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Drug Resistance in Mycobacterium tuberculosisMechanisms of Drug Resistance in TB: Molecular Mechanisms Challenging Fluoroquinolones and Pyrazinamide Effectiveness FREE TO VIEW

Paolo Miotto, PhD; Daniela M. Cirillo, MD, PhD; Giovanni Battista Migliori, MD
Author and Funding Information

From the Emerging Bacterial Pathogens Unit (Drs Miotto and Cirillo), Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan; and WHO Collaborating Centre for TB and Lung Diseases (Dr Migliori), Fondazione S. Maugeri, Care and Research Institute, Tradate, Italy.

CORRESPONDENCE TO: Giovanni Battista Migliori, MD, WHO Collaborating Centre for Tuberculosis and Lung Diseases, Fondazione Salvatore Maugeri, Care and Research Institute, via Roncaccio 16, 21049, Tradate (VA), Italy; e-mail: giovannibattista.migliori@fsm.it


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Chest. 2015;147(4):1135-1143. doi:10.1378/chest.14-1286
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Physicians are more and more often challenged by difficult-to-treat cases of TB. They include patients infected by strains of Mycobacterium tuberculosis that are resistant to at least isoniazid and rifampicin (multidrug-resistant TB) or to at least one fluoroquinolone (FQ) and one injectable, second-line anti-TB drug in addition to isoniazid and rifampicin (extensively drug-resistant TB). The drug treatment of these cases is very long, toxic, and expensive, and, unfortunately, the proportion of unsatisfactory outcomes is still considerably high. Although FQs and pyrazinamide (PZA) are backbone drugs in the available anti-TB regimens, several uncertainties remain about their mechanisms of action and even more remain about the mechanisms leading to drug resistance. From a clinical point of view, a better understanding of the genetic basis of drug resistance will aid (1) clinicians to provide quality clinical management to both drug-susceptible and drug-resistant TB cases (while preventing emergence of further resistance), and (2) developers of new molecular-based diagnostic assays to better direct their research efforts toward a new generation of sensitive, specific, cheap, and easy-to-use point-of-care diagnostics. In this review we provide an update on the molecular mechanisms leading to FQ- and PZA-resistance in M tuberculosis.

Figures in this Article

TB, with > 8.6 million cases and 1.3 million deaths in 2012, is a first-class health priority.1 The emergence of drug resistance is hampering the efforts of the international community to control and eliminate TB.24 Physicians are more and more often challenged by difficult-to-treat cases of multidrug-resistant TB (ie, active TB cases infected by Mycobacterium tuberculosis [MTB] strains that are resistant to isoniazid and rifampicin) and extensively drug-resistant TB (ie, TB caused by strains resistant to at least one fluoroquinolone [FQ] and one injectable, second-line anti-TB drug in addition to isoniazid and rifampicin). Patients with drug-resistant or extensively drug-resistant TB need long, toxic, and expensive drug treatment, and the proportion of unsatisfactory outcomes is still high.2,5,6 The development of novel effective regimens to shorten treatment involves new and/or repurposed drugs. Several of these new regimens include FQs and pyrazinamide (PZA) as backbone drugs.7 Despite this renewed interest in the two groups of antibiotics, several uncertainties remain about their mechanisms of action and the mechanisms leading to drug resistance.7

In this review, we provide an update on the molecular mechanisms leading to FQ resistance (FQ-R) and PZA resistance (PZA-R) in MTB. A deeper knowledge of the genetic basis of drug resistance will support developing new molecular assays to rapidly detect and correctly treat drug-resistant cases.

Poor treatment compliance was historically considered as the driving factor in the emergence of drug resistance.8 However, the importance of pharmacokinetics and pharmacodynamics in producing this multifaceted phenomenon has been emphasized.9 Indeed, beyond the paradigm “adequate blood concentrations provide adequate penetration in affected tissues,”10 the complexity of TB pathology plays a critical role: Structurally different compartments within the lesions (and the spatial distribution of each single drug) determine different antibiotic exposure during treatment and contribute in selecting resistant mutants.11,12 Drug resistance in MTB is solely mediated by chromosomal mutations.13 There is evidence that low levels of antibiotics increase mutagenesis in a wider range of antibacterial resistance genes and drug efflux systems. Low bacterial-drug exposure leads to the induction of efflux pumps and to the appearance of reversible phenotypic resistance in a few days.14 In a few weeks, the resistant phenotype is fixed by the development of chromosomal mutations. The molecular bases of this phenomenon are not completely clear.

