Microbes evolve diverse chemical strategies to survive in restrictive environments. Mycobacterium tuberculosis (Mtb) infection is a notable example of microbial persistence in a harsh milieu. Mtb causes tuberculosis (TB), a disease that kills more than 1.3 million people annually (1). On page 589 of this issue (2), Ruetz et al. describe how the immune system fights back against Mtb by stealing a page from the bacterial chemical warfare playbook.
When attacked by macrophages—immune cells that kill bacteria by engulfing them in an acidic intracellular compartment—Mtb undergoes metabolic changes that allow it to subsist in severely nutrient-limited conditions (3). The creative strategies that Mtb uses to hide in this persistent state are still being discovered. For example, recent analysis of lipid profiles in patient-derived Mtb strains revealed that the pathogen coats itself with a lipid- and nucleotide-derived antacid molecule (4), which serves as chemical body armor for the macrophage-entrenched bacterium.
Mtb-infected immune cells respond by diverting a common aerobic metabolite, cis-aconitate, to large-scale production of the host immunomodulator itaconate (5). Although the antibacterial properties of itaconate have been known for more than 30 years, the recent discovery that it accumulates to millimolar concentrations in activated macrophages sparked renewed interest in its molecular mechanism. Itaconate can be appended to the key metabolic cofactor coenzyme A (CoA) to yield itaconyl-CoA (I-CoA). Itaconate and I-CoA resemble intermediates in bacterial pathways for lipid and amino acid catabolism, and their ability to inhibit metabolic enzymes in other bacteria suggested a possible route to itaconate-mediated growth inhibition in pathogens (5). However, the precise target(s) of itaconate and modes of inhibition in Mtb were unknown.
MYCOBACTERIUM TUBERCULOSIS (MTB) METHYLMALONYL–COENZYME A (MM-COA) MUTASE USES A LONG TUNNEL TO BIND AND ORIENT ITS NATIVE SUBSTRATE MM-COA OR THE IRREVERSIBLE INACTIVATOR ITACONYL-COA (I-COA) NEAR A VITAMIN B12–DERIVED COFACTOR. MM-COA AND I-COA EACH TRIGGER FORMATION OF A 5′-DEOXYADENOSYL (5′-DA•) INTERMEDIATE.
GRAPHIC: C. BICKEL/SCIENCE
Ruetz et al. now address both questions by showing that I-CoA strongly inhibits Mtb methylmalonyl-CoA mutase (MCM) by undergoing covalent attachment to the enzyme’s vitamin B12–derived cobalamin (Cbl) cofactor. MCM promotes the carbon-skeleton rearrangement that converts its namesake substrate to succinyl-CoA. The reaction transforms a compound produced by breakdown of amino acids and lipids into an intermediate that feeds directly into the tricarboxylic acid cycle (6). MCM uses adenosylcobalamin (AdoCbl) to generate a reactive 5′-deoxyadenosyl radical (5′-dA•) intermediate (see the figure). In the native transformation, the enzyme carefully controls the radical, using it catalytically to abstract hydrogen (H•) from a substrate methyl group. Formation of the radical enables rearrangement of the carbon skeleton, and H• is returned by the 5′-dAH to allow the AdoCbl cofactor to be regenerated. This intricate choreography depends, in part, on the enzyme’s long access tunnel, which accommodates and recognizes the CoA appendage. Only when the substrate is bound does the enzyme promote 5′-dA• formation.
Ruetz et al. show that the immunometabolite I-CoA disguises itself in the active site of Mtb MCM and turns the enzyme’s potent chemistry against itself, leading to an irrevocable attack on the AdoCbl cofactor. I-CoA and the native MCM substrate have nearly identical shapes. When I-CoA is bound to MCM, this similarity tricks the enzyme into forming its 5′-dA• intermediate. However, ICoA directs its terminal olefin (rather than the sp3-hybridized carbon that, in methylmalonyl-CoA, donates H•) toward 5-dA•. The authors demonstrated that this feature of the Mtb I-CoA complex with MCM results in radical addition to the cofactor, forming a new covalent C–C bond in the active site. This permanently traps the cofactor in an inactive state. The inhibition strategy, called mechanism-based suicide inactivation, is also the basis for some of our most potent pharmaceuticals, because it minimizes off-target effects and cells can recover the affected pathway only through new enzyme synthesis.
Most organisms that require vitamin B12 use dedicated pathways to repair their cobalamin enzyme cofactors, which are difficult to synthesize and acquire (7). Ruetz et al. examined whether I-CoA–inhibited MCM can be targeted by vitamin B12 repair machinery, which transfers the inactivated cofactor to a second protein for replacement of the deoxyadenosyl ligand and cofactor reactivation. The authors showed that I-CoA–bound MCM is recognized by B12 repair machinery, but the inhibited enzyme does not exchange its inactivated AdoCbl for a new cofactor. Failure to deliver new cofactors to MCM can cause the repair system to break apart AdoCbl waiting to be loaded onto enzymes (8), suggesting that itaconate could ultimately induce B12 deficiency in Mtb (9).
MCM is one of two vitamin B12 enzymes in humans, where it is used to prevent accumulation of toxic lipid and protein catabolites. Ruetz et al. show that human MCM is also susceptible to irreversible inactivation by itaconate, raising questions about how human cells protect themselves from the immunometabolite during Mtb infection. Recent work suggests that the human immune system takes additional inspiration from bacteria to shield its own B12 enzymes from itaconate attack. Microbes that synthesize and excrete toxic chemicals to kill neighboring cells typically produce their own internal resistance proteins. A 2017 study (9) identified a human itaconate breakdown pathway involving an orphan enzyme, citramalyl-CoA lyase (CLYBL), in detoxification of I-CoA before it can inhibit human B12 enzymes. Loss of CLYBL leads to B12 deficiency, which is now linked by the new study to I-CoA suicide inhibition of human MCM and inactivation of human cobalamin repair pathways. Mtb and other pathogens have analogous itaconate resistance pathways (10).
How Mtb acquires vitamin B12 during infection is unknown, but the pathogen might rely on host B12 for survival. Itaconate could affect TB disease progression through nutritional immunity, a phenomenon in which the immune system limits access to necessary transition metals for intracellular pathogens (11). Certain human populations have a high incidence (3 to 6%) of biallelic mutations in the CLYBL gene. In these individuals, loss of the human itaconate detoxification pathway and subsequent B12 deficiency in macrophages could further enhance the ability of the immune system to fight TB through cobalt cofactor deprivation (9).
Whereas Cbl enzymes are rare, radical chemistry is common in microbes. Radical S-adenosyl-methionine (SAM) enzymes use an iron-sulfur cluster and SAM instead of AdoCbl to generate 5′-dA• for aliphatic H• abstraction. Although they rely on a simpler cofactor, these more widespread bacterial proteins might be similarly vulnerable to mechanism-based 5′-dA• capture by host or microbe-derived small molecules.
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