Abstract
p-Aminosalicylic acid (PAS) is an important compound for treating multidrug-resistant tuberculosis (TB). Previous studies showed that thyA mutations are often related to PAS resistance in clinical isolates. We performed a systematic analysis of isolate genotypes and detected mutations in three folate pathway genes (folC, thyA, and ribD) in 61.1% (127/208) of PAS-resistant isolates, including 11 double mutants. This result expands our knowledge about the distribution and frequency of mutations related to PAS resistance in mycobacterial clinical isolates.
TEXT
Tuberculosis (TB) is a major global health problem that affected 8.6 million new patients and caused 1.3 million deaths worldwide in 2012 (1). The prevalence of multidrug-resistant TB resistant to isoniazid and rifampin poses a great challenge to TB control and necessitates more complicated and longer treatment regimens. The compound p-aminosalicylic acid (PAS), one of the first effective antibiotics for treating TB, remains a primary component of many multidrug-resistant TB therapies (2, 3).
PAS is a prodrug that targets the folate pathway of Mycobacterium tuberculosis (4). Mutations in the dihydrofolate synthase FolC, which is required for the bioactivation of PAS, confer PAS resistance (5). In addition, mutations causing an overexpression of RibD, which can act as an alternative dihydrofolate reductase (DHFR/DfrA), confer resistance (5, 6). Finally, loss-of-function mutations in thymidylate synthase ThyA, which is a major consumer of tetrahydrofolate, result in PAS resistance (7, 8).
Other studies reported that one-third of PAS-resistant isolates had thyA mutations; however, little is known about the antibiotic resistance mechanism(s) in the remaining samples (9, 10). Here, we first randomly selected PAS-resistant isolates to determine their genotypes for folC, thyA, and ribD. Two hundred eight isolates were selected from the Beijing Bio-Bank of Clinical Resources on Tuberculosis, which included isolates from approximately 6,400 patients from 2006 to 2012. These patients received routing clinical care and were from provinces and regions in northern China, most of whom were undergoing retreatment or had relapsed. Drug susceptibility testing (DST) for 10 anti-TB drugs was performed with these isolates according to World Health Organization guidelines (11). This study was approved by the ethics review committee of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences, and Peking Union Medical College.
Resistance to PAS was determined by cultivating bacteria in the presence of the critical concentration of 1.0 mg/liter PAS on Lowenstein-Jensen medium (12). All isolates were putatively identified as M. tuberculosis by 16S rRNA gene sequencing (13). Molecular characterization of the TB strains was performed by the spoligotyping method based on M. tuberculosis-specific direct repeat (DR) region (14).
Sequencing analysis of the PAS resistance-related genes from the isolates was performed as previously described (10). Briefly, the coding and flanking regions of thyA (Rv2764c), folC (Rv2447c), and ribD (Rv2671) were amplified by PCR, and the sequences were compared with those from the wild-type strain M. tuberculosis strain H37Rv.
Among the 208 isolates, 127 (61.1%) samples contained mutations in at least one of the three genes (Table 1); 116 isolates were single-gene mutants (affecting folC, thyA, or ribD), and 11 isolates were double-gene mutants (affecting folC and thyA or folC and ribD; Fig. 1). Mutations in folC were most frequently detected, occurring in 72 (34.8%) isolates. Mutations of thyA and ribD were detected in 54 (26.0%) and 12 (5.8%) isolates, respectively.
TABLE 1.
