Genetic evaluation of relationship between mutations in rpoB and resistance of Mycobacterium tuberculosis to rifampin

Background Rifampin is a first line antituberculosis drug active against bacilli in logarithmic and stationary phase, which interferes with RNA synthesis by binding to bacterial RNA polymerase. Tubercle bacilli achieve resistance to rifampin by accumulation of mutations in a short-81 bp region of the rpoB gene. Among many mutations identified in the rpoB gene, few were verified by molecular genetic methods as responsible for resistance to rifampin (RMP). Results In this study eight different mutations identified in an 81 bp section of a "hot spot" region of the rpoB gene of RMP resistant Mycobacterium tuberculosis clinical strains were evaluated in respect to drug resistance. It was found that: mutations in positions 526 (H/D), 516 (D/V) and 531 (S/L) result in high level resistance to rifampin; mutations in positions 516 (D/Y), 515 (M/I), 510 (Q/H) or a double mutation in codons 512 (S/I) and 516 (D/G) relate to low level of resistance. Gene rpoB carrying mutations in codon 513 (Q/L) introduced into an M. tuberculosis laboratory strain did not cause resistance to rifampin, however the same gene introduced into two different clinical strains did, with the level of resistance depending on the host strain. Conclusion Mutations in an 81 bp "hot spot" region of the rpoB of M. tuberculosis lead to different levels of resistance to rifampin. Some mutations in this "hot spot" region of rpoB require a specific genetic background for the host strain to develop resistance to rifampin. Therefore, the identification of such mutations in a clinical M. tuberculosis strain is not enough to classify the given strain as resistant to rifampin.


Background
Tuberculosis (TB) is a devastating infectious disease causing high mortality and morbidity worldwide with 8 million new TB cases and 2-3 million deaths annually. The situation of TB is made even worse by the rising emergence of drug resistant strains of Mycobacterium tuberculo-sis. Multi-drug resistant TB (MDR-TB) is defined as resistant to at least isoniazid (INH) and rifampin (RMP), the two most active first-line drugs against TB. MDR-TB treatment takes up to 2 years with second line drugs, which are expensive and have side effects. In 2006 US Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) drew attention to the emergence of M. tuberculosis with extensive drug resistance to second-line antituberculosis drugs (XDR). XDR-TB is resistant to at least INH and RMP among the firstline drugs and to at least one of three injectable secondline anti-tuberculosis drugs used in TB treatment (capreomycin, kanamycin, amikacin) [1]. Thus, the treatment of such tuberculosis is becoming seriously limited, sometimes returning TB control to the pre-antibiotic era [1]. Tuberculosis chemotherapy started in 1944, when streptomycin (SM) was administered for the first time to a critically ill TB patient. Later, TB treatment was enriched with paraaminosalicylic acid (PAS-1949), INH (1952), pyrazinamide (PZA-1954), ethambutol (EMB-1962) and RMP (1963). It was identified that monotherapy generates drug-resistant mutants within a few months, endangering the success of antibiotic treatment. This problem was overcome by using combinations of drugs with as many as four drugs recommended nowadays by CDC and WHO [2].
The key antituberculosis drug commonly used in the treatment of tuberculosis is RMP. The loss of RMP as an effective drug leads to a need for a longer duration of therapy and often to a lower cure rate [3][4][5][6]. Drug resistance in M. tuberculosis is caused by mutations of various chromosomal genes, as identified for MDR occurrence due to the sequential accumulation of mutations in different genes that provide resistance to individual drugs. The individual molecular mechanisms of resistance have been identified for all first-line drugs and the majority of second-line drugs [7]. In M. tuberculosis, resistance to RMP results from mutations in the β-subunit of RNA polymerase, which is encoded by the rpoB gene [8]. Approximately 95% of RMP-resistant strains carry mutations within an 81-bp region containing codons 507 through 533 of the rpoB gene [8][9][10]. The single mechanism of resistance and narrow distribution of mutations make rpoB-81 bp region very attractive for molecular detection of resistance to RMP [11,12]. However, within several dozen different mutations detected in the rpoB-81 bp region of RMPresistant M. tuberculosis strains [for review see [13]], very few were tested by cloning and complementation assays. Mutated rpoB genes (S531L; H526Y; D516V) were introduced into the RMP sensitive M. tuberculosis H 37 Rv strain, resulting in acquired drug resistance of the host strain [14]. These authors observed that the level of acquired resistance was higher for mutants carrying mutations in codons 531 and 526 compared to mutation in codon 516. In this paper a genetic model was constructed allowing for a relatively simple verification of the relationship between the presence of a given mutation in rpoB-81 bp region and the RMP resistance of the host strain carrying such a mutation. Some rpoB mutations revealed drug-resistance only in selected M. tuberculosis strains suggesting that genetic background of the host is important for the development of resistance to RMP.

