Prodrug Pyrazinamide Research Articles

Prussian blue (PB) modified gold nanoparticles as a SERS-based sensing platform for capturing and detection of pyrazinoic acid (POA)

Pyrazinoic acid (POA) is a metabolite of the anti-tuberculosis drug pyrazinamide (PZA), and its detection can be used to assess the resistance of Mycobacterium tuberculosis in cultures, as only sensitive strains of the bacteria can metabolize PZA into POA. Prussian blue is a well-known metal-organic framework compound widely used in various sensing platforms such as electrochemical, photochemical, and magnetic sensors. In this study, we present a novel sensing platform based on Prussian blue-modified gold nanoparticles (AuNPs) designed to enhance the affinity of POA towards the sensing surface and to capture POA molecules from aqueous solutions. This SERS-based method allows for the selective enrichment of POA, which can be detected in both pure aqueous solution and in the presence of its pro-drug PZA. The limit of detection (LOD) for POA was estimated to be 1.08 μM in pure aqueous solution and 0.18 mM in the presence of PZA. Furthermore, the precision of the SERS method was verified by the relative standard deviation (RSD) of 3.34–12.02% for three parallel samples using different matrices, i.e. aqueous solution, spiked river water and spiked simulated saliva. The recoveries of the samples ranged from 92.65 to 118.51%. These all demonstrate the potential application of the proposed detection scheme in medical research.

Insights from the molecular mechanism of pyrazinamide to mutated pyrazinamidase linked to the pncA gene in clinical isolates of Mycobacterium tuberculosis

This study aims to conduct a comprehensive molecular dynamics strategy to evaluate whether mutations found in pyrazinamide monoresistant (PZAMR) strains of Mycobacterium tuberculosis (MTB) can potentially reduce the effectiveness of pyrazinamide (PZA) for tuberculosis (TB) treatment. Five single point mutations of pyrazinamidase (PZAse), an enzyme which is responsible for the activation of prodrug PZA into pyrazinoic acid, found in MTB clinical isolates, namely His82Arg, Thr87Met, Ser66Pro, Ala171Val, and Pro62Leu, were analyzed by the dynamics simulations both in the apo state (unbound state) and in the PZA bound state. The results showed that the mutation of His82 to Arg, Thr87 to Met, and Ser66 to Pro in PZAse affects the coordination state of the Fe2+ ion, which is a cofactor required for enzyme activity. These mutations change the flexibility, stability, and fluctuation of His51, His57, and ASP49 amino acid residues around the Fe2+ ion, culminating in an unstable complex and dissociation of PZA from the PZAse binding site. However, mutations of Ala171 to Val and Pro62 to Leu were found to have no effect on the complex’s stability. Based on the results, PZAse mutations of His82Arg, Thr87Met, and Ser66Pro culminated in weak binding affinity for PZA and caused significant structural deformations that led to PZA resistance. Future structural and functional studies, as well as investigations into other aspects of drug resistance in PZAse, will require experimental clarification. Communicated by Ramaswamy H. Sarma

Role of Post Approval Clinical Trials for Drug

Anti-tuberculosis drugs, the main aim for improving current treatment should be to optimize the use of the two current drugs, rifampicin and the pro-drug pyrazinamide, which are responsible to a similar extent for the entire sterilizing activity of current therapy. The rifamycin activity could be improved by increasing the dose size of rifampicin or by daily dosing with long acting rifapentine. Increasing the dose size of pyrazinamide is limited by toxicity but an alternative approach is to use inhalation with pyrazinoic acid, as an adjunct to standard oral therapy. This would acidify pulmonary lesions, thus increasing the bactericidal activity of the orally administered pyrazinamide. Because pyrazinoic acid is the active moiety, it should also increase overall pyrazinamide activity and, because most resistance arises in the pncA gene that converts pyrazinamide to pyrazinoic acid, it should act on most pyrazinamide resistant strains. Inhalation technology allows delivery of drug to lesions rapidly and without first pass toxicity. The properties of drug containing microparticles and nanoparticles during inhalation and storage are reviewed. Spray-dried larger Trojan particles in which the smaller encapsulated particles can reside should be able to improve localisation within alveoli and avoid some storage problems.

