Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc; 1; :aa; 3; . While the cytochrome bc; 1; :aa; 3; is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc; 1; :aa; 3; to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment

Toward a single dose cure for Buruli ulcer

A single dose of Q203 (Telacebec), a phase 2 clinical candidate for tuberculosis, eradicates; Mycobacterium ulcerans; in a mouse model of Buruli ulcer infection without relapse up to 19 weeks posttreatment. Clinical use of Q203 may dramatically simplify the clinical management of Buruli ulcer, a neglected mycobacterial disease

Inhibitors of energy metabolism interfere with antibiotic-induced death in mycobacteria

Energy metabolism has recently gained interest as a target space for antibiotic drug development in mycobacteria. Of particular importance is bedaquiline (Sirturo), which kills mycobacteria by inhibiting the F1F0 ATP synthase. Other components of the electron transport chain such as the NADH dehydrogenases (NDH-2 and NdhA) and the terminal respiratory oxidase bc1:aa3 are also susceptible to chemical inhibition. Because antituberculosis drugs are prescribed as part of combination therapies, the interaction between novel drugs targeting energy metabolism and classical first and second line antibiotics must be considered to maximize treatment efficiency. Here, we show that subinhibitory concentration of drugs targeting the F1F0 ATP synthase and the cytochrome bc1:aa3, as well as energy uncouplers, interfere with the bactericidal potency of isoniazid and moxifloxacin. Isoniazid- and moxifloxacin-induced mycobacterial death correlated with a transient increase in intracellular ATP that was dissipated by co-incubation with energy metabolism inhibitors. Although oxidative phosphorylation is a promising target space for drug development, a better understanding of the link between energy metabolism and antibiotic-induced mycobacterial death is essential to develop potent drug combinations for the treatment of tuberculosis.MOE (Min. of Education, S’pore)Published versio

Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus

Objectives: To delineate the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide) as an inhibitor of the NorA efflux pump and its impact on invasion of macrophages by Staphylococcus aureus. Methods: Capsaicin in combination with ciprofloxacin was tested for activity against S. aureus SA-1199B (NorA overproducing), SA-1199 (wild-type) and SA-K1758 (norA knockout). The role of NorA in the intracellular invasion of S. aureus and the ability of capsaicin to inhibit this invasion was established in J774 macrophage cell lines. The three-dimensional structure of NorA was predicted using an in silico approach and docking studies of capsaicin were performed. Results: Capsaicin significantly reduced the MIC of ciprofloxacin for S. aureus SA-1199 and SA-1199B. Furthermore, capsaicin also extended the post-antibiotic effect of ciprofloxacin by 1.1 h at MIC concentration. There was a decrease in mutation prevention concentration of ciprofloxacin when combined with capsaicin. Inhibition of ethidium bromide efflux by NorA-overproducing S. aureus SA-1199B confirmed the role of capsaicin as a NorA efflux pump inhibitor (EPI). The most significant finding of this study was the ability of capsaicin to reduce the intracellular invasion of S. aureus SA-1199B (NorA overproducing) in J774 macrophage cell lines by 2 log 10 . Conclusions: This study, for the first time, has shown that capsaicin, a novel EPI, not only inhibits the NorA efflux pump of S. aureus but also reduces the invasiveness of S. aureus, thereby reducing its virulence

Carbon metabolism modulates the efficacy of drugs targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis

The influence of carbon metabolism on oxidative phosphorylation is poorly understood in mycobacteria. M. tuberculosis expresses two respiratory terminal oxidases, the cytochrome bc1:aa3 and the cytochrome bd oxidase, which are jointly required for oxidative phosphorylation and mycobacterial viability. The essentiality of the cytochrome bc1:aa3 for optimum growth is illustrated by its vulnerability to chemical inhibition by the clinical drug candidate Q203 and several other chemical series. The cytochrome bd oxidase is not strictly essential for growth but is required to maintain bioenergetics when the function of the cytochrome bc1:aa3 is compromised. In this study, we observed that the potency of drugs targeting the cytochrome bc1:aa3 is influenced by carbon metabolism. The efficacy of Q203 and related derivatives was alleviated by glycerol supplementation. The negative effect of glycerol supplementation on Q203 potency correlated with an upregulation of the cytochrome bd oxidase-encoding cydABDC operon. Upon deletion of cydAB, the detrimental effect of glycerol on the potency of Q203 was abrogated. The same phenomenon was also observed in recent clinical isolates, but to a lesser extent compared to the laboratory-adapted strain H37Rv. This study reinforces the importance of optimizing in vitro culture conditions for drug evaluation in mycobacteria, a factor which appeared to be particularly essential for drugs targeting the cytochrome bc1:aa3 terminal oxidase.NMRC (Natl Medical Research Council, S’pore)MOH (Min. of Health, S’pore)Published versio

