Phenotypic effects of mab_1999 gene silencing in mycobacterium abscessus using CRISPR interference

Abstract

Cell division is an essential process for an organism that is orchestrated by many proteins, and its disruption can lead to cell death. The cell division process could be a promising target for a novel antibiotic, particularly against a hard-to-treat pathogen such as Mycobacterium abscessus (MAB). FtsL is a highly conserved core division protein that plays a critical role in the cell division and cell elongation process. However, its function and essentiality in MAB have not yet been characterized. This project aims to investigate the impact of mab_1999 gene repression, the predicted ftsL homolog, on MAB growth and cell division using Clustered Regularly Interspaced Short Palindromic Repeat interference (CRISPRi). Repression of mab_1999 resulted in growth defects and elongated cell morphology, indicating impaired septation in MAB. Notably, despite reduced gene expressions, growth arrest was not observed, and the elongated cells partially recovered over time suggesting adaptive or compensatory mechanisms. However, the mab_1999 knockdown did not result in increased sensitivity to antibiotics. This study also revealed a potential role for mab_1999 in hypoxic conditions, since reduced expression of mab_1999 led to growth defects under gradual oxygen depletion. However, exposure to nutrient limited conditions (Sauton), detergent stress (SDS), or heat stress did not result in significantly reduced survival. Collectively, the current findings demonstrate the utility of CRISPRi to study gene functions in MAB. Furthermore, this study also revealed the presence of fstL homolog in MAB, which is involved not only in cell division, but also potentially in hypoxic stress response.Keywords: Mycobacterium abscessus, ftsL, CRISPRi, cell division