Background: Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. Results: The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. Conclusions: Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

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 3β-hydroxysteroid dehydrogenase (3β-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3β-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 3β-HSD activity with the 17β-[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 3β-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

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

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cells–glial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of host–pathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A_2AR involved in neuroimmune response, lipid metabolism, and neuron–glia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. leprae–Schwann cell interaction.Methods: M. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. Enzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR analysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzyme-linked immunosorbent assay.Results: We demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A_2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A_2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB.Conclusion: These findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A_2AR in Schwann cells, decreases A_2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae and M. lepromatosis . Brazil consistently ranks among the countries with the highest number of leprosy cases. Data from our group showed that M. leprae upregulates the mevalonate pathway (MP), contributing to the accumulation of cholesterol-ester-enriched lipid droplets in infected macrophages, and that the inhibition of this pathway by statins decreases bacterial intracellular viability. It has already been shown that part of the deleterious effect of statins on M. leprae survival is related to the reduced cholesterol levels, which M. leprae oxidizes to generate reductive power. According to the literature, statins, by inhibiting MP, increase the production of the inflammatory cytokine IL-1β through the negative modulation of the biosynthesis of the isoprenoid geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), which are responsible for protein prenylation. In the present study, we tested the hypothesis that part of the effect of statins on the intracellular viability of M. leprae comes from their impact on IL-1β production via decreased prenylation. We demonstrate that GGPP is essential in macrophage –M. leprae interaction since the MP inhibitors, pamidronate and GGTI-298, which inhibit the enzymes farnesyl pyrophosphate synthase and geranylgeranyl transferase-1, respectively, decreased the intracellular viability of M. leprae , measured by RT-qPCR. MP inhibitors increased IL-1β production by activating the inflammasome, but this effect was reversed with GGPP. IL-1β levels were inversely related to bacterial survival. In conclusion, our findings highlight the potential role of protein geranylgeranylation in M. leprae pathogenesis and suggest new therapy options for leprosy. IMPORTANCE: Mycobacterium leprae , the bacterium that causes leprosy, survives and replicates inside macrophages. Statins, which inhibit the mevalonate pathway, promote bacterial killing in macrophages by affecting cholesterol and isoprenoid production. Cholesterol is crucial for M. leprae survival in macrophages, which explains the microbicidal effect of statins on the bacteria. However, the role of isoprenoid inhibition in statin-induced bacterial killing has not been explored. Isoprenoid groups are added to about 2% of the mammalian proteins, ensuring their proper function. This study focused on geranylgeranyl pyrophosphate (GGPP) and found that inhibiting GGPP formation or protein prenylation in infected macrophages triggered IL-1β production, thereby controlling mycobacterial infection. The findings highlight the importance of protein prenylation in M. leprae and suggest new therapeutic strategies for leprosy.

Although the current multidrug therapy (MDT) for leprosy is very successful, the long treatment duration and the emergence of antibiotic-resistant strains demand for new alternative drugs. One potential target for drug development against pathogenic mycobacteria is their need to degrade host cholesterol during infection. Mycobacterium leprae , due to its degenerate genome, has preserved only the first step of cholesterol catabolism, in which cholesterol is oxidized to cholestenone by the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD). M. leprae avidly produces cholestenone in vivo, and this metabolic activity seems to play an important role in bacterial pathogenesis. In this study, six 6-azasteroid analogs were developed with the potential to inhibit 3β-HSD, and their metabolic stability and in vitro and in vivo inhibitory activity against M. leprae were investigated. Pharmacologic properties indicated lower metabolic liabilities for azasteroid 5 in comparison to its progenitors, azasteroids 1 and 2, resulting in improved accumulation and extended t _1/2 values in mice. Azasteroid 5 also partially inhibits M. leprae 3β-HSD in vitro . When tested in the Shepard’s mouse footpad model of leprosy, azasteroid 5 showed effective bacterial killing that was accelerated when combined with a subinhibitory dose of rifampicin and exhibited an absence of detectable hepatotoxic effects. We concluded that 6-azasteroids derivatives are promising new antimicrobial candidates for leprosy treatment. IMPORTANCE: Leprosy remains a significant global health challenge, particularly in underserved regions. While multidrug therapy (MDT) has been effective, its prolonged duration and the emergence of antibiotic-resistant strains emphasize the urgent need for novel therapeutic strategies. Recent advances in understanding Mycobacterium leprae ’s unique biology have identified cholesterol metabolism as a critical pathway for bacterial survival and pathogenesis, offering a promising new target for drug development. Building on insights from tuberculosis research, azasteroids—compounds known for their potential to disrupt mycobacterial cholesterol metabolism—are now being explored as candidates for leprosy treatment. These molecules inhibit M. leprae ’s cholesterol oxidation, impairing bacterial persistence within the host. This innovative approach could lead to more effective, faster-acting therapies, overcoming current treatment limitations and resistance. Such efforts represent a vital step forward in reducing the burden of leprosy and empowering affected communities worldwide.