Abstract
Mycobacterium tuberculosis is able to utilize cholesterol as a carbon source, and this ability is linked to its virulence in macrophages and in the mouse model of infection. The M. tuberculosis cytochrome P450 Cyp125 plays a key role in cholesterol metabolism being involved in the first steps of its degradation. Cyp125 is a cholesterol hydroxylase which is essential for cholesterol catabolism in M. bovis BCG and some strains of M. tuberculosis. We generated an unmarked, in-frame deletion of Cyp125 in M. tuberculosis H37Rv. The deletion strain was able to grow as well as wild-type in medium containing glucose as the carbon source. The Cyp125 deletion strain was more sensitive to growth inhibition by clotrimazole consistent with the ability of Cyp125 to bind azoles with high affinity. The deletion strain showed no difference in sensitivity to nitric oxide or hydrogen peroxide and was not attenuated for growth inside THP-1 human macrophage-like cells. These data suggest that the attenuation of virulence seen in operon deletion strains is not linked to the lack of Cyp125 alone.
Highlights
Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for the death of over 1 million people and 9 million new cases per year [1]
We constructed an in-frame, unmarked deletion strain in the M. tuberculosis H37Rv (London Pride) background [33] using a two-step homologous recombination method
Since the deletion strain was obtained on 7H10 medium with OADC supplement i.e. glucose as the carbon source, it confirmed that the knockout strain was able to utilize this carbon source
Summary
Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for the death of over 1 million people and 9 million new cases per year [1]. The current treatment of tuberculosis is time consuming and with the current treatment procedures there is an increase in the number of both multidrug resistant (MDR-TB) and extensively drug resistant (XDR-TB) M. tuberculosis strains [1]. This indicates a need for an increased understanding of the biology and pathogenic mechanisms of the bacterium in order to develop new therapeutic strategies. In relation to other bacteria this is a relatively high number, since very few prokaryotes encode any Cyp homologues; for example, Escherichia coli has none [4,5]. M. leprae has only one, which may represent the minimal requirement for a mycobacterium, since it has undergone substantial reductive evolution of its genome [6]
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