Mycobacterium tuberculosis PanD Structure-Function Analysis and Identification of a Potent Pyrazinoic Acid-Derived Enzyme Inhibitor.

A common strategy employed in antibacterial drug discovery is the targeting of biosynthetic processes that are essential and specific for the pathogen. Specificity in particular avoids undesirable interactions with potential enzymatic counterparts in the human host, and it ensures on-target toxicity. Synthesis of pantothenate (Vitamine B5), which is a precursor of the acyl carrier coenzyme A, is an example of such a pathway. In Mycobacterium tuberculosis (Mtb), which is the causative agent of tuberculosis (TB), pantothenate is formed by pantothenate synthase, utilizing D-pantoate and β-Ala as substrates. β-Ala is mainly formed by the decarboxylation of l-aspartate, generated by the decarboxylase PanD, which is a homo-oliogomer in solution. Pyrazinoic acid (POA), which is the bioactive form of the TB prodrug pyrazinamide, binds and inhibits PanD activity weakly. Here, we generated a library of recombinant Mtb PanD mutants based on structural information and PZA/POA resistance mutants. Alterations in oligomer formation, enzyme activity, and/or POA binding were observed in respective mutants, providing insights into essential amino acids for Mtb PanD's proper structural assembly, decarboxylation activity and drug interaction. This information provided the platform for the design of novel POA analogues with modifications at position 3 of the pyrazine ring. Analogue 2, which incorporates a bulky naphthamido group at this position, displayed a 1000-fold increase in enzyme inhibition, compared to POA, along with moderately improved antimycobacterial activity. The data demonstrate that an improved understanding of mechanistic and enzymatic features of key metabolic enzymes can stimulate design of more-potent PanD inhibitors.

Pyrazinamide triggers degradation of its target aspartate decarboxylase

Pyrazinamide is a sterilizing first-line tuberculosis drug. Genetic, metabolomic and biophysical analyses previously demonstrated that pyrazinoic acid, the bioactive form of the prodrug pyrazinamide (PZA), interrupts biosynthesis of coenzyme A in Mycobacterium tuberculosis by binding to aspartate decarboxylase PanD. While most drugs act by inhibiting protein function upon target binding, we find here that pyrazinoic acid is only a weak enzyme inhibitor. We show that binding of pyrazinoic acid to PanD triggers degradation of the protein by the caseinolytic protease ClpC1-ClpP. Thus, the old tuberculosis drug pyrazinamide exerts antibacterial activity by acting as a target degrader, a mechanism of action that has recently emerged as a successful strategy in drug discovery across disease indications. Our findings provide the basis for the rational discovery of next generation PZA.

Molecular Specific and Sensitive Detection of Pyrazinamide and Its Metabolite Pyrazinoic Acid by Means of Surface Enhanced Raman Spectroscopy Employing In Situ Prepared Colloids

The prodrug pyrazinamide (PZA) is metabolized by the mycobacteria to pyrazinoic acid (POA), which is expelled into the extracellular environment. PZA resistance is highly associated to a lack of POA efflux. Thus, by detecting a reduction of the concentration of POA in the extracellular environment, by means of lab-on-a-chip (LoC)-SERS (surface-enhanced Raman spectroscopy), an alternative approach for the discrimination of PZA resistant mycobacteria is introduced. A droplet-based microfluidic SERS device has been employed to illustrate the potential of the LoC-SERS method for the discrimination of PZA resistant mycobacteria. The two analytes were detected discretely in aqueous solution with a limit of detection of 27 µm for PZA and 21 µm for POA. The simultaneous detection of PZA and POA in aqueous mixtures could be realized within a concentration range from 20 μm to 50 μm for PZA and from 50 μm to 80 μm for POA.