The development of mutations conferring drug resistance to MTB is likely due to multiple factors: Reactive oxygen species-induced mutagenesis, together with both the advantage provided under selective pressure due to the anti-TB treatment and the general physiologic advantages provided by such mutations (as supported by the emergence of resistance-conferring mutations even in absence of antibiotic exposure), drive their fixation in the infecting bacterial population.1519 Here, we analyze the mechanisms of drug resistance developed by MTB to specific drugs.

Molecular Mechanisms of Drug Resistance in MTB

We focused our attention on mutations already reported in the literature to provide a higher degree of confidence in the relationship between chromosomal mutations and phenotypic drug resistance.

Fluoroquinolones:

FQs belong to the quinolone class of antibiotics inhibiting bacterial DNA gyrase and topoisomerase IV,20 enzymes required for vital processes such as replication, transcription, recombination, and chromosomal supercoiling (Fig 1).2124 The main cause of FQ-R is the disruption of the binding site for the drug in the DNA-gyrase enzyme (encoded by the gyrA and gyrB genes).25 A completely different mechanism proposed to explain FQ-R involves pentapeptide proteins.26 The mycobacterial pentapeptide protein MfpA would protect DNA gyrase from FQ activity27: Overexpression and binding of MfpA to the GyrA subunit of DNA gyrase would inhibit FQ DNA binding, blocking the formation of the GyrA-FQ complex. However, in contrast to the observations in Escherichia coli, where pentapeptide proteins protect the DNA gyrase from FQ, MfpA in MTB was found to enhance the inhibitory activity of FQs on the topoisomerase.28

Figure Jump LinkFigure 1 –  Mechanisms of action of FQs. Mycobacterium tuberculosis lacks topoisomerase IV homologs, and DNA gyrase appears to be the sole target for FQs. DNA gyrase is an adenosine triphosphate-dependent enzyme that cleaves and reseals double-stranded DNA, thereby introducing negative supercoils into DNA. The enzyme consists of two subunits, GyrA and GyrB, which form an α2β2 heterotetramer complex in the active enzyme. The α-subunit is the site at which FQs bind to form the tyrosyl-DNA-gyrase complex. FQs distribute in the cellular region of the granulomas; three different general mechanisms of complex assembly have been proposed: (1) FQ binds to the enzyme following the interaction between gyrase and DNA; (2) FQ binds to the gyrase following the DNA cleavage; and (3) FQ binds to the gyrase and the complex interacts and cleaves the DNA. When FQs bind to the gyrase, the enzyme is not further able to rejoin cleaved strands, resulting in the formation of double-stranded DNA breaks and cell death in either a protein synthesis-dependent or protein synthesis-independent manner.2224 The bacterial cell wall is shown in yellow. Red stars = FQ; DNA-Gyr complex = covalent interaction of the DNA gyrase to the DNA; DNA-Gyr interaction = noncovalent binding of the DNA gyrase to the DNA; FQ = fluoroquinolone; Gyr = DNA gyrase (blue symbols); SOS = DNA stress-response pathway involving LexA and RecA that activates the expression of SOS response genes, including DNA repair enzymes.Grahic Jump Location

Tao and colleagues29 first described the role of a small GTPase in the emergence of drug resistance in mycobacteria. Indeed, the small GTPase named MfpB contributes to resistance against FQs via its involvement with MfpA in the protection of DNA gyrase in M smegmatis. However, the contribution of the MfpA/MfpB proteins to FQ-R in clinical isolates of MTB has not yet been evaluated.

While several mycobacterial efflux pumps have already been associated with reduced susceptibility to FQ, the drug-uptake mechanism (transporters) also could play an important role in FQ-R. Indeed, during dormancy, the cell-wall permeability is decreased and antibiotic uptake is significantly decreased.30 In addition to the diffusion through the lipid-rich membrane, porin-mediated FQ uptake has been demonstrated in MTB.30 It has been hypothesized that this alternative pathway could be inhibited by endogenous polyamines in macrophages, thus causing a reduction in the intracellular FQ accumulation and decreased antimycobacterial activity. A decreased outer membrane permeability together with cell-wall thickening in nonreplicating MTB can contribute to the phenotypic drug resistance to FQ in dormant bacilli, since polyamines are involved in several physiologic and pathologic processes, including inflammation, oxidative stress, and host-pathogen interactions.31,32

