Abstract
Mycobacterium tuberculosis, the pathogen that causes tuberculosis, presumably utilizes fatty acids as a major carbon source during infection within the host. Metabolism of even-chain-length fatty acids yields acetyl-CoA, whereas metabolism of odd-chain-length fatty acids additionally yields propionyl-CoA. Utilization of these compounds by tubercle bacilli requires functional glyoxylate and methylcitrate cycles, respectively. Enzymes involved in both pathways are essential for M. tuberculosis viability and persistence during growth on fatty acids. However, little is known about regulatory factors responsible for adjusting the expression of genes encoding these enzymes to particular growth conditions. Here, we characterized the novel role of PrpR as a transcription factor that is directly involved in regulating genes encoding the key enzymes of methylcitrate (methylcitrate dehydratase [PrpD] and methylcitrate synthase [PrpC]) and glyoxylate (isocitrate lyase [Icl1]) cycles. Using cell-free systems and intact cells, we demonstrated an interaction of PrpR protein with prpDC and icl1 promoter regions and identified a consensus sequence recognized by PrpR. Moreover, we showed that an M. tuberculosis prpR-deletion strain exhibits impaired growth in vitro on propionate as the sole carbon source. Real-time quantitative reverse transcription-polymerase chain reaction confirmed that PrpR acts as a transcriptional activator of prpDC and icl1 genes when propionate is the main carbon source. Similar results were also obtained for a non-pathogenic Mycobacterium smegmatis strain. Additionally, we found that ramB, a prpR paralog that controls the glyoxylate cycle, is negatively regulated by PrpR. Our data demonstrate that PrpR is essential for the utilization of odd-chain-length fatty acids by tubercle bacilli. Since PrpR also acts as a ramB repressor, our findings suggest that it plays a key role in regulating expression of enzymes involved in both glyoxylate and methylcitrate pathways.
Highlights
Invasive pathogens, like Mycobacterium tuberculosis, subsist on nutrients from their host and persist for decades, ensuring effective transmission of bacteria
In order to establish the role of the rv1129c-encoded M. tuberculosis PrpR protein (PrpRMt) in the regulation of prpDC transcription, we first sought to determine whether this protein interacts with the prpDC promoter region
An interaction of 6HisPrpRMt with pprpDR was confirmed by surface plasmon resonance (SPR) (Figure 2B), which showed that response unit (RU) values were proportional to protein concentration (0.05–1.2 mM)
Summary
Like Mycobacterium tuberculosis, subsist on nutrients from their host and persist for decades, ensuring effective transmission of bacteria. Accumulating evidence suggests a crucial role of fatty acids as a major carbon and energy source for this pathogen within host tissues This usage pattern presumably reflects the increased availability of lipids in infected cells [1] and is consistent with the unique feature of the M. tuberculosis genome, which contains more than 200 genes involved in fatty acid degradation [2]. Cholesterol uptake and degradation processes appear to be essential for M. tuberculosis persistence in infected animals and growth within macrophages [3,6,7,8,9]. Consistent with these observations, genes encoding b-oxidation enzymes, together with isocitrate lyase (icl1) and methylcitrate synthase (prpC), which are involved in anaplerotic metabolic cycles, are upregulated during infection of macrophages and mice, as are genes involved in cholesterol metabolism [10,11]
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