Solution structure and backbone dynamics for S1 domain of ribosomal protein S1 from Mycobacterium tuberculosis.

The pro-drug pyrazinamide is hydrolyzed to pyrazinoic acid (POA) in its use for the treatment of tuberculosis. As a molecule with bactericidal activity, POA binds to the C-terminal S1 domain of ribosomal protein S1 from Mycobacterium tuberculosis (MtRpsACTD_S1) to inhibit trans-translation. Trans-translation is a critical component of protein synthesis quality control, and is mediated by transfer-messenger RNA. Here, we have determined the solution structure of MtRpsACTD_S1(280-368), and analyzed its structural dynamics by NMR spectroscopy. The solution structure of MtRpsACTD_S1(280-368) mainly consists of five anti-parallel β strands, two α helices, and two 310 helices. Backbone dynamics reveals that the overall structure of MtRpsACTD_S1(280-368) is rigid, but segment L326-V333 undergoes large amplitude fluctuations on picosecond to nanosecond time scales. In addition, residues V321, H322, V331 and D335 with large Rex values exhibit significant chemical or conformational exchange on microsecond to millisecond time scale. Titration of the truncated MtRpsACTD_S1(280-368) with POA shows similar characteristics to titration of MtRpsACTD_S1(280-438) with POA. In addition, diverse length fragments of MtRpsACTD_S1 show various HN resonance signals, and we find that the interaction of MtRpsA(369-481) with MtRpsACTD_S1(280-368) [Kd = (4.25 ± 0.15) mM] is responsible for the structural difference between MtRpsACTD_S1(280-368) and MtRpsACTD_S1. This work may shed light on the underlying molecular mechanism of MtRpsACTD recognizing and binding POA or mRNA, as well as the detailed mechanism of interactions between MtRpsACTD_S1(280-368) and the additional C-terminal MtRpsA(369-481).

PYRAZINAMIDE/PYRAZINOIC ACID RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS: RECENT FINDINGS AND IMPLICATIONS FOR IMPROVING THE TREATMENT OF TUBERCULOSIS

Abstract. Pyrazinamide (PZA) is unique in that it is a component of the first line therapy for drug sensitive tuberculosis and in most current and experimental treatments also for multi drug resistant tuberculosis. Furthermore, PZA has been shown to help to ensure lasting cure and prevent relapse in shorter multi drug regimens. PZA is a prodrug. Mycobacterial tuberculosis (MTB) PncA enzyme activates the anti-mycobacterial prodrug PZA by transforming it into pyrazinoic acid (POA). The majority of clinical PZA resistant isolates contain mutations within the pncA gene and therefore remain sensitive to POA as they no longer activate PZA. Resistance to the active compound POA requires an alternative resistance mechanism and in vitro selected spontaneous MTB POA resistant mutants typically acquire a range of mutations in panD or mutations in one of a series of genes most of which are associated with the regulation of the bacterial stringent response. Clinically isolated PZA resistant MTB strains resistant to PZA and POA with mutations in any of these genes are unusual. Thus, it is likely the stringent response is critical for MTB in vivo and a damaged stringent response results in at least a reduction in fitness. Various lead compounds that disrupt the MTB stringent response have been identified that might form the basis for drugs with activity against latent mycobacteria with the potential to shorten tuberculosis treatment. Here we discuss the role of latency in the lifecycle of MTB and possible links to the activity PZA with a focus on potential new targets and drugs.

Insights into the Mechanisms of the Pyrazinamide Resistance of Three Pyrazinamidase Mutants N11K, P69T, and D126N.

