Tuberculosis (TB) is one of the top 10 causes of death worldwide. Mycobacterium tuberculosis (Mtb) is transmitted from one individual to the next through aerosols [1]. Upon TB infection, the pathogen can either be successfully cleared from the lungs as a result of the innate immune response or acquired T-cell immune response, remain latent in the body or progress to active TB when replication is not controlled [2]. Latent TB refers to the clinical observation of infection without apparent disease [2]. It is believed that a subpopulation of mycobacteria called persisters, are the underlying cause of latent TB. These bacterial populations have the ability to survive inside the host for extended periods of time and reactivate to cause active disease [3]. Persisters are defined as a small, viable, but non-replicating (VBNR) drug-tolerant population [4, 5]. As a result of this population, lengthy antibiotic treatment may be required to completely eradicate the infection, subsequently giving rise to genetically resistant progeny [6]. Some of the challenges associated with studying persisters are that they undergo no, or very limited, replication and are likely to be present in very low numbers [7].
To address these limitations, a new technique termed Fluorescence Dilution (FD), previously used to identify a AR population of Salmonella typhimurium in infected macrophages [8,9], has been developed for use in mycobacteria [4]. The technique allows identification of VBNR Mtb following macrophage uptake and that Mtb exhibit large heterogeneity in their growth rate inside host cells, which could have major implications for infection outcome. This reporter allows sorting of the actively replicating (AR) and VBNR populations, allowing for subsequent analysis of pure populations.
Macrophages are considered a favourite niche for Mtb to divide and persist within the host [9]. Macrophages are heterogeneous and their phenotype and functions are regulated by their surrounding micro-environment [10]. Macrophage polarisation is a process whereby macrophages are activated at a certain time to serve a specific function. Two major macrophage subpopulations with different functions include classically activated or inflammatory (M1) and alternatively activated or anti-inflammatory (M2) macrophages [11]. M1 macrophages are typically induced by Th1 cytokines, such as IFN-γ and TNF-α, or by bacterial lipopolysaccharide (LPS) recognition [10]. M1 macrophages remove pathogens during infection through activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system and generation of reactive oxygen species (ROS).
In contrast, M2 macrophages have anti-inflammatory functions by switching gene expression toward anti-inflammatory molecules. They are polarized by Th2 cytokines IL-4 and IL-13. M2 macrophages have potent phagocytosis capacity, scavenge debris and apoptotic cells, promote tissue repair and wound healing [12]. However, many intracellular pathogens have the ability to kill infected macrophages and subvert host pathways to inhibit apoptosis and instead induce necrosis. Mtb suppress macrophages to avoid cytotoxic functions and repolarize M1 polarized macrophages toward an M2 phenotype [13]. Mtb induces macrophages to produce IL-10 in favour of bacterial survival and growth inside macrophages by blocking the phagosome maturation [14].
Recently, a pioneering single-cell RNA-sequencing (scRNAseq) study revealed considerable heterogeneity in the macrophage response to the intracellular growth of a fluorescent Salmonella strain [15]. The study indicates that heterogeneity in gene expression amongst infected macrophages creates diverse environments for Salmonella to either persist without triggering immune signalling or exploit its host and continue to actively-replicate. In addition, the macrophages harbouring VBNR Salmonella display a pro-inflammatory M1 polarization state. These M1 macrophages containing VBNR bacteria evade recognition by intracellular immune receptors. In contrast, macrophages containing AR bacteria displayed an anti-inflammatory M2 polarisation state. These AR intracellular Salmonella overcome host defence by reprogramming macrophage polarization from M1 to M2 polarisation [15].
The work proposed in my study aims to utilise a similar approach to that above, but with the aim to characterise both the host and the pathogen at the same point in time (dual host-pathogen RNAseq). Dual RNAseq allows simultaneous capturing of the global transcriptome of the host and pathogen [16]. This technology allows comparison of macrophages containing AR with macrophages containing VBNR Mtb. My study exploits the FD reporter in combination with flow cytometry and fluorescence-activated cell sorting to determine the M1/M2 polarisation of macrophages containing AR and VNBR bacteria and characterise host biosignatures associated with these bacterial phenotypes using dual RNAseq analyses.
My study aims to investigate mechanisms by which the organism evades the immune system to establish latent infection. In addition, identification of latency associated antigens that could be used for further investigation and selection of latency antigen candidates based on their ability to stimulate immune cells to induce antibodies capable of mobilizing immune responses, killing the pathogen or interfering with its normal function or growth. Understanding the mechanisms Mtb exploits to alter their intracellular replications rates to evade and alter the immune system to their advantage, could lead to the development of new approaches for vaccine development and diagnoses.
