Inhibiting Mycobacterium tuberculosis Biotin Protein Ligase to target bacterial virulence factors: a novel therapeutic strategy.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is an infectious disease primarily affecting the lung, and is widespread in low- and middle-income countries. TB is treatable but major challenges remain in TB eradication due to long treatment duration, drug side effects, and emergence of drug-resistant strains. Thus, there is an urgent need to develop new drugs targeting novel metabolic pathways in the bacteria. Biotin protein ligase (BPL), the terminal enzyme in M. tuberculosis (Mtb) biotin metabolism, is essential for the bacterial survival. We have previously demonstrated that inhibition of BPL is microbicidal, prevents disease relapse in mice, and augments the chemotherapeutic efficacy of the first line anti-TB drugs rifampicin and ethambutol by enhancing intrabacterial accumulation. We have also demonstrated that BPL inhibition results in significant alteration of the Mtb cell wall (CW) architecture. This is an exciting aspect since Mtb CW is its Achille’s heel and has been targeted since the beginning of TB drug development. Currently, two of the four first line antimycobacterial drugs target the cell wall. Lipids constitute 60% of the dry weight of Mtb and 15% of its genome is dedicated to lipid metabolism associated genes. Most importantly, CW lipids play a key role in modulating host immune response against Mtb to aid bacterial survival. Taken together, these aspects position BPL as a promising novel drug target for Mtb treatment that is ripe for further investigation. The biogenesis of complex lipids is dependent on activation of Acetyl/Propionyl CoA carboxylases by biotinylation. These enzymes are involved in the first step of fatty acid biosynthesis and their optimal activity is essential for lipid biosynthesis in Mtb. Therefore, we hypothesize that chemical and/or genetic inhibition of BPL could alter CW lipids that act as virulence effectors and are crucial for early interaction between Mtb and the host immune system. This could aid bacterial clearance via enhanced activation of host antimicrobial immune responses. Consistent with the hypothesis, our preliminary data indicate that partial genetic inhibition of BPL (BPL leaky strain) elicits a robust pro-inflammatory immune response against Mtb in murine macrophages mediated via altered TLR2 engagement. These alterations in immune response are mediated by changes in the CW associated and secreted bacterial effectors. Specifically, we have identified changes in complex Mtb lipids including Phthiocerol dimycocerosate (PDIM) and Sulpholipids (SL) in the BPL hypomorph that alter phagolysosome fusion in Mtb infected macrophages. Building on these preliminary data, this proposal will characterise global alterations in bacterial effectors and macrophage responses that enhance immune activation in response to BPL inhibition and elucidate the mechanisms involved. Mtb actively supresses robust macrophage activation using bacterial effectors and our preliminary data indicate that BPL mutants of Mtb lose the ability to evade host anti-mycobacterial responses in this way. Since effective macrophage responses are the cornerstones of robust immune activation, we will use aerosol challenge studies in a murine model of Mtb infection to understand the impact of alterations in macrophage response and on other immune cells in the tissue milieu. This study will present a novel paradigm, where inhibiting a bacterial enzyme potentiates bacterial clearance both indirectly (via augmentation of host antimicrobial responses) and directly (via bactericidal effects).