General features of PAS-resistant isolates in M. tuberculosis
| No. of isolates | Mutation in drug resistance-related genesa |
Drug resistance profile forb: |
Molecular genotypec |
|||||
|---|---|---|---|---|---|---|---|---|
| folC | ribD | thyA | MDR-TB | Non-MDR-TB | Spoligotype | SIT | Lineage | |
| 75 | 54 (53) | 21 (19) | 000000000003771 | 1 | Beijing family | |||
| 1 | 1 | 000000000003071 | 1941 | Beijing family | ||||
| 1 | 1 | 777717777600371 | Orphan | |||||
| 3 | 1 | 2 | 777777477763771 | Orphan | ||||
| 1 | 1 | 777777777760000 | 1793 | T1 | ||||
| 1 | ACT→CCT (T20P) | TGG→CGG (W101R) | 1 | 000000000003771 | 1 | Beijing family | ||
| 1 | GAG→CAG (E40Q) | 1 | 000000000003771 | 1 | Beijing family | |||
| 4 | GAG→GGG (E40G) | 4 | 000000000003771 | 1 | Beijing family | |||
| 2 | ATC→ACC (I43T) | CAC→AAC (H75N) | 2 | 000000000003771 | 1 | Beijing family | ||
| 14 | ATC→ACC (I43T) | 13 | 1 | 000000000003771 | 1 | Beijing family | ||
| 2 | ATC→AGC (I43S) | 2 | 000000000003771 | 1 | Beijing family | |||
| 1 | ATC→AGC (I43S) | 1 | 771777777760711 | Orphan | ||||
| 1 | ATC→GCT (I43A) | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | ATC→GCT (I43A) | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | ATC→GCT (I43A) | ACC→GCC (T22A) | 1 | 000000000003771 | 1 | Beijing family | ||
| 5 | ATC→GCT (I43A) | 5 | 000000000003771 | 1 | Beijing family | |||
| 4 | CGG→CCG (R49P) | CAC→AAC (H75N) | 4 | 000000000003771 | 1 | Beijing family | ||
| 2 | CGG→CCG (R49P) | 2 | 000000000003771 | 1 | Beijing family | |||
| 7 | CGG→TGG (R49W) | 7 | 000000000003771 | 1 | Beijing family | |||
| 1 | CTG→GTG (L56V) | −11 nt G→A | 1 | 000000000003771 | 1 | Beijing family | ||
| 1 | CGG→TGG (R91W) | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | AGC→GGC (S150G) | 260C(nt) deletion | 1 | 000000000003771 | 1 | Beijing family | ||
| 6 | AGC→GGC (S150G) | 6 | 000000000003771 | 1 | Beijing family | |||
| 1 | AGC→TGC (S150C) | 1 | 000000000003771 | 1 | Beijing family | |||
| 10 | GAG→GCG (E153A) | 8 | 2 | 000000000003771 | 1 | Beijing family | ||
| 5 | GAG→GGG (E153G) | 1 | 4 | 000000000003771 | 1 | Beijing family | ||
| 1 | GCG→GTG (A420V) | ACC→GCC (T202A) | 1 | 777777607760771 | 42 | LAM9 | ||
| 3 | −11 nt G→A | 3 | 777777777700000 | 237 | Unknown | |||
| 7 | −11 nt G→A | 7 | 000000000003771 | 1 | Beijing family | |||
| 1 | GGA→CGA (G8R) | 1 | 000000000003771 | 1 | Beijing family | |||
| 3 | 111T(nt) deletion | 3 | 000000000003771 | 1 | Beijing family | |||
| 1 | 217CACGAGCAC(nt) insertion | 1 | 000000000003131 | Orphan | ||||
| 1 | 372T(nt) insertion | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | 472C(nt) deletion | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | TAT→TGT (Y36C) | 1 | 000000000003771 | 1 | Beijing family | |||
| 13 | CAC→AAC (H75N) | 11 | 2 | 000000000003771 | 1 | Beijing family | ||
| 1 | GGA→TGA (G76stop) | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | GTC→TTC (V77F) | 1 | 000000000003771 | 1 | Beijing family | |||
| 2 | TGG→TGT (W83C) | 2 | 000000000003771 | 1 | Beijing family | |||
| 5 | TGG→TAG (W83stop) | 5 | 000000000003771 | 1 | Beijing family | |||
| 3 | GGG→AGG (G91R) | 3 | 000000000003771 | 1 | Beijing family | |||
| 1 | TGG→TGA (W98stop) | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | TCC→CCC (S105P) | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | CGG→CAG (R126Q) | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | TTC→GTC (F152V) | 1 | 376777774220471 | N | ||||
| 1 | TTC→GTC (F152V) | 1 | 777777776760771 | 1626 | T1 | |||
| 1 | TGT→TAT (C161T) | 1 | 000000000003771 | 1 | Beijing family | |||
| 3 | CAC→CGC (H207R) | 3 | 000000000003771 | 1 | Beijing family | |||
| 1 | ATT→GTT (I211V) | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | CCG→CTG (P224L) | 1 | 000000000003771 | 1 | Beijing family | |||
| 1 | CGG→CCG (R235P) | 1 | 000000000003771 | 1 | Beijing family | |||
| Total (n = 208) | 166 | 42 | ||||||
Unless otherwise stated, the sequences were wild type. For mutant genotypes, they are displayed as the substitution of a single nucleotide and the corresponding amino acid substitution. nt, nucleotide; −11 nt, substitution of the 11th nucleotide upstream from the start codon.
