Pantothenate and Pantetheine Antagonize the Antitubercular Activity of Pyrazinamide

Abstract

Pyrazinamide (PZA) is a first-line tuberculosis drug that inhibits the growth of Mycobacterium tuberculosis via an as yet undefined mechanism. An M. tuberculosis laboratory strain that was auxotrophic for pantothenate was found to be insensitive to PZA and to the active form, pyrazinoic acid (POA). To determine whether this phenotype was strain or condition specific, the effect of pantothenate supplementation on PZA activity was assessed using prototrophic strains of M. tuberculosis. It was found that pantothenate and other β-alanine-containing metabolites abolished PZA and POA susceptibility, suggesting that POA might selectively target pantothenate synthesis. However, when the pantothenate-auxotrophic strain was cultivated using a subantagonistic concentration of pantetheine in lieu of pantothenate, susceptibility to PZA and POA was restored. In addition, we found that β-alanine could not antagonize PZA and POA activity against the pantothenate-auxotrophic strain, indicating that the antagonism is specific to pantothenate. Moreover, pantothenate-mediated antagonism was observed for structurally related compounds, including n-propyl pyrazinoate, 5-chloropyrazinamide, and nicotinamide, but not for nicotinic acid or isoniazid. Taken together, these data demonstrate that while pantothenate can interfere with the action of PZA, pantothenate synthesis is not directly targeted by PZA. Our findings suggest that targeting of pantothenate synthesis has the potential to enhance PZA efficacy and possibly to restore PZA susceptibility in isolates with panD-linked resistance.

INTRODUCTION

Pyrazinamide (PZA) is one of four first-line drugs used in standard short-course combination therapy for tuberculosis (TB). Due to its role in reducing relapse rates and in shortening treatment duration from 9 to 6 months (1, 2), PZA is anticipated to be an irreplaceable component of future first-line TB drug regimens (3). However, the durability of PZA and other therapeutic agents is being challenged by the emergent spread of drug-resistant strains of Mycobacterium tuberculosis. In order to maintain comparable short-course drug regimens, it is important to identify suitable alternatives to PZA. To do so, it is necessary to understand the mechanistic basis for the susceptibility and resistance of M. tuberculosis to PZA and its potential surrogates.

Under standard laboratory growth conditions, PZA lacks measurable activity against M. tuberculosis due to the ability of the bacilli to eliminate cytoplasmic pyrazinoic acid (POA) by an as yet unidentified efflux mechanism (4). Conditions that promote POA accumulation, such as low pH (≤6.0) (5), anaerobiosis (6), and coincubation with electrochemical gradient uncouplers (7) or efflux pump inhibitors (4), have been shown to significantly enhance the susceptibility of M. tuberculosis to PZA. While M. tuberculosis can be found within hypoxic compartments during infection (8), the contribution of these factors to in vivo PZA susceptibility has yet to be experimentally confirmed.

PZA is a prodrug that is hydrolyzed to the presumed active form of POA by the M. tuberculosis pyrazinamidase/nicotinamidase PncA (9). PncA is an enzyme of the NAD salvage pathway (10, 11). Due to the dispensability of NAD salvage in M. tuberculosis metabolism, spontaneous loss-of-function mutations in the pncA gene can arise without a notable fitness cost to the bacilli. As such, pncA loss-of-function mutations show a high correlation with clinical PZA resistance (12, 13). Moreover, a direct association between loss-of-function mutations in pncA and PZA resistance in animal models of infection has been established (14). While POA is effective in vitro against PncA-deficient strains of M. tuberculosis, it lacks activity in vivo, presumably due to its high rate of clearance (15).

