Abstract:
Tuberculosis (TB) is a communicable disease that is caused by the bacillus Mycobacterium tuberculosis (M. tb). TB is one of the top causes of mortality worldwide, and it is estimated that one quarter of the world’s population is infected with M. tb and is at risk of developing TB disease. The human immune system attempts to contain a M. tb infection by forming hypoxic granulomas, that halt bacterial replication. TB treatment via antibiotics is increasingly difficult due to drug resistance.
RNase HI is essential for growth of M. tb under lab conditions, and functions to cleave RNA from RNA-DNA hybrid R-loops. Without this endonuclease activity, R-loops inhibit essential RNA and DNA polymerase activity, ergo, no transcription of mRNA. RNase HI is therefore a novel drug target against replicating M. tb. The work in this thesis aim to assess the metabolic state of non-replicating cultures and whether this is altered by decreased RNase HI activity.
Microscopy and gas-chromatography mass-spectrometry were used to observe differences between RNase HI knockout and wildtype strains in a hypoxic model. These include differences in cell morphologies such as ovoid and V-shaped cells as well as metabolic profiles which indicate the metabolic state of non-replicating mutants.
These findings show: the importance of RNase HI in hypoxic conditions and the consequences on carbon flux upon deletion of the enzyme. Media specific growth and pH changes under different levels of oxygen stress. Morphological differences between the RNase HI knockout and wildtype strains under aerobic and hypoxic stages are also described.