M. tuberculosis relies on trace oxygen to maintain energy homeostasis and survive in hypoxic environments

The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, we find here that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy. M. tuberculosis can metabolize oxygen efficiently at least two orders of magnitude below the concentration predicted to occur in hypoxic lung granulomas. Despite a difference in apparent affinity for oxygen, both the cytochrome bcc:aa3 and cytochrome bd oxidase respiratory branches are required for hypoxic respiration. Simultaneous inhibition of both oxidases blocks oxygen consumption, reduces ATP levels, and kills M. tuberculosis under hypoxia. The capacity of mycobacteria to scavenge trace levels of oxygen, coupled with the absence of complex regulatory mechanisms to achieve hierarchal control of the terminal oxidases, may be a key determinant of long-term M. tuberculosis survival in hypoxic lung granulomas.</p

M. tuberculosis relies on trace oxygen to maintain energy homeostasis and survive in hypoxic environments

Summary: The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, we find here that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy. M. tuberculosis can metabolize oxygen efficiently at least two orders of magnitude below the concentration predicted to occur in hypoxic lung granulomas. Despite a difference in apparent affinity for oxygen, both the cytochrome bcc:aa3 and cytochrome bd oxidase respiratory branches are required for hypoxic respiration. Simultaneous inhibition of both oxidases blocks oxygen consumption, reduces ATP levels, and kills M. tuberculosis under hypoxia. The capacity of mycobacteria to scavenge trace levels of oxygen, coupled with the absence of complex regulatory mechanisms to achieve hierarchal control of the terminal oxidases, may be a key determinant of long-term M. tuberculosis survival in hypoxic lung granulomas

Multifunctional Wound Curation Dressing Material FemuFrostAn Antioxidant-Loaded Nanoemulsion Frosted Patch of Poly(vinyl alcohol) and Hyaluronic Acid

The wound curation dressing material should own explicit elements to aggrandize wound cessation. The cryogel of poly(vinyl alcohol) (PVA) and hyaluronic acid (HA) is deemed to promote the angiogenesis, production of extracellular matrix components, granulation, and epithelialization. The research aims to tailor and evaluate the composite PVA/HA cryogel ingrained ferulic acid-loaded nanoemulsion patch labeled as PH-FemuFrost to improve the therapeutic properties and mechanical strength of the patches. The PH-FemuFrost exhibited a water uptake capacity of 268 ± 15.07%, porosity of 70.52 ± 7.4%, and 48.62 ± 2.2% in vitro degradation. The texture analysis revealed the improved mechanical properties of PH-FemuFrost in terms of burst strength and stiffness. The PH-FemuFrost exhibited in vitro antioxidant and antimicrobial activity against Staphylococcus aureus and Candida albicans species. The wound healing efficiency of PH-FemuFrost patches was significantly increased than blank PVA-HA patches. The groups treated with PH-FemuFrost exhibited a dense network of collagen type 1 in comparison to negative and PVA-HA groups. The normal skin and healed skin exhibited parallel arrangement of type I collagen fibers toward the skin. The levels of inflammatory mediators such as IL-6 (p value p value 0.0098), and TNF-α levels (p value < 0.0001) of PH-FemuFrost is significantly reduced compared to the negative group

Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection

The recent discovery of small molecules targeting the cytochrome bc 1 : aa 3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc 1 : aa 3 consists of a menaquinone:cytochrome c reductase ( bc 1 ) and a cytochrome aa 3 -type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc 1 : aa 3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc 1 : aa 3 , as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis.MOH (Min. of Health, S’pore