Several models have recently been proposed to explain the mode of PZA action on M. tuberculosis. The first of these models comes from studies involving the PZA structural analog 5-chloropyrazinamide (5-Cl PZA), which was found to be a potent inhibitor of the mycobacterial fatty acid synthase I (FAS-I) (16–18). While M. tuberculosis FAS-I activity was found to also be compromised by PZA treatment (16, 18, 19), in situ evidence for this inhibition has been confounded by the inability to overexpress FAS-I in M. tuberculosis (16). The model has also been challenged by a report in which inhibition of purified FAS-I by POA was not observed, and FAS-I inhibition in whole cells was suggested to be a downstream consequence of POA-mediated organic acid stress (17). Further work is critical to resolve direct FAS-I targeting by POA. A second model suggested that POA promotes acidification of the M. tuberculosis cytoplasm through a proton-shuttling mechanism (7). In this model, bacilli residing in an acidic environment pump POA anion out to the extracellular environment. A fraction of the excluded POA becomes protonated and reenters the cell by passive diffusion. Once in the cytoplasm, the proton dissociates from POA, and the cycle continues until the cytoplasmic pH equilibrates with that of the extracellular environment. Inconsistent with this model, the acid dependence of PZA activity can be abolished by incubation under alkaline pH (5), by overexpression of pncA (19), or by preventing POA from cycling in and out of the cell by the addition of efflux pump inhibitors (4). Moreover, it has been demonstrated that the primary in vivo niches for M. tuberculosis, phagocyte lysosomes and tubercular granulomas, do not acidify when live bacilli are present (20, 21). Thus, while acidic culture conditions can promote the cytoplasmic accumulation of POA, low pH can be fully uncoupled from the activity of the drug against an essential cellular target. The third model to be proposed suggested that POA inhibits trans-translation, a process that many bacteria employ to liberate ribosomes that have stalled on nonstop mRNA transcripts (22). This model was based in part on an apparent interaction between the ribosomal protein RpsA and POA and the identification of an rpsA polymorphism in a previously described PZA-resistant M. tuberculosis clinical isolate, DHMH 444. Inconsistent with this model, strain DHMH 444 was previously shown to be fully susceptible to POA (23) and susceptible to PZA in a murine model of infection (14). Moreover, trans-translation has been shown to be nonessential for growth under conditions that promote PZA susceptibility, and a strain defective for trans-translation was found to be fully susceptible to PZA (24). Thus, inhibition of trans-translation is not likely to explain PZA-mediated inhibition of M. tuberculosis growth. The most recent model to be proposed suggested that PZA inhibits the M. tuberculosis aspartate decarboxylase PanD and thereby inhibits synthesis of the essential metabolic cofactors pantothenate and coenzyme A (CoA) (Fig. 1) (25). This model was based on the identification of panD missense mutations in spontaneous PZA-resistant M. tuberculosis laboratory isolates that did not have loss-of-function mutations in pncA. As FAS-I is pantothenate dependent and coenzyme A plays a crucial role in carbon and energy metabolism, this model provides a potential explanation for the pleiotropic affects of PZA on M. tuberculosis. However, additional genetic and biochemical studies are essential to delineate a mechanistic link between PanD and PZA action. While the models described above are not mutually exclusive, many questions remain in refining our understanding of the PZA mode of action.

FIG 1.

Pantothenate/coenzyme A biosynthetic pathway of M. tuberculosis. The boxes represent proteins that catalyze the respective reactions depicted by arrows. The series of reactions from pyruvate to pantoate are abbreviated by a triple arrow. The H37Rv genes that encode the proteins are shown in italics. Metabolites that were found to antagonize the antitubercular activity of PZA are shown in boldface.

Upon evaluating PZA activity against various laboratory strains of M. tuberculosis, we found that supplementation of cultures with β-alanine, pantothenate, or pantetheine could antagonize the action of PZA and some of its structural analogs. These data are consistent with the previous indication of a link between pantothenate synthesis and PZA action. In this study, we found that an M. tuberculosis strain with panC (encoding pantoate β-alanine ligase) and panD deleted (Fig. 1) could be cultivated with pantetheine at a concentration that did not antagonize PZA activity. This finding uncouples pantothenate synthesis and PZA action, indicating that panothenate-mediated antagonism occurs via a novel mechanism that is independent of PanD. These observations suggest that inhibition of the pantothenate/coenzyme A biosynthetic pathway has the potential to improve PZA potency and restore PZA susceptibility in isolates with pantothenate-linked resistance.

MATERIALS AND METHODS

Bacterial strains and growth media.

M. tuberculosis strains H37Rv, H37Ra, mc²7000 (H37Rv ΔRD1 ΔpanCD), Erdman, and CDC1551 were gifts from W. R. Jacobs, Jr., of the Albert Einstein College of Medicine. The strains were grown in Middlebrook 7H9 medium (Difco) supplemented with 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC) (Difco), 0.2% (vol/vol) glycerol, and 0.05% (vol/vol) tyloxapol. d,l-Pantothenate (50 μg ml⁻¹) or d-pantethine (7.2 μg ml⁻¹) was added for growth of M. tuberculosis strain mc²7000.

Compounds.

All chemicals were purchased from Sigma-Aldrich unless otherwise stated. PZA (MP Biomedicals), nicotinamide (Alfa Aesar), n-propyl pyrazinoate (nPPA) (a gift from J. T. Welch, State University of New York [SUNY], Albany, NY), 5-Cl PZA (a gift from J. T. Welch), and isoniazid (INH) were dissolved in dimethyl sulfoxide (DMSO). POA and nicotinic acid (Alfa Aesar) were dissolved in water, the pH was adjusted to 5.8 using NaOH, and the solutions were filter sterilized using a 0.2-μm syringe filter. β-Alanine (Alfa Aesar), d,l-pantothenate, d-pantetheine, l-aspartic acid, carnosine, taurine, 3-aminopropanol, propanoic acid, propylamine, 4-aminobutyric acid, and glycine (Fisher) were dissolved in water and filter sterilized. d-Pantolactone was hydrolyzed to d-pantoate via alkaline saponification (26) and then neutralized and filter sterilized.

Antimycobacterial susceptibility determinations.

Antimycobacterial susceptibility was determined by measuring the optical densities of cultures at 600 nm (OD₆₀₀). PZA, POA, nicotinamide, and nicotinic acid susceptibility testing was performed using medium adjusted to pH 5.8. For INH, 5-Cl PZA, and nPPA susceptibility testing, the pH of the growth medium was not adjusted from pH 6.8. At the concentrations used, the compounds that were tested for antagonism did not measurably alter the pH of the growth medium. The MIC (MIC₉₀) for antimycobacterial compounds was defined as the minimum concentration required for inhibition of at least 90% of growth relative to the no-antimycobacterial control. The minimum antagonistic concentration (MAC₁₀) was defined as the minimum concentration of antagonist required to enable greater than 10% growth in the presence of antimycobacterial compounds relative to the no-antimycobacterial control. For MIC₉₀ and MAC₁₀ determinations, M. tuberculosis strains were first grown to mid-exponential phase in standard growth medium and diluted to an OD₆₀₀ of 0.01 in 5 ml of 7H9 medium in 30-ml square Nalgene bottles (Fisher). Antimycobacterial compounds and/or antagonists were added to the final concentrations indicated in the text. Cultures were incubated at 37°C with shaking on a rotary platform at 100 rpm for the specified amount of time. All results presented are from a minimum of three independent determinations.

Antagonism growth assays.

The growth kinetics of M. tuberculosis strains were assessed in the presence of antimycobacterial compounds and select antagonists under the conditions described above. Exponentially growing cells were diluted to an OD₆₀₀ of 0.01 in supplemented 7H9 medium containing 200 μg ml⁻¹ PZA or 400 μg ml⁻¹ POA with or without 1 mM pantothenate, β-alanine, or pantetheine. The cultures were incubated at 37°C with shaking on a rotary platform at 100 rpm. Growth was monitored by following the OD₆₀₀ over time.

RESULTS

Metabolites of the pantothenate/coenzyme A biosynthesis pathway antagonize PZA and POA activity in M. tuberculosis.

While conducting drug susceptibility assays, it was found that the M. tuberculosis H37Rv derivative mc²7000 (ΔpanCD ΔRD1) was insensitive to PZA (Table 1). To determine whether this lack of PZA susceptibility was strain or condition specific, the effect of pantothenate supplementation on the PZA susceptibilities of different prototrophic laboratory strains of M. tuberculosis was assessed (Table 1). It was found that the addition of pantothenate to the growth medium increased the PZA MIC₉₀ by 4-fold for strains H37Ra and H37Rv and by greater than 8-fold for strains Erdman and CDC1551 (Table 1). Thus, the PZA insensitivity of M. tuberculosis mc²7000 was due to the presence of exogenous pantothenate in the growth medium and is likely to occur for any strain of M. tuberculosis supplemented with sufficient levels of pantothenate.

TABLE 1.

MICs of PZA for various M. tuberculosis strains in the absence or presence of exogenously supplied pantothenate

Strain	Relevant characteristics	PZA MIC90a (μg ml−1)
		No pantothenate	200 μM pantothenate
H37Rv	Virulent laboratory strain	200	800
mc27000	H37Rv ΔpanCD ΔRD1	NDb	>800
H37Ra	Spontaneously attenuated derivative of H37Rv	100	400
Erdman	Virulent laboratory strain	100	>800
CDC1551	Virulent laboratory strain	100	>800

^aMIC₉₀, defined as the minimum concentration that inhibits ≥90% of growth relative to the no-drug control over 3 weeks of incubation in supplemented 7H9 medium, pH 5.8.

^bND, not determined.

To further characterize this effect, the PZA MIC₉₀s for M. tuberculosis strain H37Ra were determined over a range of pantothenate concentrations. PZA antagonism was found to occur in a dose-dependent manner (Fig. 2). As normal whole-blood pantothenate concentrations are typically in the low micromolar range (27), it is worth noting that 7 μM pantothenate was sufficient to enable growth of M. tuberculosis at a PZA concentration above the proposed resistance breakpoint of 300 μg ml⁻¹ (28).

FIG 2.

Dose response for pantothenate-mediated antagonism of the antitubercular activity of PZA. Pantothenate and PZA were added to cultures of M. tuberculosis H37Ra at various concentrations. PZA MICs were determined following 3 weeks of incubation at 37°C. *, PZA MIC was greater than 800 μg ml⁻¹.

To assess the kinetics of antagonism, the growth of M. tuberculosis strains H37Ra and H37Rv was monitored during exposure to PZA or POA in medium supplemented with pantothenate or β-alanine. Pantothenate enabled initiation of growth in the presence of PZA at an earlier time than β-alanine (Fig. 3A and C). However, initiation of growth in the presence of pantothenate and PZA was markedly delayed relative to the no-drug control (Fig. 3A and C). In contrast, both pantothenate and β-alanine antagonized POA immediately, resulting in growth kinetics similar to those of the no-drug controls (Fig. 3B and D). The starkly different response times for PZA and POA suggest that PZA is likely antagonized following its conversion to POA.

FIG 3.

Growth kinetics of M. tuberculosis strains supplemented with pantothenate and β-alanine during exposure to PZA and POA. The growth of M. tuberculosis strains H37Rv (A and B), H37Ra (C and D), and mc²7000 (E and F) in 7H9 medium containing OADC (10%), glycerol (0.2%), and tyloxapol (0.05%) with 1 mM pantothenate (solid diamonds), 1 mM β-alanine (solid squares), or no additional supplementation (solid circles) is shown. (E and F) Strain mc²7000 was additionally supplemented with 7.2 μg ml⁻¹ pantethine. The cultures were treated with either 200 μg ml⁻¹ PZA (A, C, and E) or 400 μg ml⁻¹ POA (B, D, and F) in the presence of 1 mM pantothenate (open diamonds), 1 mM β-alanine (open squares), or no additional supplementation (open circles).

