Protocol for multi-modal single-cell RNA sequencing on M. tuberculosis-infected mouse lungs.

To elucidate how different immune cells contribute to control or progression of M. tuberculosis (Mtb) infection, we developed a technique to perform multi-modal single-cell RNA sequencing (scRNA-seq) from in vivo Mtb-infected lung macrophages. This protocol simultaneously acquires the transcriptome, surface marker expression, and bacterial phenotype of each infected cell. We describe steps for sorting Mtb-infected cells and staining with CITE-seq antibodies, as well as for methanol fixation and generation of scRNA-seq libraries. This protocol can be used on tissues derived from murine, nonhuman primate, and human infections. For complete details on the use and execution of this protocol, please refer to Pisu et al. (2021).1.

Iron limitation in M. tuberculosis has broad impact on central carbon metabolism.

Mycobacterium tuberculosis (Mtb), the cause of the human pulmonary disease tuberculosis (TB), contributes to approximately 1.5 million deaths every year. Prior work has established that lipids are actively catabolized by Mtb in vivo and fulfill major roles in Mtb physiology and pathogenesis. We conducted a high-throughput screen to identify inhibitors of Mtb survival in its host macrophage. One of the hit compounds identified in this screen, sAEL057, demonstrates highest activity on Mtb growth in conditions where cholesterol was the primary carbon source. Transcriptional and functional data indicate that sAEL057 limits Mtb's access to iron by acting as an iron chelator. Furthermore, pharmacological and genetic inhibition of iron acquisition results in dysregulation of cholesterol catabolism, revealing a previously unappreciated linkage between these pathways. Characterization of sAEL057's mode of action argues that Mtb's metabolic regulation reveals vulnerabilities in those pathways that impact central carbon metabolism.

Single cell analysis of M. tuberculosis phenotype and macrophage lineages in the infected lung.

In this study, we detail a novel approach that combines bacterial fitness fluorescent reporter strains with scRNA-seq to simultaneously acquire the host transcriptome, surface marker expression, and bacterial phenotype for each infected cell. This approach facilitates the dissection of the functional heterogeneity of M. tuberculosis-infected alveolar (AMs) and interstitial macrophages (IMs) in vivo. We identify clusters of pro-inflammatory AMs associated with stressed bacteria, in addition to three different populations of IMs with heterogeneous bacterial phenotypes. Finally, we show that the main macrophage populations in the lung are epigenetically constrained in their response to infection, while inter-species comparison reveals that most AMs subsets are conserved between mice and humans. This conceptual approach is readily transferable to other infectious disease agents with the potential for an increased understanding of the roles that different host cell populations play during the course of an infection.

Dual RNA-Sequencing of Mycobacterium tuberculosis-Infected Cells from a Murine Infection Model.

Dual RNA-sequencing is a powerful technique to assess both bacterial and host transcriptomes in an unbiased way. We developed a protocol to perform Dual RNA-seq on in vivo-derived macrophage populations infected with Mycobacterium tuberculosis. Here, we provide a practical step-by-step guide to execute the protocol on Mtb-infected cells from a murine infection model. Our protocol can also be easily applied to perform Dual RNA-seq on in vitro-derived cells as well as different Mtb-infected host cell types. For complete details on the use and execution of this protocol, please refer to Pisu et al. (2020).

Dual RNA-Seq of Mtb-Infected Macrophages In Vivo Reveals Ontologically Distinct Host-Pathogen Interactions.

Dissecting the in vivo host-pathogen interplay is crucial to understanding the molecular mechanisms governing control or progression of intracellular infections. In this work, we explore the in vivo molecular dynamics of Mtb infection by performing dual RNA-seq on Mycobacterium tuberculosis-infected, ontogenetically distinct macrophage lineages isolated directly from murine lungs. We first define an in vivo signature of 180 genes specifically upregulated by Mtb in mouse lung macrophages, then we uncover a divergent transcriptional response of the bacteria between alveolar macrophages that appear to sustain Mtb growth through increased access to iron and fatty acids and interstitial macrophages that restrict Mtb growth through iron sequestration and higher levels of nitric oxide. We use an enrichment protocol for bacterial transcripts, which enables us to probe Mtb physiology at the host cell level in an in vivo environment, with broader application in understanding the infection dynamics of intracellular pathogens in general.

The Deconstructed Granuloma: A Complex High-Throughput Drug Screening Platform for the Discovery of Host-Directed Therapeutics Against Tuberculosis.

Mycobacterium tuberculosis (Mtb) continues to be a threat to Global Public Health, and its control will require an array of therapeutic strategies. It has been appreciated that high-throughput screens using cell-based assays to identify compounds targeting Mtb within macrophages represent a valuable tool for drug discovery. However, the host immune environment, in the form of lymphocytes and cytokines, is completely absent in a chemical screening platform based on infected macrophages alone. The absence of these players unnecessarily limits the breadth of novel host target pathways to be interrogated. In this study, we detail a new drug screening platform based on dissociated murine TB granulomas, named the Deconstructed Granuloma (DGr), that utilizes fluorescent Mtb reporter strains screened in the host immune environment of the infection site. The platform has been used to screen a collection of known drug candidates. Data from a representative 384-well plate containing known anti-bacterial compounds are shown, illustrating the robustness of the screening platform. The novel deconstructed granuloma platform represents an accessible, sensitive and robust high-throughput screen suitable for the inclusive interrogation of immune targets for Host-Directed Therapeutics.

The Alternative Sigma Factors SigE and SigB Are Involved in Tolerance and Persistence to Antitubercular Drugs.

The emergence and spread of drug-resistant Mycobacterium tuberculosis strains possibly threaten our ability to treat this disease in the future. Even though two new antitubercular drugs have recently been introduced, there is still the need to design new molecules whose mechanisms of action could reduce the length of treatment. We show that two alternative sigma factors of M. tuberculosis (SigE and SigB) have a major role in determining the level of basal resistance to several drugs and the amount of persisters surviving long-duration drug treatment. We also demonstrate that ethambutol, a bacteriostatic drug, is highly bactericidal for M. tuberculosis mutants missing either SigE or SigB. We suggest that molecules able to interfere with the activity of SigE or SigB not only could reduce M. tuberculosis virulence in vivo but also could boost the effect of other drugs by increasing the sensitivity of the organism and reducing the number of persisters able to escape killing.

The ESX-3 secretion system is necessary for iron and zinc homeostasis in Mycobacterium tuberculosis.

ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions.