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Title: Inactivation of Fructose-1,6-Bisphosphate Aldolase Prevents Optimal Co-catabolism of Glycolytic and Gluconeogenic Carbon Substrates in Mycobacterium tuberculosis}
Article:
Abstract:
Metabolic pathways used by Mycobacterium tuberculosis( Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1, 6-bisphosphate aldolase( FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb's ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout( Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb's ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb's FBA in vivo.
Introduction:
Metabolism is an important aspect of all host–pathogen interactions[1]. Mycobacterium tuberculosis( Mtb) has evolved to replicate and survive for decades within diverse host granulomas, including caseating, fibrotic and cavitating lesions, each providing a different environment[2],[3]. Mtb's ability to adapt to multiple environments may in part be attributed to its metabolic flexibility and its capacity to efficiently catabolize multiple carbon sources simultaneously[4]–[6]. Fatty acids and lipids are major carbon sources available to Mtb in vivo[7]–[10], but Mtb also has access to glucose and glycolytic 3-carbon compounds, requires trehalose import for virulence and can utilize CO2 as source of carbon[6],[11]–[13]. Mtb lacks classical carbon catabolite repression and it remains to be identified how co-catabolism of multiple carbon sources is regulated to achieve optimal growth[4]. Knowledge about Mtb's metabolism benefits the understanding of tuberculosis pathogenesis and can identify potential new targets for chemotherapeutic interventions, as Mtb mutants lacking metabolic enzymes are among the most attenuated in the mouse model of tuberculosis[8],[10],[14]–[17]. Fructose bisphosphate aldolase( FBA) is central to glycolysis and gluconeogenesis( Figure S1) and has been the focus of structural, enzymatic, and drug developmental studies[18]–[22]. Evidence supporting FBA's requirement for optimal growth came from the failure to isolate mutants with transposon insertions in fba, the gene encoding FBA, in standard in vitro culture conditions[23]. Also, previous attempts to delete fba from the Mtb chromosome by homologous recombination have failed[18],[19]. A conditional fba mutant generated in the attenuated H37Ra strain revealed that growth in either glucose- or succinate-containing media was strictly dependent upon induction of FBA expression, demonstrating that FBA is an essential enzyme for both glycolysis and gluconeogenesis[19]. Moreover, FBA is expressed in Mtb during mouse and guinea pig infections, and increased amounts were identified in culture filtrates during hypoxia, a condition which persistent Mtb is thought to encounter in the host[19],[24]. FBA has recently been shown to be secreted by Mtb and to bind host plasminogen, potentially enabling FBA to play a metabolism-independent role in host-pathogen interactions[19]. Importantly, Mtb FBA differs from human FBA by its reaction mechanism. FBAs catalyze the reversible cleavage of fructose-1, 6-bisphosphate( FBP) to yield dihydroxyacetone phosphate( DHAP) and glyceraldehyde 3-phosphate( G3P). However, class I FBAs such as the human enzyme produce a Schiff-base reaction intermediate whereas class II FBAs, to which the Mtb enzyme belongs, require a divalent metal cation such as Zn2+ to stabilize the enolate intermediate[25]. Although some bacteria express both class I and class II enzymes, Mtb lacks an annotated class I FBA[19],[26]. Class I FBA activity in Mtb has been reported in earlier studies, but could not be detected by others[19],[27],[28]. The difference in catalytic mechanism of FBA from humans compared to that of Mtb enabled the design of bacteria-targeting class II-specific FBA inhibitors and work is ongoing to improve their efficacy[20],[22],[29],[30]. Our goal was to investigate the importance of Mtb's FBA in acute and chronic mouse infections and to further characterize the basis for its in vitro essentiality. To achieve these goals we generated Mtb strains in which FBA expression was tightly regulated by a tunable dual-control( DUC) genetic switch that combines transcriptional silencing and controlled protein depletion[31]. We found that essentiality of FBA was condition-dependent, and could be overcome by availability of two carbon sources entering metabolism above and below the FBA-catalyzed step. However this was dependent on the specific ratio of the glycolytic and gluconeogenic carbon sources, because metabolism of butyrate by a strain lacking fba was dependent on Mtb's ability to efficiently co-catabolize glucose. Mtb relied on FBA for both growth during acute and persistence during chronic mouse infections.
Discussion:
The experiments reported here enhance our understanding of Mtb carbon metabolism and are relevant to tuberculosis drug development. Mtb fba has previously been shown to be required for growth on standard media[19],[33] and FBA inhibitors are under development[22],[29],[30], but to our knowledge no FBA-specific inhibitor has been effective at inhibiting growth of live Mtb. Furthermore, it was unknown whether FBA is essential for growth or survival of Mtb in vivo. Using a genetic approach we evaluated FBA as a potential drug target by assessing its essentiality for growth and persistence during mouse infections. Depletion of FBA after establishment of chronic infection led to complete clearance of viable Mtb in mouse lungs and spleens, which promotes FBA as a potential therapeutic target for killing of persistent bacilli. Unexpectedly, FBA was not essential in all conditions in vitro, and a conditional fba mutant was critical to identify conditions in which Δfba could be isolated. This deletion mutant was able to replicate when provided with combinations of carbon substrates entering metabolism above and below the FBA-catalyzed reaction. Thus, it will be crucial to determine the efficacy of FBA inhibitors against live Mtb in carbon conditions where Mtb is vulnerable to FBA inactivation in contrast to standard mycobacterial liquid culture medium, which contains a combination of glucose and glycerol making FBA dispensable. Earlier studies have highlighted Mtb's ability to co-catabolize multiple carbon sources in vitro and in macrophages[4]–[6]. This is in contrast to other bacteria such as E. coli, which exhibits diauxic growth in the presence of multiple carbon sources resulting from their sequential metabolism[34],[35]. Here, we provide further evidence for Mtb's co-catabolism and offer insight into its metabolic regulation. Growth of WT Mtb on glucose and on butyrate, which requires β-oxidation for conversion into acetyl-CoA mimicking the fate of long chain fatty acids, was enhanced in a dose responsive manner by addition of the respective other carbon source. Growth of Δfba only occurred with two carbon substrates, feeding into either side of the FBA reaction. Additionally, deletion of FBA rendered Mtb extraordinarily sensitive to the relative concentrations of carbon sources in the medium. In media containing glucose and butyrate, Δfba was unable to efficiently utilize glucose to enhance its growth while growth of WT scaled with glucose. Metabolism of increasing amounts of butyrate was also restricted shown by Δfba's limited growth despite the presence of 0. 2% glucose. FBA thus facilitates efficient butyrate catabolism through its ability to metabolize glucose and FBA inactivation resulted in suboptimal co-catabolism of glucose and butyrate. The mechanism of death of Δfba in carbon-unbalanced media remains to be determined but may be due to buildup of FBA substrates at higher concentrations of glucose or butyrate, as well as regulatory mechanisms allowing Mtb to sense carbon levels and respond accordingly. Indeed, the inability of Δfba to grow on standard Mtb plate medium containing glucose, glycerol and oleic acid was overcome by the addition of 0. 2% additional glucose, which was not required for growth in nearly identical medium lacking oleic acid. Inactivation of FBA in E. coli prevented growth with glucose but not with glycerol and succinate as carbon substrates[36],[37]. Accumulation of sugar phosphates including the FBA substrate fructose-1, 6-bisphosphate( FBP) is thought to be the cause of this growth inhibition, which is supported by suppressor mutations that prevent FBP buildup and allow growth on glucose[38]. With our metabolite extraction protocol we did not detect FBP, however other sugar phosphates including hexose-P, sedoheptulose-P and pentose-P increased dramatically in Δfba cultured on glucose, while triose-P and PEP accumulated on glycerol. The accumulation of these metabolites was not observed when both glucose and glycerol were available to Δfba, except for the triose-P pools, which remained elevated but much less than in the glycerol condition. It is thus possible that the accumulation of phosphorylated metabolites is cause for the death of Δfba in conditions where glycolytic and gluconeogenic carbon flow is unbalanced, such as with single carbon substrates or in mixes with an abundance of a glycolytic or gluconeogenic substrate. How potent would an FBA inhibitor need to be to affect Mtb growth? We measured the vulnerability of Mtb to FBA depletion in single glycolytic and gluconeogenic carbon sources where FBA is essential. Although depleting FBA by 97% did not affect growth with butyrate as sole carbon source, it prevented growth with glucose and 87% inhibition was sufficient to reduce growth with glucose. These data reveal a differential susceptibility of Mtb to FBA depletion, depending on the available carbon source. Antibiotic targets vary widely in how much inhibition is required to stop replication or induce death and not all effective drug targets are highly vulnerable to inhibition[39]. Specific target-drug interactions can contribute to the efficacy of a compound. We cannot predict how effectively FBA must be depleted to abolish replication in vivo, but the WT-like phenotype of FBA-DUC without doxy suggests that 13% of WT FBA levels are sufficient for normal growth and persistence in mice. The fast killing of Mtb following further FBA depletion in mouse lungs and spleens suggests that it is an effective target during acute and chronic mouse infections. During infections FBA may also have an additional, metabolism-independent function in Mtb's interaction with the host as it can be secreted and bind to human plasminogen[19]. The requirement of FBA for growth and persistence in mice suggests that in vivo Mtb either faces single carbon sources or lacks access to the growth permissive ratio of carbon sources that can compensate for the lack of FBA. This is likely due to an abundance of fatty acids and lipids which are predominant carbon sources available to Mtb in vivo[7]–[10]. It is unknown if FBA is essential for growth and persistence of Mtb in humans; experimental animal models that more closely mimic human TB pathology[40] would help address this question. Given that human granulomas contain lipid-rich foamy macrophages and build up lipids in the caseum[41],[42], it is plausible that Mtb requires FBA also during human infection.
The development of new chemotherapies targeting Mycobacterium tuberculosis ( Mtb ) will benefit from genetic evaluation of potential drug targets and a better understanding of the pathways required by Mtb to establish and maintain chronic infections . We employed a genetic approach to investigate the essentiality of fructose-1 , 6-bisphosphate aldolase ( FBA ) for growth and survival of Mtb in vitro and in mice . A conditional fba mutant revealed that Mtb requires FBA for growth in the acute phase and for survival in the chronic phase of mouse infections . In vitro essentiality of fba was strictly condition-dependent . An FBA deletion mutant ( Δfba ) required a balanced combination of carbon substrates entering metabolism above and below the FBA-catalyzed reaction for growth and died in media with single carbon sources and in mouse lungs . Death of Δfba in vitro was associated with the perturbation of intracellular metabolites . These studies highlight how a conditional fba mutant helped identify conditions in which FBA is dispensable for growth of Mtb , evaluate FBA as a potential target for eliminating persistent bacilli and offer insight into metabolic regulation of carbon co-catabolism in Mtb .