Investigating the effect of a Mycobacterium tuberculosis inhA promoter mutation in cell envelope composition and morphology
Isoniazid (INH) resistance in Mycobacterium tuberculosis (M.tb) compromises the effectiveness of global tuberculosis treatment programs, with the inhA promotor mutation (IPM) serving as a driver of low-level drug resistance. IPMs are seen more frequently in multi-drug-resistant isolates and are considered a gateway to the development of extensive drug resistance. IPMs contribute resistance to INH and ethionamide which are first and second-line drugs respectively that target mycolic acid (MA) synthesis. MA are characteristic components of mycobacterial species’ cell wall and contribute to the impermeability of cell wall by virtue of its hydrophobicity. IPMs result in a transcriptional upregulation of inhA, and mabA, which are genes involved in MA synthesis, and hemZ which is involved in heme synthesis through the incorporation of iron. It is unknown how the upregulation of genes involved in MA synthesis may affect the cell wall, therefore, this project aimed to investigate molecular and microscopic differences in the cell wall between IPM and wild-type (WT) strain. To investigate the influence of IPM on overall cell wall morphology multiple complementary techniques were employed. Scanning electron microscopy (SEM) was implemented to visualise extracellular surface features and determine cell length, while transmission electron microscopy (TEM) was used to examine ultrastructural cell wall layer distances. To further understand the architecture and organisation of M.tb cell wall we assessed membrane fluidity using DPH, a membrane fluorescent probe. In parallel, a preliminary investigation of cell wall integrity using propidium iodide was performed. Finally, to determine the influence of IPM on MA, two extraction protocols were compared and validated. The resulting extracts were firstly evaluated by thin-layer chromatography, followed by mass spectrometry and nuclear magnetic resonance spectrometry to confirm MA extraction. We found that even though two genetically identical mutants were used, their experimental outcome often differed. SEM analysis revealed no distinctive morphological features of mutants vs wild-type. However, a wide variety of cellular morphologies were observed across the strains. We highlight the presence of an extracellular matrix which surround bacterial clumps as well as the presence of divisional scars. TEM analysis confirms that cryogenic fixation with freeze substitution dehydration yielded improved electron microscopy images over conventional fixation with alcohol dehydration. Automated analysis of cell wall layers from TEM images proved inefficient due to variability. Therefore, manual measurements were taken and compared. However, no significant differences were found between strains. Membrane fluidity and integrity studies alluded that IPMs might have an altered cell envelope organisation as the mutants had slightly elevated DPH and PI signals compared to the WT. Evaluation of MA extractions showed that organic solvent extraction was superior to an enzymatic extraction. TLC of extracted compounds provided initial separation and confirmation of MA, but mass spectrometry proved challenging due to the hydrophobicity, high molecular mass, and structural diversity. In contrast, NMR spectroscopy successfully confirmed the presence of MA in our sample extract. Our results suggested that there was no difference between IPM and wild-type strains in terms of general morphology, the thickness of cell wall layers or membrane fluidity. Chemical characterization of the lipids produced should be explored further.
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