Bacterial strains and growth conditions
The M. tuberculosis strains examined for this study were isolated from TB patients in Poland in 2000 during the second national survey of drug resistance [12,15]. Eight clinical strains identified as drug resistant, carrying different mutations in the rpoB gene, and two susceptible strains identified as drug sensitive, which did not carry any mutation in rpoB, were selected. Moreover, a control laboratory strain M. tuberculosis H 37 Ra, was included in this study. Primary isolation, differentiation, and drug susceptibility testing were performed with Lowenstein-Jensen (LJ) medium and the BACTEC 460-TB system (Becton-Dickinson, Sparks, Md.), as reported earlier [15]. All mycobacterial strains used in this study were cultured in Middlebrook 7H9 broth supplemented with OADC (albumin-dextrose-sodium chloride) and with kanamycin (25 μg/ml), or hygromycin (10 μg/ml), when required. Mycobacterial transformants were selected on Middlebrook 7H10 agar plates enriched with OADC containing kanamycin (Km) or hygromycin (Hyg).

Gene cloning strategies
Standard molecular biology protocols were used for all cloning procedures [16]. All PCR products were obtained using thermostable ExTaq polymerase (Takara) and cloned initially into pGemT vector (Promega), sequenced, and then released by digestion with appropriate restriction enzymes before cloning into the final vectors. Some restriction enzymes recognition sites were incorporated into the sequence of primers. The primers and plasmids used in this work are listed in Table 1 and 2, respectively. To engineer various rpoB genes of M. tuberculosis controlled by a natural promoter, a basal pRpoZero vector was constructed (Fig. 1). The vector contained the 5' end of rpoB until a natural BstEII restriction enzyme recognition site (681 plus 950 bp of upstream region) which was connected to the 3' fragment of the gene starting with a natural BstEII restriction enzyme recognition site (1122 plus 218 bp of downstream region). The resultant construct was used for cloning of the inner BstEII-BstEII fragment (1716 bp) of rpoB genes from various M. tuberculosis clinical strains resistant to RMP. The correct orientation of cloned BstEII fragments was verified by digestion with PvuII endonuclease. Next, the cloned genes controlled by their natural promoter, carrying given mutations or wild type sequence in the hot spot region were relocated into the pMV306 integration vector. The resultant constructs (pMRP1-9) were electrotransformed into RMP susceptible strains, and the integration of DNA was monitored by Km selection and verified by PCR. Alternatively, the investigated rpoB genes were relocated without putative pro-moter sequence into pMV306P hsp integration vector under control of strong promoter (P hsp65 ). The resultant constructs (pMHRP1-9) were electrotransformed into RMP susceptible strains, and the integration of DNA was monitored by Hyg selection and verified by PCR.

Susceptibility testing
Susceptibility testing was conducted using the proportion method on Youmans' liquid medium supplemented with 10% OADC with seven concentrations of RMP (50, 25, 12.5, 6.2, 1.5, 0.75, 0.37 μg/ml). The growth was determined after 21 days of incubation. The results were verified by Alamar Blue Assay [17][18][19] and by plating bacteria on Middlebrook 7H10 supplemented with OADC and various concentrations of RMP.

