Abstract
Tuberculosis (TB) is a curable disease caused primarily by the bacterium Mycobacterium tuberculosis (Mtb), yet it continues to claim a life every 20 seconds globally. While New Zealand has a relatively low overall burden of TB, Māori and Pasifika communities are disproportionately affected compared to New Zealand Europeans, with incidence rates of 6 and 16 cases per 100,000, respectively, versus 1 case per 100,000 among New Zealand Europeans.
Unique Mtb strains appear to be endemic to these communities, with the "Southern Cross" strain being strongly associated with Pacific peoples.
Prior to this study, the Aung Lab conducted Oxford Nanopore DNA sequencing and identified two subvariants of the Southern Cross strain, previously thought to be highly clonal. One of these subvariants contains a 2-kb deletion in the esxRS locus, a region homologous to esxGH, which is part of the ESX-3 secretion system. Mtb possesses five Type VII secretion systems, known as ESX systems (ESX1-5), which play key roles in host-pathogen interactions and immune evasion.
Previous studies have shown that deletion of esxGH can lead to upregulation of esxRS, suggesting a potential regulatory link between these regions. It is therefore hypothesised that the deletion of esxRS in this subvariant may result in compensatory upregulation of esxG and esxH, leading to altered intracellular survival compared to wild-type Mtb. This study aims to explore this hypothesis by characterising the subvariant both transcriptionally, using RNA sequencing, and functionally, through macrophage infection assays.
To investigate this, we utilised CRISPR interference (CRISPRi) to generate knockdown strains of the model avirulent Mtb strain mc²6230, targeting the genes esxR, esxS, and an upstream region known as PPE47. These knockdown strains were subjected to RNA sequencing to assess differential gene expression. However, preliminary analysis of the RNA-seq data revealed a mixed culture with BLAST reads indicating the presence of Cellulosimicrobium cellulans alongside Mtb.
A macrophage infection assay was also performed using PMA-differentiated THP-1 cells infected with wild-type and knockdown Mtb strains at a multiplicity of infection (MOI) of 10, to assess differences in intracellular survival. This showed the esxS knockdown had increased intracellular survival when compared to wild-type Mtb. However, due to our mixed culture, the results cannot be validated, as Cellulosimicrobium cellulans is capable of surviving within macrophages.
Future work will ensure obtaining pure Mtb cultures. Once this is achieved, the experimental pipeline established in this project can be used to comprehensively investigate the transcriptional profiles and functional characteristics of the Southern Cross Mtb variants.