Abstract
The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.
Highlights
New drugs active against persistent Mycobacterium tuberculosis are needed
M. tuberculosis Aldolase Is an Essential Glycolytic and Gluconeogenetic Enzyme of M. tuberculosis—The requirement of Fructose-bisphosphate aldolases (FBAs)-tb for M. tuberculosis growth was examined genetically by knocking out the chromosomal copy of fba-tb in the presence or absence of a rescue copy of this gene carried by an integrative plasmid
Attempts to inactivate fba-tb in M. tuberculosis H37Ra and M. tuberculosis H37Rv in the absence of a rescue copy of this gene yielded no candidate mutant at the last selection step of the procedure, suggesting that, unlike the situation in Neisseria meningitidis [40] but similar to those in E. coli, Streptomyces, and Candida albicans [41,42,43], the class II FBA gene of M. tuberculosis is required for growth even under optimal laboratory growth conditions where both glucose and oleic acid are present in the culture medium
Summary
New drugs active against persistent Mycobacterium tuberculosis are needed. Production of Active Form of FBA-tb and Evidence for Its Surface Exposure in M. tuberculosis—With the aim of studying the expression and subcellular localization of FBA-tb in M. tuberculosis grown under different in vitro and in vivo conditions, the FBA-tb protein was produced and purified from E. coli, and polyclonal anti-FBA-tb antibodies were raised in rabbit.
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