Study highlights glycolysis as a potential therapeutic target to combat Mycobacterium tuberculosis

How Mycobacterium tuberculosis confuses the human immune system is uncertain, although evidence has pointed to host immunometabolism -; Intrinsic links between metabolism in immune cells and their immune function. pathogen M. Tuberculosis Infected myeloid cells are known to disrupt a metabolic pathway called glycolysis, including macrophages, through an obscure mechanism.

A more accurate understanding of these pathogenic mechanisms may provide a target against the bacterium that causes 1.6 million deaths in 2021, along with 10 million new TB cases each year.

Now a study has been published Nature communication Researchers at the University of Alabama at Birmingham and the Africa Health Research Institute, or AHRI, have shown how M. tuberculosis disrupts the homeostasis of the high-energy molecule NADH and reprograms glycolysis in myeloid cells. This highlights glycolysis as a potential therapeutic target to combat the world’s leading infectious disease killer.

Glycolysis is the pathway that converts glucose to pyruvate while forming the high-energy molecules ATP and NADH. But the pathway can run in both directions, and researchers took advantage of this to implement more selective methods to block glycolytic flux. Previous experiments have taken a more sledgehammer-like approach, such as using an inhibitor that blocks glucose uptake in myeloid cells.

The reverse process of lactate fermentation is catalyzed by the enzyme lactate dehydrogenase or LDH. LDH has four subunits, a mixture of LDHA and LDHB subunits. While LDH is composed mostly of the LDHA subunit, a subunit that is predominantly expressed in myeloid cells, it preferentially converts pyruvate to lactate and NADH to NAD.⁺. However, an LDH made of LDHB subunits favors the opposite reaction.

The role of LHDA in tuberculosis pathogenesis is unknown. UAB and AHRI researchers looked at lung tissues from tuberculosis patients and found that myeloid, bronchial epithelial cells and lymphocytes stain positive for LDHA because they are involved in distinct immunological phenomena such as granuloma formation and alveolitis.

These data implicate LDHA as an important metabolic protein in the immune response of human tuberculosis lesions.”

Adri Stein, PhD, is senior author of the study

Knowing that NADH/NAD⁺ By regulating glycolysis at a defined step, Stein and colleagues hypothesized that NAD(H)-mediated glycolytic flux in myeloid cells may affect the host M. Protects against tuberculosis infection. To test this, they created mice that lacked the LDHA subunit in myeloid cells. The glycolytic capacity of these cells decreases because altered LDH function, composed of only LDHB subunits, reduces their ability to regenerate NAD.⁺ from NADH in the presence of pyruvate.

LDHA-deficient mice, while M. Those infected with a low dose of tuberculosis were more susceptible to infection and had a significantly reduced survival time. In addition, LDHA-deficient mice had worse lung gross pathology and histopathology. Moreover, wild-type mice initially M. As a protective immune response elicited a robust inflammatory response against tuberculosis infection, while LDHA-deficient mice had a marked absence of primary inflammation.

“This suggests that LDHA is necessary for protection against tuberculosis and that glycolytic flux in myeloid cells is essential for M. tuberculosis infection and disease control,” Stein said.

Despite the evidence of a reduced immune response, when the researchers measured gene expression in the lungs of LDHA-deficient mice, they found that mRNAs associated with inflammatory processes were most enriched, particularly a strong interferon-gamma gene set. “The robust interferon-gamma gene expression in more susceptible mice with a blunted immune response is particularly striking because interferon-gamma is an essential antimycobacterial cytokine that is thought to be protective in tuberculosis,” Stein said.

This problem was solved by bioenergetics experiments that showed that mouse macrophages require LDH and its LDH-mediated NAD.⁺ Regeneration for the metabolic response to interferon-gamma.

From NAD⁺ M. As depletion appeared to be central to the glycolytic inhibition caused by tuberculosis, the researchers asked whether NAD was added.⁺ The precursor, nicotinamide, will alter the ability of macrophages to mount an immune response.

Nicotinamide m. found to increase the glycolytic capacity of macrophages derived from tuberculosis-infected bone marrow. The researchers hypothesized that nicotinamide acts as a host-directed therapy by increasing glycolysis in M. tuberculosis-infected macrophages by converting NAD to NAD(H).⁺ rescue route

They found that nicotinamide was an effective treatment for tuberculosis in in vitro experiments in which they infected luciferase-expressing macrophages. M. Tuberculosis. Infected macrophages showed that nicotinamide dose-dependently reduced luminescence 48 hours after infection, and that this reduction in pathogenic bacteria was dependent on glycolysis. In a mouse model, feeding mice nicotinamide for four weeks, either three days or 28 days after infection, showed a tenfold reduction in M. tuberculosis burden in the lungs and also reduced lung inflammation.

Nicotinamide was first described in the 1940s as a treatment for tuberculosis, via a different mechanism; But it was largely abandoned when more effective drugs were discovered during the golden age of antibiotics.

However, the tuberculosis landscape has changed dramatically over the past 60 years. The incidence of tuberculosis has increased to more than 10 million new cases annually, and the pathogen has developed resistance to frontline drugs that displace nicotinamide, Stein said.

“We have provided further evidence of the host-dependent effects of nicotinamide, the metabolic requirements for its activity, and the recent demonstration of its efficacy as a treatment for tuberculosis, using two treatment methods in vivo,” Stein said of the current study. . “Logically, nicotinamide satisfies many of the criteria for an optimal novel tuberculosis treatment regimen as defined by the World Health Organization. It is inexpensive, orally bioavailable, shelf-stable, remarkably safe and tolerable, and it is well studied and routinely used in humans.” Various For hints. Ultimately, these properties make nicotinamide attractive as an ancient tool in the modern setting.”

An unanswered question remains, Stein said. M. How does tuberculosis reduce NAD(H) levels? One partial explanation, the researchers say, is that the tuberculosis necrotizing toxin M. tuberculosis, or TNT, can be secreted by an NAD⁺ Glycohydrolase. In 2015 UAB’s Michael Niederwess, Ph.D. This toxin reported by 132 years of research on M. The first toxin found in tuberculosis. TNT significantly reduced NAD in wild type M. tuberculosis⁺ Abundance in infected macrophages.

Stein is a UAB professor of microbiology and oversees labs at UAB and AHRI in Durban, KwaZulu Natal, South Africa, an area that is the epicenter of the global tuberculosis epidemic. Niederweis is a professor in the UAB Department of Microbiology.

First author of the study, “NAD(H) homeostasis underlies host protection mediated by glycolytic myeloid cells in tuberculosis,” is Hayden T. Packle, MD, Ph.D., UAB Department of Microbiology

Stein and Packle were co-authored by Krishna C. Think, vinyl p. Reddy, Sajid Nadeem, Ritesh R. Sevalkar and Joel N. Glasgow, UAB Department of Microbiology; Anupam Aggarwal, UAB Department of Medicine, Division of Nephrology; and Kivershen Nargan, Kapongo Lumamba and Threnesan Naidoo, AHRI, University of KwaZulu Natal, Durban, South Africa. Naidu also has an appointment at Walter Sisulu University, Eastern Cape, South Africa

Support came from National Institutes of Health grants Al111940, AI134810, AI137043, AI138280, A127182 and DK079337. Part funding came through Wellcome Strategic Core Award 201433/Z/16/A.