In an effort to discover the mechanism of resistance offered by Mycobacterium tuberculosis (Mtb) toward the pyrazinamide (PZA) drug, an extensive molecular dynamics strategy was employed. PZA is a first-line prodrug that effectively cuts therapy time by 33% (from 9 to 6 months). Pyrazinamidase enzyme (PZase), encoded by the pncA gene, is responsible for the activation of prodrug PZA into pyrazinoic acid (POA). POA is toxic and potently inhibits the growth of latent Mtb even at low pH values. PZA resistance is caused by three genes pncA, rpsA, and panD. Among them, the pncA gene contributes 72-99% to the resistance. Hence, the present study focused on the novel mutations N11K, P69T, and D126N in the pncA gene. In the present study, the possible mechanism of these three mutations was studied through molecular dynamics simulation and docking techniques. Our in-depth analysis and results are in strong agreement with our experimental observation. The binding pocket analysis showed that mutations decrease the volume of the active site and hinder the correct orientation of PZA drug in the active site. Moreover, the Patchdock score was found to be low as compared to WT showing the disturbance of shape complementarity between PZase and PZA drug. These mutations were found to disturb the position of the Fe2+ ion. Among the mutations, D126N allosterically disturbed the position of the Fe2+ ion. MMGBSA analyses showed that these mutations decrease the binding affinity toward the PZA drug. In conclusion, mutations N11K, P69T, and D126N result in weak binding affinity with PZA and also cause significant structural deformations that lead to PZA resistance. This study provides useful information that mutations in other than active parts may also cause protein folding and ligand displacement effects, altering the biological functions.

Analysis of mutations in pncA reveals non-overlapping patterns among various lineages of Mycobacterium tuberculosis

Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug, resistance to which occurs primarily due to mutations in pncA (Rv2043c) that encodes the pyrazinamidase enzyme responsible for conversion of pro-drug PZA into its active form. Previous studies have reported numerous resistance-conferring mutations distributed across the entire length of pncA without any hotspot regions. As different lineages of Mycobacterium tuberculosis display a strong geographic association, we sought to understand whether the genetic background influenced the distribution of mutations in pncA. We analyzed the whole genome sequence data of 1,480 clinical isolates representing four major M. tuberculosis lineages to identify the distribution of mutations in the complete operon (Rv2044c-pncA-Rv2042c) and its upstream promoter region. We observed a non-overlapping pattern of mutations among various lineages and identified a lineage 3-specific frame-shift deletion in gene Rv2044c upstream of pncA that disrupted the stop codon and led to its fusion with pncA. This resulted in the addition of a novel domain of unknown function (DUF2784) to the pyrazinamidase enzyme. The variant molecule was computationally modelled and physico-chemical parameters determined to ascertain stability. Although the functional impact of this mutation remains unknown, its lineage specific nature highlights the importance of genetic background and warrants further study.

Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD.

Previously, we showed that a major in vitro and in vivo mechanism of resistance to pyrazinoic acid (POA), the bioactive component of the critical tuberculosis (TB) prodrug pyrazinamide (PZA), involves missense mutations in the aspartate decarboxylase PanD, an enzyme required for coenzyme A biosynthesis. What is the mechanism of action of POA? Upon demonstrating that treatment of M.bovis BCG with POA resulted in a depletion of intracellular coenzyme A and confirming that this POA-mediated depletion is prevented by either missense mutations in PanD or exogenous supplementation of pantothenate, we hypothesized that POA binds to PanD and that this binding blocks the biosynthetic pathway. Here, we confirm both hypotheses. First, metabolomic analyses showed that POA treatment resulted in a reduction of the concentrations of all coenzyme A precursors downstream of the PanD-mediated catalytic step. Second, using isothermal titration calorimetry, we established that POA, but not its prodrug PZA, binds to PanD. Binding was abolished for mutant PanD proteins. Taken together, these findings support a mechanism of action of POA in which the bioactive component of PZA inhibits coenzyme A biosynthesis via binding to aspartate decarboxylase PanD. Together with previous works, these results establish PanD as a genetically, metabolically, and biophysically validated target of PZA.

In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1

Through mutant selection on agar containing pyrazinoic acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined.

Missense Mutations in the Unfoldase ClpC1 of the Caseinolytic Protease Complex Are Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis.

ABSTRACTPreviously, we showed that mutations in Mycobacterium tuberculosis panD, involved in coenzyme A biosynthesis, cause resistance against pyrazinoic acid, the bioactive component of the prodrug pyrazinamide. To identify additional resistance mechanisms, we isolated mutants resistant against pyrazinoic acid and subjected panD wild-type strains to whole-genome sequencing. Eight of the nine resistant strains harbored missense mutations in the unfoldase ClpC1 associated with the caseinolytic protease complex.

Host-Mediated Bioactivation of Pyrazinamide: Implications for Efficacy, Resistance, and Therapeutic Alternatives.

Pyrazinamide has played a critical role in shortening therapy against drug-sensitive, drug-resistant, active, and latent tuberculosis (TB). Despite widespread recognition of its therapeutic importance, the sterilizing properties of this 60-year-old drug remain an enigma given its rather poor activity in vitro. Here we revisit longstanding paradigms and offer pharmacokinetic explanations for the apparent disconnect between in vitro activity and clinical impact. We show substantial host-mediated conversion of prodrug pyrazinamide (PZA) to the active form, pyrazinoic acid (POA), in TB patients and in animal models. We demonstrate favorable penetration of this pool of circulating POA from plasma into lung tissue and granulomas, where the pathogen resides. In standardized growth inhibition experiments, we show that POA exhibits superior in vitro potency compared to PZA, indicating that the vascular supply of host-derived POA may contribute to the in vivo efficacy of PZA, thereby reducing the apparent discrepancy between in vitro and in vivo activity. However, the results also raise the possibility that subinhibitory concentrations of POA generated by the host could fuel the emergence of resistance to both PZA and POA. In contrast to widespread expectations, we demonstrate good oral bioavailability and exposure in preclinical species in pharmacokinetic studies of oral POA. Baseline exposure of oral POA can be further increased by the xanthine oxidase inhibitor and approved gout drug allopurinol. These promising results pave the way for clinical investigations of oral POA as a therapeutic alternative or an add-on to overcome PZA resistance and salvage this essential TB drug.

Differential Cocrystallization Behavior of Isomeric Pyridine Carboxamides toward Antitubercular Drug Pyrazinoic Acid

Pyrazinoic acid, the active form of the antitubercular pro-drug Pyrazinamide, is an amphiprotic molecule containing carboxylic acid and pyridine groups and therefore can form both salts and cocrystals with relevant partner molecules. Cocrystallization of pyrazinoic acid with isomeric pyridine carboxamide series resulted in a dimorphic mixed-ionic complex with isonicotinamide and in eutectics with nicotinamide and picolinamide, respectively. It is observed that with alteration of the carboxamide position, steric and electrostatic compatibility issues between molecules of the combination emerge and affect intermolecular interactions and supramolecular growth, thus leading to either cocrystal or eutectic for different pyrazinoic acid–pyridine carboxamide combinations. Intermolecular interaction energy calculations have been performed to understand the role of underlying energetics on the formation of cocrystal/eutectic in different combinations. On the other hand, two molecular salts with piperazine and cyto...

Metal-ion effects on the polarization of metal-bound water and infrared vibrational modes of the coordinated metal center of Mycobacterium tuberculosis pyrazinamidase via quantum mechanical calculations.

Mycobacterium tuberculosis pyrazinamidase (PZAse) is a key enzyme to activate the pro-drug pyrazinamide (PZA). PZAse is a metalloenzyme that coordinates in vitro different divalent metal cofactors in the metal coordination site (MCS). Several metals including Co2+, Mn2+, and Zn2+ are able to reactivate the metal-depleted PZAse in vitro. We use quantum mechanical calculations to investigate the Zn2+, Fe2+, and Mn2+ metal cofactor effects on the local MCS structure, metal–ligand or metal–residue binding energy, and charge distribution. Results suggest that the major metal-dependent changes occur in the metal–ligand binding energy and charge distribution. Zn2+ shows the highest binding energy to the ligands (residues). In addition, Zn2+ and Mn2+ within the PZAse MCS highly polarize the O–H bond of coordinated water molecules in comparison with Fe2+. This suggests that the coordination of Zn2+ or Mn2+ to the PZAse protein facilitates the deprotonation of coordinated water to generate a nucleophile for catalysis as in carboxypeptidase A. Because metal ion binding is relevant to enzymatic reaction, identification of the metal binding event is important. The infrared vibrational mode shift of the C=Nε (His) bond from the M. tuberculosis MCS is the best IR probe to metal complexation.

Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.

Mycobacterium canettii is intrinsically resistant to both pyrazinamide and pyrazinoic acid

Sir, Pyrazinamide is part of the standard short-course regimen that enables the treatment of drug-susceptible tuberculosis (TB) within 6 months (in combination with isoniazid, rifampicin and ethambutol). It is the only first-line TB drug that is being considered as part of future regimens, which have just completed Phase 2A trials [pyrazinamide in combination with bedaquiline (also known as Sirturo, TMC207 or R207910), PA-824 or PA-824/moxifloxacin]. These are designed to shorten the treatment duration for drug-susceptible TB, as well as multidrugresistant and extensively drug-resistant TB, which is paramount to reduce both the further spread of TB and the significant direct and opportunity costs to patients. The ability of these regimens to treat TB effectively might be compromised when applied to members of the Mycobacterium tuberculosis complex (MTBC) that are intrinsically resistant to one or more of the aforementioned drugs. In this context, Mycobacterium canettii is of particular importance, given that we have recently raised the possibility that this taxon might be intrinsically resistant to PA-824. In addition, it is intrinsically resistant to pyrazinamide despite lacking non-synonymous mutations in pncA (Rv2043c), encoding the pyrazinamidase that converts the prodrug pyrazinamide into its active form pyrazinoic acid. – 6 Recently, it has been shown that pyrazinoic acid inhibits the ribosomal protein S1 RpsA (Rv1630), thereby affecting transtranslation. When interrogating the recently published genome of the first M. canettii strain isolated (CIPT 140010059), we found two non-synonymous mutations in rpsA (T5A and T210A) in addition to two synonymous changes (P9P and E457E), which might constitute the basis for the intrinsic pyrazinamide resistance, particularly given that the Thr at codon 5 was previously found to be mutated in a pyrazinamide-resistant strain without pncA mutations. To test this hypothesis, we sequenced rpsA and pncA in a collection of 71 pyrazinamide-susceptible and -resistant isolates that encompassed representatives of the global diversity of the MTBC [M. tuberculosis genotypes Beijing, Cameroon, Delhi/ Central Asian, East African Indian, Ghana, Haarlem, Latin-American-Mediterranean, New-1, S-type, Turkey, Uganda I and II, Ural and X-type; Mycobacterium africanum West African I (a and b) and West African II; Mycobacterium bovis; M. canettii; Mycobacterium caprae; Mycobacterium microti; and Mycobacterium pinnipedii (see the Methods section and Table S1, available as Supplementary data at JAC Online)]. The three M. canettii strains from our reference collection shared the aforementioned mutations with CIPT 140010059 and displayed pyrazinoic acid MICs of 200 mg/L compared with 50 mg/L for the pyrazinamidesusceptible H37Rv reference strain (Table 1), suggesting that the mutations in rpsA or in an as yet unknown target of pyrazinamide/pyrazinoic acid were responsible for its intrinsic resistance. In contrast, we found that the four M. bovis isolates, which were intrinsically resistant to pyrazinamide, had pyrazinoic acid MICs that were lower or equal to the MIC for the H37Rv reference strain. This is in line with prior results that found that BCG is fully susceptible to pyrazinoic acid and becomes susceptible to pyrazinamide upon complementation of the mutated pncA (H57D) with a wild-type copy, thereby restoring the ability to convert pyrazinamide into pyrazinoic acid. Interestingly, the M. bovis strains harboured a non-synonymous rpsA mutation (A440T) that was shared with both M. caprae isolates in this study, as well as previously sequenced M. bovis isolates (AF2122/97 and 06-01518) and all BCG strains sequenced to date (China, Denmark 1331, Mexico 1931, Moreau RDJ, Pasteur 1173P2, Russia, Tice and Tokyo 172). However, none of the M. africanum, M. microti or M. pinnipedii isolates in this study had this particular mutation, suggesting it was a phylogenetically informative marker for the M. caprae/M. bovis/BCG group of animal-adapted strains, rather than a marker for pyrazinamide resistance. We found a further non-synonymous mutation in rpsA (D37A) that did not correlate with pyrazinamide resistance in one M. tuberculosis Cameroon strain. Finally, we identified the known synonymous R212R mutation in rpsA, which had been proposed as a marker for lineage 2 (East Asian) strains, whereas a second study had concluded that this was not a lineage-specific mutation. The fact that only the six Beijing isolates in our collection harboured this mutation supported the conclusion of the former study. In the Republic of Djibouti, M. canettii is responsible for 11% of active TB cases, which are clinically and radiographically indistinguishable from TB caused by other members of the MTBC. However, more data regarding its prevalence and mode of transmission are required. Our observation that M. canettii was resistant to pyrazinoic acid and harboured rpsA mutations suggested that altered trans-translation might be the mechanistic basis for the intrinsic pyrazinamide resistance of this pathogen.

