Deciphering the molecular basis of the global emergence of non-tuberculous Mycobacteria as important human respiratory pathogens

Mycobacterium avium belongs to the group Nontuberculous Mycobacterium (NTM), which are acid fast, environmental bacteria belonging to the family Mycobacteriaceae. They are facultative intracellular bacilli causing opportunistic infections in individuals with pre-existing conditions such as respiratory and immune disorders. There has been a steady increase in the number of NTM infection reports in the past four decades and many of them show resistance to common anti-mycobacterial drugs. It is known that the bacteria manipulate the host immune response to their advantage; however, the intracellular trafficking and survival strategies are poorly understood.<br/><br/>To address this, we have developed an image-based, single cell assay using a fluorescence dilution (FD) system to measure replication and persistence of M. avium in human THP-1 macrophage-like cells and assess new drug treatments. Accuracy of fluorescence signal as proxy of intracellular replication was confirmed against the gold standard technique of CFU plating. Moreover, the assay was validated using a panel of known anti-mycobacterial drugs, reproducing efficacy profiles similar to those reported in the literature. Based on our results, the fluorescence dilution system is an effective high throughput assay to test anti-mycobacterial drugs and host directed therapies.<br/><br/>To dissect host responses to infection and identify potential targets for host-directed therapy, 10x single cell RNA sequencing (scRNA-seq) of M. avium infected THP-1 macrophages was performed, revealing for the first time the heterogeneity in the host response; i.e, several large cell clusters showing different degrees of an inflammatory response, as well as a small cluster of infected cells showing a strong type I interferon response, suggesting a dynamic interplay of the immune signals at the single cell level.<br/>Based on the differentially expressed genes identified in the scRNA-seq analysis, small molecule host targeted inhibitors were used in the FD assay, which led to the identification of importazole, a nuclear transport inhibitor, as a compound that inhibits intracellular replication. Further mechanistic studies showed that importazole has direct antibacterial activity against M. avium, causing a reversible increase in bacterial cell length and inhibition of replication, suggesting that the compound could inhibit bacterial cell division.<br/><br/>Together, this work established and successfully employed a new image-based screening system to measure replication and persistence of M. avium in combination with scRNA-seq of infected macrophages, identifing the drug importazole as new inhibitor for M. avium replication, opening new avenues for the development of new therapies for M. avium infections.<br/><br/><i>Thesis embargoed until 31 July 2027</i>.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

Queen's University Belfast

Year dc:date.issued

2022

Author dc:creator

• Nagalekshmi, Karthika

Advisors dc:contributor.advisor

• O'Kane, Cecilia
• Schroeder, Gunnar

• Non tuberculous mycobacteria
• NTM
• host-pathogen

Language dc:language

eng

Identifier

oai:pure.qub.ac.uk/portal:studenttheses/40fc95f1-8862-4361-8397-34ab76d275a4

OAI identifier oai:identifier

oai:pure.qub.ac.uk/portal:studenttheses/40fc95f1-8862-4361-8397-34ab76d275a4