FQ-R in MTB is mainly due to the acquisition of point mutations within the quinolone resistance-determining region (QRDR) of the gyrA gene (codons 74 to 113), accounting for nearly 90% of FQ resistance in MTB.33 According to a meta-analysis, codons 90, 91, and 94 are the most mutated sites, accounting for > 50% of resistant cases.33,34 The S95T mutation is the only mutation occurring in the QRDR of gyrA that is not associated with FQ-R in MTB.33,34

In general, mutations in the QRDR of gyrA are responsible for high-level phenotypic cross-resistance to FQs in clinical isolates.34 However, FQ-R due to mutations outside the QRDR of gyrA and/or harboring in gyrB are reported in several studies.33,35 At least four mutations (G247S, A384V, T80A, A90G) in FQ-susceptible and FQ-resistant isolates were reported to be polymorphisms and do not play any role in FQ-R.35,36 With few exceptions, mutations outside the gyrA QRDR do not lead to FQ-R. More than 15 mutations have been identified in the gyrB gene alone, and approximately 50% of mutations have been identified outside the gyrB QRDR. Among them, D533A, D500A, and the double mutation N538T-T546M were the only mutations not exhibiting any significant increase in the minimum inhibitory concentration (MIC) level for the four FQs tested (ciprofloxacin, ofloxacin, levofloxacin, and moxifloxacin). Interestingly, different substitutions confer different levels or patterns of resistance. Cross-resistance to all FQs seems to be associated only with N538D and E540V. D500H and D500N mutations confer resistance to ofloxacin and levofloxacin; N538K and E540D substitutions confer resistance to moxifloxacin only. The mutations located outside the QRDR either had no effect or only slightly increased the MIC levels for the FQs tested.35

Novel findings are improving our understanding of the relationships between DNA-gyrase mutations and drug resistance. The D87G substitution in the gyrA gene causing FQ-R reduces negative supercoiling and results in large-scale changes to gene expression, altering broad antimicrobial susceptibility, as well as fitness and evolutionary adaptability.16 Among those identified as being responsive to gyrA mutation are genes involved in a global response to stress (rpoE, rpoS, and recA), showing that the gyrA mutation itself influenced susceptibility to antibiotics other than quinolones, without further mutations. Further studies may elucidate the link between mutations conferring drug resistance, stress response, and bacterial fitness.

Pyrazinamide:

PZA has remarkable sterilizing activity on the so-called persistent bacterial population.37 Although the antimycobacterial activity of PZA has been known for 7 decades, its mechanism of action is not yet fully understood (Fig 23840). The main mechanism of PZA-R in mycobacteria relies on the inactivation of the nicotinamidase/pyrazinamidase enzyme (PncA, encoded by the pncA gene), physiologically involved in the metabolism of nicotinamide and responsible of the conversion of PZA to its active form (pyrazinoic acid).38,41 In general, the frequency of mutations at pncA in PZA-resistant isolates ranges between 72% and 98%.37 Mutations affecting the pncA gene are mainly missense, single nucleotide changes causing amino acid substitutions. However, nonsense mutations, insertions, and deletions are not rare. Few substitutions occur also in the promoter region of the gene. The variety of the mutations occurring in the pncA gene of PZA-resistant isolates is demonstrated by > 400 different single nucleotide polymorphisms (SNPs) described in 78 published studies. Further complexity is added by insertions and deletions, and by the fact that new studies, such as that by Napiórkowska and colleagues,42 are describing additional mutations. Although these mutations are scattered across entire gene (encoding a protein of 187 amino acids), some degree of clustering seems to occur in three regions of the PncA protein: positions 3-17, 61-85, and 132-142.37 Indeed, loss of pyrazinamidase activity is associated with mutations affecting the active site (eg, D8, W68), disrupting the protein core (eg, Q10, I6, V44, V139, M175, F94, H137) and directly or indirectly interfering with the coordination of the iron (II) ion (eg, D49, H51, H57, H71).43,44 The iron (II) ion is required for the correct binding and positioning of PZA at the active site of the PncA enzyme, and depletion of metal ions abrogates pyrazinamidase activity.43,44 It should be noted that some mycobacterial species, including M bovis and M canettii, are intrinsically resistant to PZA.45 Enzymatic activity seems unaffected by mutations at specific codons, including 102, 171, and 12.4648 Recently, mutations unrelated to drug resistance have been identified and proposed as phylogenetic markers49; thus, a better correlation between SNPs and drug-resistant phenotype is needed.