The level of RMP resistance depends on the site and kind of substitution identified in the rpoB gene
The epidemiological studies carried out in many clinical laboratories worldwide have revealed several dozen mutations present in the rpoB gene of RMP resistant M. tuberculosis strains [12,14,[20][21][22][23]. According to our knowledge, only three specific mutations of rpoB have been verified so far by molecular cloning techniques [14]. The complementation of RMP sensitive M. tuberculosis strain with rpoB gene carrying given mutation is not simply due to the gene length (3519 bp). One step amplification of gene together with its putative promoter based on M. tuberculosis genomic DNA as a template and its cloning is rather tough for investigators. To avoid this problem we have engineered pRpoZero vector carrying a 950 bp putative promoter region followed by 5'(721 bp) and 3' (1258 bp) rpoB gene fragments of an RMP-sensitive M. tuberculosis H 37 Ra strain (Fig. 1). The missing inner part of the rpoB gene flanked with natural BstEII restriction sites contains an 81-bp mutable region. The BstEII fragment (1716 bp) of rpoB gene can be easily amplified based on genomic DNA isolated from investigated M. tuberculosis RMP-resistant strains and cloned in frame to complete the rpoB gene in the pRpoZero system.
In this study we have selected eight M. tuberculosis RMPresistant clinical strains carrying different mutations in rpoB gene [12] ( Table 3). The PCR generated BstEII inner fragments of the rpoB gene were verified by sequencing and were cloned into the pRpoZero vector. The correct orientation of insert was confirmed by PvuII restriction analysis. Subsequently, the constructed genes, together with Construction strategy of integration (pMRP1-9; pMHRP1-9) and self-replicating (pMERP1-9) plasmids carrying wild type and mutated rpoB genes under control of own (pMRP1-9) and heat shock (pMHRP1-9; pMERP1-9) promoter Figure 1 Construction strategy of integration (pMRP1-9; pMHRP1-9) and self-replicating (pMERP1-9) plasmids carrying wild type and mutated rpoB genes under control of own (pMRP1-9) and heat shock (pMHRP1-9; pMERP1-9) promoter. Description in the text.

The overproduction of mutated RpoB does not cause high level of resistance to RMP
We could not exclude that the different resistance of M. tuberculosis hosts carrying identical mutations in rpoB depends on different expression of RpoB controlled by unknown regulatory proteins. For example, the raised expression of target molecule (InhA) due to accumulations of mutations in promoter region is one of the known mechanisms of resistance to INH. As questions arose as to whether expression of mutated rpoB genes under control of the heat shock promoter (P hsp60 ) resulted in increased resistance of M. tuberculosis to RMP, the wild type rpoB and its mutated copies were cloned under control of the heat shock promoter as described in Methods.
Although we did not have antibodies to test the level of expression for RpoB, the expression system is known to be very efficient [24,25]. The self-replicating constructs (pMERP1-9, Fig. 1) appeared to be very unstable when introduced into M. tuberculosis host (data not shown). Therefore the vectors (pMHRP1-9), which are able to integrate into attB site of mycobacterial chromosomal DNA, carrying wild type and mutated rpoB under P hsp60 promoter were constructed and electroporated into M. tuberculosis H 37 Ra. The presence of the relevant DNA introduced into the attB site of chromosomal DNA was verified by PCR amplification. The resultant recombinant strains were subjected to RMP resistance analysis by the proportional method. The results of RMP-MIC analysis obtained for strains carrying mutated rpoB genes under control of the strong P hsp60 promoter were similar to strains carrying the same rpoB genes under control of their natural promoter (Table 4). These observations suggest that the RMP-resistance of M. tuberculosis strains carrying rpoB mutated genes was not dependent on the rpoB expression level but resulted from the host genetic background that influence the drug-resistance phenotype.

