Cholesterol metabolism: a potential therapeutic target in Mycobacteria

Abstract

Tuberculosis (TB), although a curable disease, is still one of the most difficult infections to treat. Mycobacterium tuberculosis infects 10 million people worldwide and kills 1.5 million people each year. Reactivation of a latent infection is the major cause of TB. Cholesterol is a critical carbon source during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into lipid virulence factors. The M. tuberculosis genome contains a large regulon of cholesterol catabolic genes suggesting that the microorganism can utilize host sterol for infection and persistence. The protein products of these genes present ideal targets for rational drug discovery programmes. This review summarizes the development of enzyme inhibitors targeting the cholesterol pathway in M. tuberculosis. This knowledge is essential for the discovery of novel agents to treat M. tuberculosis infection.

Linked articles: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.

Figure 1

Diagram of the mycobacterial cell envelope. The cytoplasmic membrane of mycobacteria is encapsulated by a layer of peptidoglycans. The peptidoglycan backbone is attached to arabinogalactan through an unusual disaccharide‐phosphate‐linker region. The arabinogalactan is a branched‐chain polysaccharide consisting of a proximal galactose chain linked to a distal arabinose chain. Mycolic acids are covalently linked to the arabinogalactan‐peptidoglycan co‐polymer and are an essential component of the cell wall. Extractable lipids (free lipids) are shown in red. Another major component non‐covalently associated to the mycobacterial cell wall is the immunogenic lipoarabinomannan (LAM), which is attached to the cytoplasmic membrane by a phosphatidylinositol anchor. The porin MspA mediates the uptake of small and hydrophilic nutrients such as sugars and phosphates, whereas hydrophobic compounds diffuse directly across the cell wall. PIM, phosphatidylinositol mannoside; CF, cord factor; TMM, trehalose monomycolate; PDIM, phthiocerol dimycocerosates; LM, lipomannan, SL: sulfolipids. Virulence factors that could be affected by the cholesterol pathway are shown in red and marked with stars.

Figure 2

Mechanisms of action for current and investigational tuberculosis drugs. Targets of current drugs include cell‐wall synthesis (isoniazid, ethionamide, ethambutol and cycloserine), folate synthesis (p‐aminosalicylate), transcription (rifampin), translation (aminoglycosides), DNA metabolism (fluoroquinolones) and the cell membrane (pyrazinamide). New compounds in clinical trials target other bacterial functions. TMC207 seems to inhibit the ATP synthase complex. SQ‐109 inhibits cell‐wall synthesis, and linezolid and PNU‐100480 affect protein synthesis. Delamanid (OPC‐67863) and pretomanid (PA‐824) are prodrugs, the activation of which depends on the same cellular enzyme. TBA‐354, a nitroimidazole that is part of the same class of drugs as delamanid and pretomanid. The ultimate targets of these compounds remain unknown. Moxifloxacin, rifapentine and delamanid (green background) are in Phase III clinical trials, whereas the rest are either in Phase I or II. The asterisks denote projects that are being developed or co‐developed by the GTB Alliance.

Figure 3

Strategies used by M. tuberculosis to modulate phagosome maturation. After internalization, the bacterium uses an array of effector molecules, including the lipids phosphatidylinositol mannoside (PIM) and lipoarabinomannan (LAM) to arrest phagosome maturation. Retention of the TACO protein prevents bacterial delivery to the lysosome. TACO is a host protein that is known to be associated with cholesterol. Cholesterol has also been shown to play a role in the entry of mycobacteria into the macrophage. PE‐proteins are (Pro‐Glu) repetitive glycine‐rich proteins. MP, mycobacterial phagosome.

Figure 4

The role of essential putative operons in the intracellular survival of M. tuberculosis in macrophages (mce4, igr and the hsaACDB) in cholesterol metabolism. The diagram shows the established roles of the mce4, igr and the nat operons in cholesterol uptake and metabolism. The effect of deleting each of these operons or genes within the operon on the growth of M. tuberculosis in cholesterol and/or the effect on attenuation of infection in macrophage or in a mouse model is shown in black text. Cholesterol is transported into M. tuberculosis via the Mce4 transport system. The igr operon consists of six genes, the most important of which is cytochrome P450 (cyp125). It also comprises two acyl‐CoA dehydrogenases (fadE28 and fadE29), two conserved hypothetical proteins (Rv3541‐2c) and a putative lipid carrier protein (ltp2). Inactivation of the igr operon resulted in growth defects in cholesterol attenuation in a mouse model, an effect that is prevented by mutating the sterol uptake Mce4 system. The nat operon consists of six genes, including a NAT, four hsaACDB genes and a hypothetical protein. The nat gene product NAT utilizes Pr‐CoA, a cholesterol degradation product. Products of uncertain identity are shown as question marks. The dashed arrows indicate a multistep process. Gene products with enzymic activity are shown in red, the NAT enzyme is shown in green, hypothetical proteins in grey and transport proteins in blue.

Figure 5

Proposed degradation pathways of the cholesterol aliphatic side chain and ring nucleus. The carbon atoms derived from the ring nucleus are converted to CO₂ via the tricarboxylic acid cycle (TCA), whereas the Pr‐CoA produced from the degradation of the side chain is assimilated into mycobacterial lipids (e.g. PDIM). The enzymes and the cholesterol metabolites are described in the text. The aliphatic side chain (pink) degradation is shown with no background while the sterol ring (blue) degradation is shown with a grey background. The fate of rings C and D (black) is still unknown. Numbers correspond to the intermediates mentioned in the text. Steps catalysed by enzymes discussed in this review are shown in red arrows.

Figure 6

Identified inhibitors of key enzymes in the cholesterol pathway in M. tuberculosis.