Figure Jump LinkFigure 2 –  Mechanisms of action of PZA. Despite the importance of PZA in the treatment of TB, its mechanism of action is probably the least understood of all anti-TB drugs. The prodrug PZA has been reported to accumulate mainly in the caseum of granulomas and it is active only at acidic pH, after its conversion to POA. The main mechanism of mycobacterial killing by PZA was demonstrated to be the depletion of cellular adenosine triphosphate reserves caused by the inhibitory role of the pyrazinoic acid on the proton motive force interfering with adenosine triphosphate synthesis.38 The process can be summarized as follows: (A) Membrane transport systems (passive or facilitated diffusion seem to play a role) are involved in transporting the prodrug into the TB bacillus; (B) the PZase activity of PncA transforms the prodrug into the active compound POA; (C) in acid conditions, POA is converted to HPOA; (D) HPOA enters and kills the TB bacterial cell by reducing membrane potential and affecting membrane transport (the membrane in yellow in the figure); (E) trans-translation inhibition by directly targeting the rpsA gene (30S ribosomal protein S1) was demonstrated in 2011.39 Other factors affecting/influencing the activity of PZA are, in addition, acid pH, culture age, starvation and microaerophilic/anaerobic conditions. The main mechanisms of resistance are the following: (1) Interspersed mutations in the pncA gene (encoding the PZase enzyme; see phase B in the figure) represent the most frequently involved mechanism (72%-98%); (2) failure of PZA uptake by resistant strains (block of phase A in the figure); (3) mutations in the direct target rpsA (block of phase E in the figure)39; (4) mutations in panD encoding aspartate decarboxylase are associated with PZA resistance (the mechanism is still unknown).40 HPOA = protonated pyrazinoic acid; POA = pyrazinoic acid; PZA = pyrazinamide. (Figure adapted from Zhang and Mitchison.37)Grahic Jump Location

In 2011, Shi and colleagues39 showed how pyrazinoic acid interferes with trans-translation by inhibiting the 30S ribosomal protein S1. trans-Translation is dispensable during active growth, but becomes important for bacteria in managing stalled ribosomes or damaged mRNA and proteins under stress conditions.50 Few studies considered rpsA mutations in clinical isolates and further analyses are needed to better understand their role in PZA-R.51,52 Zhang and colleagues40 provided further insight into the PZA mechanism of action by showing novel mutations in the panD gene. panD encodes aspartate α-decarboxylase, an enzyme involved in synthesis of β-alanine, which, in turn, is required for pantothenate and coenzyme A synthesis.53 The possibility that PZA may inhibit pantothenate and coenzyme A synthesis (thereby interfering with diverse metabolic functions, such as energy production and fatty acid metabolism) needs to be addressed in future studies, together with the report on the frequency of these mutations in clinical isolates.40

Molecular Diagnostic Tests

Molecular diagnostics for fast detection of drug resistance in MTB gained increasing attention in the last 20 years. Although molecular drug-susceptibility testing (DST) proved to be cost effective for specific applications for some first-line drugs, for second-line drugs, the gaps in understanding the molecular bases of resistance are restricting the development of effective tools.5359

For FQs, several line probe-based assays and microarray-based assays have been developed,6064 but only few of them received the in vitro diagnostics certification mark. In 2013, a World Health Organization group evaluated the line probe assay developed for detecting second-line drug resistance in MTB.65 Overall, the expert group concluded that the GenoType MTBDRsl assay (Hain Lifescience GmbH) shows moderate test sensitivity for the detection of FQ-R (69.1%-99.2%), with high test specificity (94.8%-99.2%). However, based on the Grading of Recommendations Assessment, Development, and Evaluation system, the final recommendation was “very low quality of evidence,” and phenotypic DST should remain the reference standard for extensively drug-resistant TB.66 The molecular assay can be used as a rule-in test for resistant cases, but cannot replace phenotypic testing.