To determine whether pantothenate-related metabolites also antagonize PZA action, other intermediates of the CoA biosynthetic pathway were assessed. Supplementation of M. tuberculosis cultures with 1 mM β-alanine, pantothenate, or pantetheine led to a dramatic increase in the MIC₉₀s of both PZA and POA (Table 2). In addition, carnosine, a dipeptide of β-alanine and histidine, also antagonized PZA and POA activity (Table 2). In contrast, aspartate (a metabolic precursor of β-alanine) and pantoate (a cosubstrate with β-alanine in the synthesis of pantothenate) did not show measurable antagonistic activity (Table 2). Evaluation of the MAC₁₀s for this series of metabolites revealed that pantothenate, β-alanine, and pantetheine were the most potent PZA antagonists, while the activity of carnosine was relatively weak (Table 3). Aspartate and pantoate did not show measurable antagonistic activity at any of the concentrations that were tested (Table 3). These data indicate that β-alanine appears to be an essential constituent of antagonistic metabolites.

TABLE 2.

MIC₉₀s of select antimycobacterial compounds in the presence of various metabolites

^aMIC₉₀, defined as the MIC that inhibits >90% of M. tuberculosis H37Ra growth relative to the no-drug control over 2 weeks of incubation.

^bND, not determined.

TABLE 3.

Minimum concentrations of metabolites required for antagonism of PZA action against M. tuberculosis H37Ra

^aMAC₁₀, defined as the minimum concentration of antagonist that permits greater than 10% growth relative to the no-drug control.

Structural analogs of β-alanine antagonize PZA activity.

To further assess the structural requirements for the antagonism of PZA activity, we investigated the abilities of several β-alanine analogs to antagonize PZA activity in cultures of M. tuberculosis. These structural analogs differed from β-alanine by the absence of the terminal carboxyl group (taurine, 3-aminopropanol, and propylamine), the absence of the terminal amino group (propanoic acid), and an altered chain length between the carboxyl group and the amino group (4-aminobutyrate and glycine) (Table 4). While most of the analogs did not show any measurable antagonistic activity against PZA, 3-aminopropanol and propanoic acid were found to abolish PZA activity at concentrations of 6.25 and 200 μM, respectively (Table 4).

TABLE 4.

Minimum concentrations of β-alanine structural analogs required to antagonize PZA action against M. tuberculosis

^aMAC₁₀, defined as the minimum concentration of antagonist that permits greater than 10% growth relative to the no-drug control.

Susceptibility of M. tuberculosis to PZA is independent of panCD.

The ability of β-alanine to antagonize PZA and POA activity is consistent with the speculation that POA inhibits the M. tuberculosis aspartate decarboxylase encoded by panD (25). As such, it follows that any condition that can support the growth of a strain with panD deleted should antagonize PZA activity. Upon evaluating conditions that permit growth of the pantothenate-auxotrophic strain mc²7000, it was found that the strain could be cultured in the absence of pantothenate by supplementation with a subantagonistic concentration of pantetheine (Fig. 3E). Under these growth conditions, PZA susceptibility was restored for the first 3 weeks of incubation, after which growth was observed (Fig. 3E). This inhibitory effect of PZA for a strain with panD deleted indicates that PanD is not the principal target for PZA-mediated inhibition of M. tuberculosis.

As POA activity was more potently antagonized by pantothenate/CoA intermediates, 400 μg ml⁻¹ POA showed only a modest inhibitory effect on the growth of strain mc²7000 in the presence of pantetheine (Fig. 3F). Similar to what was observed for other M. tuberculosis strains, pantothenate antagonized PZA and POA activities against mc²7000 (Fig. 3E and F). However, in contrast to what was observed for strains H37Rv and H37Ra, β-alanine failed to antagonize PZA and POA activities for strain mc²7000 (Fig. 3E and F). The essential role of panCD in β-alanine-mediated antagonism of PZA and POA activity indicates an essential role for pantothenate synthesis in this process.

Metabolites of the pantothenate/CoA biosynthetic pathway antagonize some PZA structural analogs.

