Dr Jomien Mouton, Stellenbosch University - VALIDATE Fellow
Identification of latency associated antigens and biosignatures associated with Mycobacterium tuberculosis
Tuberculosis (TB) is a chronic infectious disease, caused by a bacterium called Mycobacterium tuberculosis (Mtb). It is important to realise that most people infected with Mtb do not develop active disease and that these people have no symptoms of disease. In latently infected individuals, the immune system controls the infection and the bacteria enter a state of hibernation, where they are not growing or slowly growing, but are still able to survive. In many people TB remain inactive for a lifetime, but in others, especially those with weak immune systems, with e.g. HIV/AIDS, the bacteria may awaken and cause active TB disease. These hibernating, so-called persister, bacteria are able to survive TB treatment and could contribute to the lengthy drug treatment. It is very difficult to isolate persister bacteria to study them, because these bacteria are present in low numbers and are very difficult to grow. We are therefore interested in finding ways to study persistent bacteria by using specially labelled bacteria and sophisticated, laser-based methods. To be able to do this, we have recently developed a new technique to identify and isolate persistent Mtb. This technique involves the use of two fluorescent proteins, one that allows the tracking of “live” bacteria and one that allows the accurate measurement of bacterial growth. By applying this technique, we have shown that when bacteria enter host cells immune cells (macrophages), both growing and non- or slowly growing bacteria emerge. We now aim to apply this technique to study macrophages containing either non-growing mycobacteria or growing mycobacteria. We will do this by using single-cell technologies to analyse both host and pathogen cells simultaneously to identify a unique signature associated with persister mycobacteria that could be used to assess disease risk. We also aim to identify novel molecules (antigens) associated with persister mycobacteria that are capable of inducing an immune response in latently infected individuals.
To elucidate how macrophages interact with replicating and non-replicating mycobacterium, we employed a dual-fluorescent replication reporter in combination with flow cytometry to identify and isolate host cells containing either non-replicating or replicating mycobacteria for RNA sequencing and fluorophore-conjugated antibody staining. Our results indicate that macrophages containing differentially replicating mycobacteria are distinctly associated with either M1 or M2 macrophage polarisation. The VALIDATE Fellowship has had a tremendous impact on my career development. Specifically, it allowed me to be appointed as a researcher at Stellenbosch University, which afforded me the opportunity to supervise my own students and apply for funding as a primary investigator. In addition, I have had the opportunity to serve on the Network Management Board (NMB) and expand my network by meeting new people at the annual meetings. The funding I have received as part of my fellowship has made high-end research possible that contributed to scarce skills development in RNA sequencing in South Africa.
- Mouton JM, Heunis T, Dippenaar A, Gallant JL, Kleynhans L, Sampson SL. Comprehensive characterisation of the attenuated double auxotroph Mycobacterium tuberculosis ΔleuDΔpanCD as an alternative to H37Rv. Frontiers in Microbiology 2019. 10: 1922
- Paarwater B, Mouton JM, Sampson SL, Malherbe S, Kotze L, Walzl G, du Plessis N. Inhaled particulate matter affects immune responsiveness of human lung phagocytes to mycobacteria. American Journal of Physiology-Lung Cellular and Molecular Physiology. 2021. 321(3):L566-L575
- Parbhoo T, Sampson SL, Mouton JM. Recent developments in the application of flow cytometry to advance our understanding of Mycobacterium tuberculosis physiology and pathogenesis. Cytometry Part A 2020. 97(7):783.
- Gallant J, Mouton JM, Ummels R, ten Hagen-Jongman C, Kriel N, Pain A, Warren RW, Bitter W, Heunis T, Sampson SL. Identification of gene fusion events in Mycobacterium tuberculosis that encode chimeric proteins. NAR Genomics and Bioinformatics 2020. 2(2): lqaa033.
Find out more about Dr Jomien Mouton here.