Investigating the role of sufT in Mycobacterium abscessus using CRISPRi

Chimukuche, Nyaradzai Mitchell

Investigating the role of sufT in Mycobacterium abscessus using CRISPRi

Master Thesis

2022

Abstract

Mycobacterium abscessus (M. abscessus) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections including pulmonary M. abscessus disease. Treatment of M. abscessus infections is challenging because the mycobacterium is intrinsically resistant to different classes of antibiotics, including those used to treat tuberculosis. It is important to develop new and effective drugs for M. abscessus treatment, therefore acquiring knowledge on the unique vulnerabilities of M. abscessus which can be a targeted for future drug design is required. The clustered routinely interspaced palindromic repeats interference (CRISPRi) system was recently optimized for transcription repression in mycobacteria. This study aimed to investigate the functionality of the mycobacterial CRISPRi in M. abscessus, specifically to investigate the role of the sufT homologue, MAB_2744c, in survival under iron limiting conditions and susceptibility to the metal chelator deferoxamine. To this end, 4 sgRNAs were designed, two targeting MAB_2744c (sg2744F1, sg2744F2) and two targeting the upstream gene 2745c (sg2745F1 and sg2745F2) and used to generate 4 M. abscessus sufT knock-down strains (M. abscessus attB::pLJR962-2744c-a, M. abscessus attB::pLJR962-2744c-b, M. abscessus attB::pLJR962-2745c-a and M. abscessus attB::pLJR962-2745c-b. The CRISPRi strains all grew under standard conditions, reaching exponential phase between 24 and 48 hours and a maximum OD600 of 8 after 48 hours. Reduced growth for all CRISPR interference strains was observed when comparing iron limiting conditions to iron replete conditions (2 µM), while no difference was observed for any of the strains when comparing growth with and without sgRNA expression. Expression analysis revealed that no transcriptional silencing was achieved in any of the strains, and therefore no conclusions could be made about the role of sufT during growth under iron limitation. Similarly, while comparison of biofilm formation under iron limiting and iron replete conditions confirmed that biofilm formation is reduced when iron is limiting, the role of sufT in this process could not be investigated using the CRISPRi strains. The importance of iron for the growth of M. abscessus was further confirmed by investigating its susceptibility to the metal chelator deferoxamine. This study demonstrates that the previously described mycobacterial CRISPRi system does not function optimally in M. abscessus when a single sgRNA is used and therefore the system requires further optimization for use in this organism.

Keywords

Molecular and Cell Biology

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