To determine the specificity of pantothenate-linked antimycobacterial antagonism, other compounds bearing structural similarity to PZA were examined for activity against M. tuberculosis during supplementation with metabolites of the pantothenate and CoA biosynthetic pathway. The PZA structural analogs 5-Cl PZA, nicotinamide, and nPPA were all antagonized by panthothenate supplementation (Table 2). Nicotinamide activity was also weakly (2-fold) antagonized by β-alanine and carnosine (Table 2). The MIC₉₀s of the structurally related compounds isoniazid and nicotinic acid were not altered by any of the metabolites tested (Table 2), indicating that antagonism is not general for all antitubercular agents.

DISCUSSION

Factors that influence the activity of PZA against M. tuberculosis are important considerations for optimizing TB short-course therapy. In this study, we determined that the antimycobacterial activity of PZA is antagonized by the presence of exogenous pantothenate or pantetheine. While PZA activity was also antagonized by metabolites linked to the pantothenate/CoA biosynthetic pathway, the observation that pantoate β-alanine ligase is essential for antagonism by β-alanine indicates that this effect is mediated by pantothenate or a downstream metabolite (Fig. 1).

Other metabolites that are peripherally associated with the pantothenate/CoA biosynthetic pathway were also found to antagonize PZA activity. Carnosine, a dipeptide of β-alanine and histidine, was identified as a weak antagonist. While carnosine is not directly involved in pantothenate/CoA biosynthesis, it can serve as a source of β-alanine upon hydrolysis of the aminoacyl bond. In mammals, this bond is rapidly hydrolyzed in serum by the M20 zinc dipeptidase carnosinase (29). In M. tuberculosis, Rv2522c encodes a carnosinase ortholog (30% identity) whose corresponding activity has yet to be characterized (30). In addition, 3-aminopropanol, a structural analog of β-alanine lacking the carbonyl group, was also found to antagonize PZA activity. While 3-aminopropanol has no known role in mycobacterial metabolism, M. tuberculosis encodes multiple short-chain alcohol dehydrogenases and aldehyde dehydrogenases that might convert 3-aminopropanol to β-alanine.

The mechanistic basis for pantothenate-mediated antagonism of PZA action is currently unresolved. However, as POA activity is also abolished by exogenously supplied pantothenate, the antagonism is not simply due to a failure in the activation of PZA. Based on the previous description of panD missense mutations in PZA-resistant strains that lack mutations in pncA, it has been suggested that PZA might target aspartate decarboxylase (25). However, in the present study, we found that an M. tuberculosis panD deletion mutant was still susceptible to PZA when grown with a subantagonistic concentration of pantetheine, indicating that PanD is unlikely to be a principal target for PZA. Further, we have also found that pantothenate antagonizes the activity of 5-Cl PZA, a bona fide FAS-I inhibitor (16, 17, 31). There is currently no precedent for 5-Cl PZA-mediated inhibition of pantothenate synthesis. Thus, pantothenate-mediated antagonism occurs via a mechanism that is likely target independent. We anticipate that this antagonism of structurally conserved antitubercular compounds is related to the fact that the compounds share a common localization pathway. Additional studies are required to resolve the mechanistic basis for this phenomenon.

Mammals lack the biosynthetic machinery for de novo synthesis of the pantothenate and rely on dietary intake of this essential metabolic cofactor. There are limited data on tissue-specific concentrations of pantothenate, although with normal levels of consumption (4 to 7 mg per day), human serum pantothenate concentrations are typically on the order of 2 μM (27), the level at which we begin to see antagonism of the antimycobacterial action of PZA in our in vitro assays. Despite a lack of evidence for therapeutic efficacy, trials with high-dose pantothenate supplementation as treatment for conditions such as cystinosis, acne, and hyperlipidemia have been described (32–34). In one such study, subjects were given oral doses of 70 to 1,000 mg/kg of body weight per day, resulting in plasma pantothenate concentrations of greater than 250 μM (33). It is unknown how this concentration would relate to pantothenate levels within macrophage phagosomes or the lumen of granulomas. Further investigation is necessary to determine whether there is any association between pantothenate intake levels and PZA efficacy. However, based on our observations, we speculate that excessive pantothenate supplementation could have a deleterious impact on TB short-course therapy.