The situation is even more complex for PZA and, to the best of our knowledge, no US Food and Drug Administration-approved assay or molecular assay with the in vitro diagnostics certification mark exists for the rapid detection of PZA-R in MTB. Several research groups are trying to develop a molecular assay for rapid detection of PZA-R in MTB, focusing on line probe-based and microarray-based technologies, or single-stranded conformation polymorphism and denaturing assays.6772 One of the few multicenter studies available on this topic showed a sensitivity of 89.7% and a specificity of 96.0% for line probe assays for the detection of PZA-R.63 According to a systematic review published in 2011,73 sensitivity and specificity for molecular assays could be in the range of 96% to 98% and 96% to 97%, respectively. However, the number of studies considering molecular approaches other than gene sequencing was limited, as was the number (few hundreds) of clinical isolates evaluated. Thus, due to the multiplicity of mutations found in the pncA gene, a more comprehensive analysis of circulating PZA-R is needed to better understand the potential usefulness of these molecular assays in clinics. Nevertheless, the correlation between mutations and phenotypic resistance is often uncertain due to the poor performances reported for the conventional DST on PZA.74 The presence of several phylogenetic SNPs45,75 makes the development of reliable molecular assays even more complex.

Among the limitations of current tools for molecular DST, the lack of knowledge of all the genomic regions involved in the emergence of phenotypic resistance and the limited number of targets assayed severely affect the diagnostic performances and the clinical usefulness of such tests. Large sets of bacterial genome data have been made available by next-generation sequencing technology. As in the past, sequencing of bacterial genomes probably will provide significant improvements of our understanding about the molecular basis of drug resistance in MTB. Nevertheless, the continuously evolving next-generation sequencing technology will provide novel tools for diagnostic purposes, as demonstrated by an increasing number of studies.7678

Drug Resistance, In Vitro MIC, and Clinical Outcome

The use of molecular assays led to an increasing number of studies highlighting discrepancies between conventional and genotypic DST for different drugs, including (but not limited to) first-line antibiotics.7984 Three core elements are important to understand the individual mutation’s contribution to the drug-resistance phenotype in MTB: (1) different mutations of the same genomic region can lead to different MIC values, (2) same mutations can cause variable levels of resistance to different members of the same drug class, and (3) same specific mutations can lead to different MIC levels in different strains, suggesting a role for the genetic background.85 This multiplicity of aspects can be exemplified by analyzing the relationship between mutations in the rpoB gene and rifampicin resistance. It has been demonstrated that some mutations in the rpoB gene lead to different levels of resistance to rifampicin and that some mutations require a specific genetic background for driving resistance development.86 In addition, some rpoB mutations were reported to correlate with different MIC values and susceptibility results among different testing methods.80,8789 Despite the obvious relevance of PZA and FQs in therapeutic regimens, fewer data are available. Different mutations affecting DNA gyrase cause distinct levels of resistance and cross-resistance among FQs.84,90 D94G substitution in GyrA is associated with high-level resistance, while mutations affecting codon number 91 in GyrA (or substitutions A543V, T539N, and E540A in GyrB) are associated to low-level resistance.84 Similarly, H70R, D94A, and D94N were associated with low-level resistance to levofloxacin,91 and mutations in the pncA gene are associated with different MIC levels for PZA.92

The clinical implication of these findings is highly related to the currently accepted concept of critical concentration cutoffs in determining clinical resistance. Most laboratories perform DST for MTB only at the “critical concentration” recommended for their specific testing method. Strains harboring mutations leading to a higher MIC level than wild-type strains but equal to or slightly lower than the critical concentration would be susceptible. However, molecular assays could rapidly identify the mutations conferring borderline resistance. In some cases, this information could be used in adapting the treatment regimen. For example, a significant association was found between the presence of such rpoB gene mutations and treatment failure.88,93 Cross-resistance among different members of the same drug class driven by some mutations provides further evidence.94101 Jo and colleagues102 showed the clinical impact of the use of later-generation FQs to treat ofloxacin-resistant cases. Unfortunately, gyrase-sequencing data were not available from the study, making it impossible to correlate the mutation to the treatment outcome.

Data on the differences in resistance levels support the implementation of quantitative DST for detecting specific resistance-conferring mutations in MTB. Patients infected with strains exhibiting low-level resistance could benefit from increased dosages. For this reason, better correlation between specific mutations and MIC levels, together with association between genetic variants and cross-resistance are needed. Furthermore, there is clear evidence that genetic diversity in MTB impacts the pathogenic features of isolates, but the link between epistatic interactions in different drug-resistance-conferring mutations, the strain genetic background, and compensatory mutations need further elucidation.98,103 This evidence strongly supports the development of more appropriate molecular tests that take into account the complexity of the genetic basis of drug resistance in MTB, to achieve evidence-based impact in the clinical practice. Small regulatory RNAs encoded by the intergenic chromosomal regions of MTB have been described.104107 Whole-genome sequencing data strongly support a role for these regions in the development of drug resistance in MTB. Indeed, Zhang and colleagues107 reported SNPs in intergenic regions consistently associated with drug resistance. From a clinical point of view, these observations have two main implications: Epidemiologic and phylogenetic data could alert the clinicians in terms of risk for developing drug resistance, and on the other side, developers of new molecular-based diagnostic assays cannot ignore the relevance of the genetic background when considering the molecular drug-resistance mechanisms in MTB.

Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Other contributions: Rosella Centis, MSc, and Lia D’Ambrosio, MA, provided editorial and technical support.

DST

drug-susceptibility testing

FQ

fluoroquinolone

FQ-R

fluoroquinolone resistance

MIC

minimum inhibitory concentration

MTB

Mycobacterium tuberculosis

PZA

pyrazinamide

PZA-R

pyrazinamide resistance

QRDR

quinolone resistance-determining region

SNP

single nucleotide polymorphism

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Figures

Figure Jump LinkFigure 1 –  Mechanisms of action of FQs. Mycobacterium tuberculosis lacks topoisomerase IV homologs, and DNA gyrase appears to be the sole target for FQs. DNA gyrase is an adenosine triphosphate-dependent enzyme that cleaves and reseals double-stranded DNA, thereby introducing negative supercoils into DNA. The enzyme consists of two subunits, GyrA and GyrB, which form an α2β2 heterotetramer complex in the active enzyme. The α-subunit is the site at which FQs bind to form the tyrosyl-DNA-gyrase complex. FQs distribute in the cellular region of the granulomas; three different general mechanisms of complex assembly have been proposed: (1) FQ binds to the enzyme following the interaction between gyrase and DNA; (2) FQ binds to the gyrase following the DNA cleavage; and (3) FQ binds to the gyrase and the complex interacts and cleaves the DNA. When FQs bind to the gyrase, the enzyme is not further able to rejoin cleaved strands, resulting in the formation of double-stranded DNA breaks and cell death in either a protein synthesis-dependent or protein synthesis-independent manner.2224 The bacterial cell wall is shown in yellow. Red stars = FQ; DNA-Gyr complex = covalent interaction of the DNA gyrase to the DNA; DNA-Gyr interaction = noncovalent binding of the DNA gyrase to the DNA; FQ = fluoroquinolone; Gyr = DNA gyrase (blue symbols); SOS = DNA stress-response pathway involving LexA and RecA that activates the expression of SOS response genes, including DNA repair enzymes.Grahic Jump Location
Figure Jump LinkFigure 2 –  Mechanisms of action of PZA. Despite the importance of PZA in the treatment of TB, its mechanism of action is probably the least understood of all anti-TB drugs. The prodrug PZA has been reported to accumulate mainly in the caseum of granulomas and it is active only at acidic pH, after its conversion to POA. The main mechanism of mycobacterial killing by PZA was demonstrated to be the depletion of cellular adenosine triphosphate reserves caused by the inhibitory role of the pyrazinoic acid on the proton motive force interfering with adenosine triphosphate synthesis.38 The process can be summarized as follows: (A) Membrane transport systems (passive or facilitated diffusion seem to play a role) are involved in transporting the prodrug into the TB bacillus; (B) the PZase activity of PncA transforms the prodrug into the active compound POA; (C) in acid conditions, POA is converted to HPOA; (D) HPOA enters and kills the TB bacterial cell by reducing membrane potential and affecting membrane transport (the membrane in yellow in the figure); (E) trans-translation inhibition by directly targeting the rpsA gene (30S ribosomal protein S1) was demonstrated in 2011.39 Other factors affecting/influencing the activity of PZA are, in addition, acid pH, culture age, starvation and microaerophilic/anaerobic conditions. The main mechanisms of resistance are the following: (1) Interspersed mutations in the pncA gene (encoding the PZase enzyme; see phase B in the figure) represent the most frequently involved mechanism (72%-98%); (2) failure of PZA uptake by resistant strains (block of phase A in the figure); (3) mutations in the direct target rpsA (block of phase E in the figure)39; (4) mutations in panD encoding aspartate decarboxylase are associated with PZA resistance (the mechanism is still unknown).40 HPOA = protonated pyrazinoic acid; POA = pyrazinoic acid; PZA = pyrazinamide. (Figure adapted from Zhang and Mitchison.37)Grahic Jump Location

Tables

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