MDR-TB refers to isolates resistant to isoniazid (INH) and rifampin (RIF). The numbers in parentheses show the number of resistant isolates after retesting of 9 wild-type MDR-TB and 9 non-wild-type MDR-TB isolates.
Determination of shared international types (SITs) was based on SpolDB4 database. N, data not available.
FIG 1.
Distribution of PAS-resistant isolates with different gene mutations. The numbers of isolates that harbored mutants of three genes are shown.
We detected 15 different mutations in nine codons of folC (Table 1). Most of these mutations were amino acid substitutions affecting five specific residues (positions 40, 43, 49, 150, and 153), accounting for 94.4% (68/72) of the folC mutant strains. Residue 43 was the most common mutation site, with three substitutions (I43A, I43T, and I43S) detected in 37.5% (27/72) of the folC mutant strains. The second most frequent site was residue 153, with two substitutions (E153A and E153G) accounting for 20.8% (15/72) of the folC mutant strains. Six mutations were substitutions in three residues: E40Q and E40G in residue 40, R49P and R49W in residue 49, and S150G and S150C in residue 150. The remaining four folC mutations (T20P, L56V, R91W, and A420V) were detected in four isolates.
The activation of PAS relies on FolC to form the dihydrofolate (H2Pte-Glu) analog hydroxyl dihydrofolate (H2PtePAS-Glu). The crystal structures of FolC show that the α1 to α2 loop (residues 36 to 50) is involved in dihydropteroate (H2Pte) binding, and a four-helix bundle (α1 to α2/α4 to α5) is important for interaction with hydroxyl dihydropteroate (H2PtePAS), an analog of H2Pte (15). Our results showed that most folC mutations were located in this domain, indicating that the reduced efficiency of H2PtePAS glutamination was the main cause of drug resistance in these clinical isolates.
Compared to the clinical isolates, a different distribution of folC mutations occurred in the laboratory mutants. Zhao et al. showed that 67% (29/43) of M. tuberculosis strain H37Ra and 70% (16/23) of Mycobacterium bovis BCG spontaneous PAS-resistant isolates had mutations in folC (5). Although mutations at residues 43 and 153 occurred in 58.3% (42/72) of the folC mutant strains isolated from patients, no mutations at residue 43 were detected in the spontaneous mutants. Instead, substitutions at residue 153 occurred most frequently in the H37Ra mutants, accounting for 72.4% of the folC mutants. This discrepancy is likely related to different strain backgrounds, as well as their growth differences in vivo versus in vitro.
Diversified thyA mutations were detected in our clinical isolates, which occurred throughout the gene. We detected 24 mutations in 54 isolates, consisting of 19 single nucleotide polymorphisms (SNPs) and five frameshift mutations (Table 1). Most thyA mutations were detected in one isolate only, but four mutations were detected in multiple isolates. The most common mutation was H75N, found in 17 isolates. Three other common mutations included W83stop, W83C, and G91R. The active domain of ThyA is formed by residues 21 to 209, and the dimerization domain is formed by residues 16 to 205. Structural data for bacteria indicate that the C-terminal amino acid of ThyA plays a functional role in the catalysis of reductive methylation (16). Mutations in these two functional regions were found in 94.4% (51/54) of the thyA mutants.
The loss of ThyA function is generally caused by an amino acid substitution or protein truncation. We found that 16 substitutions were caused by SNPs, which were found in 74.1% (40/54) of the thyA mutants. These substitutions were putative destabilizing mutations, as revealed by the positive changes of free folding energy computed by the PoPMuSiC program (17). In 13 (24.1%) isolates, premature stop codons were caused by three SNPs (G76stop, W83stop, and W98stop), and four frameshift mutations occurred due to three single nucleotide deletions (111T, 260C, and 472C) and one single nucleotide insertion (372T). In addition, an insertion in nucleotide 217 was found in one isolate, resulting in a repeat of three amino acids (from HEH at residues 73 to 75 to HEHHEH at residues 73 to 78). According to the three-dimensional structure of ThyA in Escherichia coli, which shows a helix formed by residues 70 to 74, this repeat would alter the secondary structure in this region (18). Thus, destabilizing mutations and protein truncation might influence the catalytic activity and substrate-binding ability of ThyA, resulting in a loss of its function.
