Corrigendum: Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

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Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

Reductive power generated by Mycobacterium leprae through cholesterol oxidation contributes to lipid and ATP synthesis

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

Altered composition and functional profile of high-density lipoprotein in leprosy patients.

The changes in host lipid metabolism during leprosy have been correlated to fatty acid alterations in serum and with high-density lipoprotein (HDL) dysfunctionality. This is most evident in multibacillary leprosy patients (Mb), who present an accumulation of host lipids in Schwann cells and macrophages. This accumulation in host peripheral tissues should be withdrawn by HDL, but it is unclear why this lipoprotein from Mb patients loses this function. To investigate HDL metabolism changes during the course of leprosy, HDL composition and functionality of Mb, Pb patients (paucibacillary) pre- or post-multidrug therapy (MDT) and HC (healthy controls) were analyzed. Mb pre-MDT patients presented lower levels of HDL-cholesterol compared to HC. Moreover, Ultra Performance Liquid Chromatography-Mass Spectrometry lipidomics of HDL showed an altered lipid profile of Mb pre-MDT compared to HC and Pb patients. In functional tests, HDL from Mb pre-MDT patients showed impaired anti-inflammatory and anti-oxidative stress activities and a lower cholesterol acceptor capacity compared to other groups. Mb pre-MDT showed lower concentrations of ApoA-I (apolipoprotein A-I), the major HDL protein, when compared to HC, with a post-MDT recovery. Changes in ApoA-I expression could also be observed in M. leprae-infected hepatic cells. The presence of bacilli in the liver of a Mb patient, along with cell damage, indicated hepatic involvement during leprosy, which may reflect on ApoA-I expression. Together, altered compositional and functional profiles observed on HDL of Mb patients can explain metabolic and physiological changes observed in Mb leprosy, contributing to a better understanding of its pathogenesis.

Altered composition and functional profile of high-density lipoprotein in leprosy patients

The changes in host lipid metabolism during leprosy have been correlated to fatty acid alterations in serum and with high-density lipoprotein (HDL) dysfunctionality. This is most evident in multibacillary leprosy patients (Mb), who present an accumulation of host lipids in Schwann cells and macrophages. This accumulation in host peripheral tissues should be withdrawn by HDL, but it is unclear why this lipoprotein from Mb patients loses this function. To investigate HDL metabolism changes during the course of leprosy, HDL composition and functionality of Mb, Pb patients (paucibacillary) pre- or post-multidrug therapy (MDT) and HC (healthy controls) were analyzed. Mb pre-MDT patients presented lower levels of HDL-cholesterol compared to HC. Moreover, Ultra Performance Liquid Chromatography-Mass Spectrometry lipidomics of HDL showed an altered lipid profile of Mb pre-MDT compared to HC and Pb patients. In functional tests, HDL from Mb pre-MDT patients showed impaired anti-inflammatory and anti-oxidative stress activities and a lower cholesterol acceptor capacity compared to other groups. Mb pre-MDT showed lower concentrations of ApoA-I (apolipoprotein A-I), the major HDL protein, when compared to HC, with a post-MDT recovery. Changes in ApoA-I expression could also be observed in M. leprae-infected hepatic cells. The presence of bacilli in the liver of a Mb patient, along with cell damage, indicated hepatic involvement during leprosy, which may reflect on ApoA-I expression. Together, altered compositional and functional profiles observed on HDL of Mb patients can explain metabolic and physiological changes observed in Mb leprosy, contributing to a better understanding of its pathogenesis. AUTHOR SUMMARY: Leprosy is a chronic disease caused by Mycobacterium leprae, which causes lesions on the skin and peripheral nerves. Some patients do not present an efficient immune response and have a disseminated infection (multibacillary, Mb). Mb patients have lipid accumulation in infected tissues that is important for microorganism survival. High-density lipoprotein (HDL) is composed of proteins and lipids and is produced in the liver. It removes excess of lipids from peripheral tissues and presents anti-inflammatory activity; however, these activities are not being properly performed in leprosy. To understand more about HDL metabolism on leprosy, the chemical composition and functionality of HDL from leprosy patients were analyzed before and after treatment with antibiotics (multidrug therapy, MDT). It was observed that HDL has an altered lipid composition in Mb patients before MDT, which may lead to an impairment of its functions. Apolipoprotein A-I (ApoA-I), the main HDL protein, seems to be highly affected during infection. These functions can be slightly recovered after MDT, but not in the levels of healthy individuals. Our data open new perspectives to elucidate the modulation of lipid metabolism in leprosy and consequently to prevent disease complications.

The Essential Role of Cholesterol Metabolism in the Intracellular Survival of Mycobacterium leprae Is Not Coupled to Central Carbon Metabolism and Energy Production.