Biochemical and Mutational Analysis of a Novel Nicotinamidase from Oceanobacillus iheyensis HTE831

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD+ salvage pathway. This paper reports a new nicotinamidase from the deep-sea extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831 (OiNIC). The enzyme was active towards nicotinamide and several analogues, including the prodrug pyrazinamide. The enzyme was more nicotinamidase (kcat/Km = 43.5 mM−1s−1) than pyrazinamidase (kcat/Km = 3.2 mM−1s−1). Mutational analysis was carried out on seven critical amino acids, confirming for the first time the importance of Cys133 and Phe68 residues for increasing pyrazinamidase activity 2.9- and 2.5-fold, respectively. In addition, the change in the fourth residue involved in the ion metal binding (Glu65) was detrimental to pyrazinamidase activity, decreasing it 6-fold. This residue was also involved in a new distinct structural motif DAHXXXDXXHPE described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales.

Mutations in panD encoding aspartate decarboxylase are associated with pyrazinamide resistance in Mycobacterium tuberculosis

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug susceptible and multidrug-resistant tuberculosis (MDR-TB). Resistance to PZA is most commonly associated with mutations in the pncA gene encoding nicotinamidase/pyrazinamidase which converts the prodrug PZA to the active form pyrazinoic acid (POA). RpsA (ribosomal protein S1) involved in trans-translation was recently shown to be a target of PZA and mutations in RpsA are found in some PZA-resistant TB strains. However, some other PZA-resistant strains lack mutations in either pncA or rpsA. To identify potential new mechanisms of PZA resistance, we isolated 174 in vitro mutants of M. tuberculosis H37Rv resistant to PZA to search for resistant isolates that do not have pncA or rpsA mutations. DNA sequencing revealed that 169 of the 174 (97.1%) PZA-resistant mutants had pncA mutations but 5 mutants lacked pncA or rpsA mutations. Whole genome sequencing analyses revealed that the 5 PZA-resistant mutants had different mutations all occurring in the same gene panD encoding aspartate decarboxylase, which is involved in synthesis of β-alanine that is a precursor for pantothenate and co-enzyme A biosynthesis. panD mutations were identified in naturally PZA-resistant Mycobacterium canetti strain and a PZA-resistant MDR-TB clinical isolate. Future studies are needed to address the role of panD mutations in PZA resistance and confirm PanD as a new target of PZA.Emerging Microbes & Infections (2013) 2, e35; doi:10.1038/emi.2013.38