The first gene reported to be related to PAS resistance was thyA, and previous studies described mutations in this gene and detected them in approximately one-third of PAS-resistant clinical isolates and laboratory mutants (9, 10). We detected a slightly lower frequency of thyA mutants in our samples, but we identified more diverse thyA mutations. We identified 20 novel mutations in addition to the four previously reported mutations (H75N, G91R, F152V, and T202A).
Our results showed that the SNP −11G→A (the 11th nucleotide substitution found upstream from the start codon) was the predominant genotype in ribD mutants, with 91.7% (11/12) of clinical isolates harboring this mutation. The amino acid substitution G8R was identified in one isolate. RibD is a riboflavin biosynthesis protein, and its C-terminal reductase domain shares strong sequence similarities with DHFR (19). The SNP −11G→A leads to the overexpression of RibD and causes resistance to DHFR inhibitors. Thus, the overexpression of this protein resulted in PAS resistance in these clinical isolates (6).
The spoligotyping results demonstrated that the Beijing family of TB strains was the dominant lineage and was represented by 195 (93.8%) of 208 isolates (Table 1). The bacterial family is one of the most widespread clades of M. tuberculosis worldwide and is often associated with multidrug resistance (20). Its distinct characteristics include a high mutation frequency, a cell wall structure that decreases intracellular concentrations of anti-TB drugs, and an increased the risk of drug resistance acquisition (21, 22). Two other TB lineages, Latin American-Mediterranean (LAM9) and T1, were represented by one and two isolates, respectively, and the remaining 10 isolates were associated with unknown lineages.
Bacterial genetic diversity has confounded studies investigating the molecular basis of drug resistance. Phylogenetic polymorphisms exist within antibiotic resistance genes, which might lead to false associations made between certain polymorphisms and phenotypic resistance (23, 24). For example, in our study, isolates carried the mutation T202A in thyA, a marker for the LAM lineage of M. tuberculosis that is not responsible for PAS resistance (8, 25). Theoretically, these false associations would be identified by detecting the occurrence of mutations in both susceptible and resistant strains from within the same genotype (26). The SNPs observed simultaneously in susceptible and resistant strains may be due to genetic diversity rather than related to drug resistance. A previous study showed no thyA mutation detected in susceptible isolates from TB patients in China (9). Here, our observation of >100 Beijing isolates with wild-type folC, ribD, or thyA indicates that this genotype likely does not have any deeply rooted polymorphisms. Therefore, mutations occurred among these isolates mainly due to drug selective pressure, and these wild-type Beijing strains act as controls to some extent, despite being PAS resistant.
Mutations in folate pathway genes were the main cause of PAS resistance in our study, which we detected in nearly two-thirds of the PAS-resistant clinical isolates. The remaining 81 resistant isolates were classified as wild-type strains, and their emergence can be explained by one of three possibilities. First, some wild-type isolates may carry low-frequency resistance mutations not detectable by Sanger sequencing. This phenomenon, known as heteroresistance, is frequently observed in clinical isolates with resistance to second-line drugs (27–29). Second, the breakpoint for DST might be too low, thereby leading to systematic false-positive resistance results (i.e., the wild-type distribution of MIC might be bisected) (30). Third, some of the putative PAS-resistant strains may be random false positives. Extensive repetition of phenotypic DST and Sanger sequencing (as done by Simons et al. [31, 32]) to resolve discrepancies between genotype and phenotype for pyrazinamide was beyond the scope of this study. We did, however, repeat DST for 9 randomly selected multidrug-resistant (MDR)-TB and 9 non-MDR-TB strains that lacked mutations in thyA, folC, and ribD, given that these strains were most likely to be false positives. We found that 20% of these strains were PAS susceptible upon retesting.
To our knowledge, this study is the first to perform a systematic analysis of mutations in genes encoding key enzymes of the folate pathway in clinical isolates and their relationship to drug resistance in TB. These findings expand our knowledge about PAS resistance. The features of PAS resistance revealed in this study will increase our understanding of the distribution and frequency of mutations in M. tuberculosis isolates with PAS resistance in TB patients from China.
ACKNOWLEDGMENTS
All the mycobacterial strains used in this project were acquired from the Beijing Bio-Bank of Clinical Resources on Tuberculosis.
This study was supported by the National Major Science and Technology Project for the Prevention and Treatment of AIDS and Viral Hepatitis and Other Major Infectious Diseases (grant 2012ZX10003002-001).
We have no conflicts of interest to declare.
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