UNLABELLED: Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae-infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigated the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD), is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-(14)C]cholesterol or [26-(14)C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side chain of cholesterol as a source of energy and carbon. However, the bacteria avidly incorporated cholesterol and, as expected, converted it to cholestenone both in vitro and in vivo. Our data indicate that M. leprae has lost the capacity to degrade and utilize cholesterol as a nutritional source but retains the enzyme responsible for its oxidation to cholestenone. Thus, the essential role of cholesterol metabolism in the intracellular survival of M. leprae is uncoupled from central carbon metabolism and energy production. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies. IMPORTANCE: Our study focused on the obligate intracellular pathogen Mycobacterium leprae and its capacity to metabolize cholesterol. The data make an important contribution for those interested in understanding the mechanisms of mycobacterial pathogenesis, since they indicate that the essential role of cholesterol for M. leprae intracellular survival does not rely on its utilization as a nutritional source. Our findings reinforce the complexity of cholesterol's role in sustaining M. leprae infection. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies.

Mycobacterium leprae intracellular survival relies on cholesterol accumulation in infected macrophages: a potential target for new drugs for leprosy treatment.

We recently showed that Mycobacterium leprae (ML) is able to induce lipid droplet formation in infected macrophages. We herein confirm that cholesterol (Cho) is one of the host lipid molecules that accumulate in ML-infected macrophages and investigate the effects of ML on cellular Cho metabolism responsible for its accumulation. The expression levels of LDL receptors (LDL-R, CD36, SRA-1, SR-B1, and LRP-1) and enzymes involved in Cho biosynthesis were investigated by qRT-PCR and/or Western blot and shown to be higher in lepromatous leprosy (LL) tissues when compared to borderline tuberculoid (BT) lesions. Moreover, higher levels of the active form of the sterol regulatory element-binding protein (SREBP) transcriptional factors, key regulators of the biosynthesis and uptake of cellular Cho, were found in LL skin biopsies. Functional in vitro assays confirmed the higher capacity of ML-infected macrophages to synthesize Cho and sequester exogenous LDL-Cho. Notably, Cho colocalized to ML-containing phagosomes, and Cho metabolism impairment, through either de novo synthesis inhibition by statins or depletion of exogenous Cho, decreased intracellular bacterial survival. These findings highlight the importance of metabolic integration between the host and bacteria to leprosy pathophysiology, opening new avenues for novel therapeutic strategies to leprosy.

Mycobacterium leprae intracellular survival relies on cholesterol accumulation in infected macrophages: a potential target for new drugs for leprosy treatment

We recently showed that Mycobacterium leprae (ML) is able to induce lipid droplet formation in infected macrophages. We herein confirm that cholesterol (Cho) is one of the host lipid molecules that accumulate in ML-infected macrophages and investigate the effects of ML on cellular Cho metabolism responsible for its accumulation. The expression levels of LDL receptors (LDL-R, CD36, SRA-1, SR-B1, and LRP-1) and enzymes involved in Cho biosynthesis were investigated by qRT-PCR and/or Western blot and shown to be higher in lepromatous leprosy (LL) tissues when compared to borderline tuberculoid (BT) lesions. Moreover, higher levels of the active form of the sterol regulatory element-binding protein (SREBP) transcriptional factors, key regulators of the biosynthesis and uptake of cellular Cho, were found in LL skin biopsies. Functional in vitro assays confirmed the higher capacity of ML-infected macrophages to synthesize Cho and sequester exogenous LDL-Cho. Notably, Cho colocalized to ML-containing phagosomes, and Cho metabolism impairment, through either de novo synthesis inhibition by statins or depletion of exogenous Cho, decreased intracellular bacterial survival. These findings highlight the importance of metabolic integration between the host and bacteria to leprosy pathophysiology, opening new avenues for novel therapeutic strategies to leprosy.

Lipid droplet formation in leprosy: Toll-like receptor-regulated organelles involved in eicosanoid formation and Mycobacterium leprae pathogenesis.

A hallmark of LL is the accumulation of Virchow's foamy macrophages. However, the origin and nature of these lipids, as well as their function and contribution to leprosy disease, remain unclear. We herein show that macrophages present in LL dermal lesions are highly positive for ADRP, suggesting that their foamy aspect is at least in part derived from LD (also known as lipid bodies) accumulation induced during ML infection. Indeed, the capacity of ML to induce LD formation was confirmed in vivo via an experimental model of mouse pleurisy and in in vitro studies with human peripheral monocytes and murine peritoneal macrophages. Furthermore, infected cells were shown to propagate LD induction to uninfected, neighboring cells by generating a paracrine signal, for which TLR2 and TLR6 were demonstrated to be essential. However, TLR2 and TLR6 deletions affected LD formation in bacterium-bearing cells only partially, suggesting the involvement of alternative receptors of the innate immune response besides TLR2/6 for ML recognition by macrophages. Finally, a direct correlation between LD formation and PGE(2) production was observed, indicating that ML-induced LDs constitute intracellular sites for eicosanoid synthesis and that foamy cells may be critical regulators in subverting the immune response in leprosy.