Discussion
All bacteria achieve resistance to RMP by mutations in a defined region of the RNA polymerase subunit β. In M. tuberculosis, approximately 95% of RMP resistant clinical isolates carry a mutation in the rpoB gene [8]. On the other hand, many isolates from M. avium and M. intracellulare present a natural resistance to RMP as a result of an efficient permeability and exclusion barrier [26,27]. Mutations in rpoB generally result in high level resistance to RMP. However, specific mutations in codons 511, 516, 518 and 522 can result in a lower resistance to RMP [14,28,29]. The role of some rpoB mutations (H526Y, S531L, D516V) in causing resistance was confirmed by genetic transformation experiments [14,30]. Several dozen other mutations identified in the rpoB gene of RMPresistant M. tuberculosis clinical isolates have never been confirmed by genetic cloning [12,[31][32][33][34][35]. Nowadays, when many genetic techniques are well developed, the knowledge about mutations connected to RMP-resistance is becoming used in the rapid identification of drug resistance [11,12,36,37]. However, the utility of these techniques depends on the precise information about the role of any given mutation in RMP resistance.
In this study we have engineered a genetic system which is helpful in the verification of the relationship between the presence of a given mutation in rpoB and RMP resistance. We have found that rpoB gene carrying either D516V or S531L mutation causes resistance to RMP when introduced into the M. tuberculosis hosts what was in agreement with previous investigations [14]. On the other hand, when mutated rpoB was introduced into drug sensitive M. tuberculosis laboratory or clinical strains, the other substitutions in position 516 (D/Y; D/G), even when supported with Q510H, M515I or S512I identified in RMP-resistant M. tuberculosis clinical strains, did not result in a significant increase of RMP-resistance. Other authors previously reported the identification of D516Y substitutions of rpoB in M. tuberculosis resistance to a high level of RMP [21,38], low level of RMP [14] and in strains sensitive to RMP [39]. Taken together, this suggests that D516Y/G substitutions in rpoB are not sufficient to result in RMP-resistance of M. tuberculosis. The substitutions in codon 526 (H/Y, D, R, L, P) were usually identified in M. tuberculosis clinical isolates highly resistant to RMP [14,23,38]. In this paper we have provided direct evidence that mutation H526D in rpoB is responsible for RMP-resistance when introduced into M. tuberculosis host. This result is similar to a previous finding verified by genetic transformation of rpoB carrying mutation H526Y [14]. By contrast, the contribution of rpoB carrying Q513L mutation to RMP-resistance was not that evident. The insertion of this gene into an M. tuberculosis H 37 Ra laboratory strain did not result in a significant level of RMP-resistance, however the insertion of the same gene was responsible for resistance to RMP of two M. tuberculosis clinical strains (MIC 12.5 and 50 μg/ml) when used as hosts. As identified in various clinical studies, the level of RMP-resistance of M. tuberculosis isolates carrying the Q513L mutation varies from 2 to 200 μg/ml [14,20,21,23,38]. The collected results suggest that rpoB carrying Q513L mutation is able to cause resistance to RMP only in selected tubercle bacilli. It is likely that this mutation can result in RMP-resistance in strains with low cell wall permeability since this exclusion barrier is responsible for natural resistance of some MAIC strains [26,27]. We also cannot exclude the possibility that other mechanisms support RMP-resistance of strains carrying Q513L mutation.
The drug resistance of M. tuberculosis can be also connected to the overproduction of a drug target due to accumulation of point mutations in a promoter region [40][41][42]. To test whether overproduction of rpoB carrying a given mutation result in higher MIC for RMP compared to a strain expressing the same gene under control of the natural promoter, rpoB genes were cloned under control of the P hsp promoter and introduced into M. tuberculosis host. The P hsp promoter, commonly used in genetics studies of mycobacteria controlling the groEL gene (Rv0440) in M. tuberculosis, has already been reported as highly active in mycobacterial cells growing in vitro [24,25]. A recent microarray study showed that the expression level of groEL in M. tuberculosis cells growing in log phase is high, but not higher than rpoB [43]. However, the arresting of M. tuberculosis growth results in 3.6-fold induction of groEL with a decrease of rpoB expression in the same conditions [44]. We have not observed higher RMP resistance when mutated rpoB genes were expressed under control of P hsp promoter in comparison to the natural promoter. It is possible that the natural level of RpoB is high enough to saturate RMP (if its concentration in cell is low). On the other hand, the extra expression of rpoB cannot help in cells accumulating high RMP level. However, to elucidate this problem an alternative expression system and precise control of protein expression would be required.
The natural resistance to RMP in some M. avium and M. intracellulare strains is known to be as a result of an efficient cell wall permeability and exclusion barrier [26,27], suggesting that these elements may be also important in M. tuberculosis. Changes in cell wall composition could affect permeability [45] decreasing the intracellular concentration of drug.

Conclusion
Among several dozen amino acid substitutions identified in an rpoB 81-bp region only a few are directly responsible for M. tuberculosis resistance to rifampin. Many others require a specific genetic background to develop resistance. Our findings lead to the conclusion that direct, molecular identification of rifampin resistant M. tuberculosis clinical isolates is possible only for strains carrying selected mutations in RpoB. The identification of other mutations suggests that investigated strains might be resistant to this drug.