggregation of the single-cell transcriptome to bulk transcriptomes. With these parameters defined, the sc-RNAseq will be applied to biologically relevant in vitro conditions in the second year, including Mtb exposed to the front-line TB drug rifampin (RIF) and Mtb undergoing desiccation stress, a model of aerosolization. Here, the clusters of subpopulation transcriptomes found by the method will be compared with orthogonal, observable, and predictable phenotypes including cultivability and RIF resistance over time. These two aims will validate baseline characteristics of the assay, lay the foundation for leveraging this method against more complex biological samples in the future, and begin defining the fundamental biology of Mtb subpopulation behavior in response to stress.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).