Role of folP1 and folP2 Genes in the Action of Sulfamethoxazole and Trimethoprim Against Mycobacteria

Abstract

The combination of trimethoprim (TMP) and sulfamethoxazole (SMX) has been shown to be active against Mycobacterium tuberculosis (Mtb) in clinical tuberculosis (TB) treatment. However, the mechanism of action of TMP-SMX against Mtb is still unknown. To unravel this, we have studied the effect of TMP and SMX by deleting the folP2 gene in Mycobacterium smegmatis (Msm), and overexpressing the Mtb and Msm folP1/2 genes in Msm. Knocking out of the folP2 gene in Msm reduced the minimum inhibitory concentration of SMX 8-fold compared with wild type. Overexpression of the folP1 genes from Mtb and Msm increased the MICs by 4- and 2-fold in Msm for SMX and TMP, respectively. We show a strong correlation between the expression of folP1 and folP2 genes and TMP-SMX resistance in mycobacteria. This suggests that a combination of FolP2 inhibitor and SMX could be used for TB treatment with a better outcome.

Introduction

Tuberculosis (TB) is a chronic disease caused by Mycobacterium tuberculosis (Mtb). The emergence of multidrug resistance (MDR), defined as resistance to at least the two main first-line anti-TB drugs, rifampicin and isoniazid; extensively drug resistance (XDR), defined as MDR strains that are also resistant to a fluoroquinolone and at least one second-line injectable agent such as amikacin, kanamycin (KAN), or capreomycin; and the more severe totally drug resistance (TDR), defined as Mtb strains resistant to all first- and second-line anti-TB drugs, is an urgent medical and public health concern, as the available anti-TB drugs exhibit limited efficacy [20, 26]. Development of new drugs is time-consuming, difficult, and expensive. However, if already existing clinically established effective drugs could be used for treatment of TB, then faster and cheaper drug development coupled with effective TB management would be attained.

Sulfamethoxazole (SMX) and trimethoprim (TMP) are such potential candidates for TB treatment, having been used in drug regimens for the treatment of various bacterial infections of the respiratory, urinary, and gastrointestinal tracts for more than 40 years [1, 10]. TMP and SMX target successive steps of the folate biosynthesis pathway. SMX inhibits the dihydropteroate synthase (DHPS) activity, which catalyzes the addition of dihydropterindiphosphate to p-aminobenzoic acid (PABA), a structural analog of SMX. The product of DHPS, 7,8-dihydropteroate (DHP), reacts with glutamate to form dihydrofolate (DHF), which is reduced to tetrahydrofolate (THF) by dihydrofolate reductase (DHFR), the target of TMP (Fig. 1). Bacteria, fungi, and plants synthesize folate de novo, but mammals lack DHPS and therefore cannot produce folate. THF is an essential co-factor involved in the transfer of a one-carbon unit and is implicated in the biosynthesis of purines and pyrimidines and in the biosynthesis and catabolism of some amino acids. The combination of TMP and SMX prevents the emergence of drug resistance and has been demonstrated to be synergistic in many bacteria [3, 10]. Vilcheze and Jacobs suggested that the folate biosynthesis pathway could be a good Mtb target for drug development [19].

Fig. 1.SMX and TMP target the folate pathway in mycobacteria. SMX: sulfamethoxazole; TMP: trimethoprim; PABA, p-aminobenzoic acid; DHP: 7,8-dihydropteroate; DHFS: dihydrofolate synthase; DHF: dihydrofolate; THF: tetrahydrofolate; DHFR: dihydrofolate reductase.

Forgacs et al. [7] reported that drug-susceptible and drug-resistant Mtb strains were susceptible to TMP/SMX with a bacteriostatic activity of 2/38 μg/ml. Subsequently, an analysis of 12 drug-susceptible Mtb clinical isolates from Australia revealed a susceptibility concentration of below 38 μg/ml to SMX [13]. A clinical case study of a patient infected with an XDR Mtb strain reported susceptibility to TMP-SMX at 1/19 μg/ml [4]. Huang et al. [9] demonstrated that SMX inhibited 80% growth in 117 Mtb clinical isolates at an MIC90 of 9.5 mg/l, regardless of their susceptibility to the first-line agents. When used in combination with rifampicin or isoniazid, SMX and TMP have been shown to be bactericidal and prevent the emergence of drug resistance in Mtb [11, 19]. Recently, 100 Mtb isolates, including 48 MDR-TB and 13 XDR-TB, were tested. All the isolates had MICs ≤ 38 mg/l of SMX, whereas it was less active inside the macrophages. This implied SMX could be a treatment option in selected MDR and XDR TB cases in the initial phase [6]. The analysis of complete genome data revealed the presence of two genes in mycobacteria, folP1 and folP2, which encode proteins that have homology to DHPS in other bacteria [5, 21]. The overall structure of FolP1 and FolP2 showed a “TIM barrel” fold (n = 8, S = 8) with eight α-helices surrounding a central barrel composed of eight parallel β-strands (Fig. 2). The structure and key residues essential for substrate binding of FolP1 and FolP2 are highly conserved, with the two proteins sharing only about 30% amino acid identity [2, 8, 12]. In mycobacteria, the folP1 gene is located within the folate operon, whereas folP2 is organized with genes belonging to fatty acid metabolism [5]. It has been shown that the product encoded by the respective folP1 gene exhibited DHPS activity in mycobacteria [12], and folP1 was found to be essential for growth [15]. However, the function of folP2 has not yet been established.

Fig. 2.Comparison of binding mode of SMX in YpDHPS and Mtb FolP1. (A) Crystal structure of the YpDHPS with SMX (green) complex (PDB: 3TZF). (B) Predicted binding mode of SMX (yellow) with Mtb FolP1. PtP in the active site is shown in sticks (grey). Key residues of the binding site are shown as lines and the hydrogen bonds are labeled as red dash lines.

This study thus sets out to explore the role of folP1/ folP2 genes in TMP-SMX resistance in mycobacteria by gene knockout, overexpression, and drug susceptibility experiments.

 

Materials and Methods

Bacterial Strains and Media

E. coli DH5α was grown at 37°C in Luria Bertani (LB) broth and agar. Mycobacterium smegmatis (Msm) mc2155, its mutants, and Mtb H37Rv were grown in Middlebrook 7H9 broth (Difco) supplemented with 10% oleic acid albumin dextrose catalase (OADC) a nd 0 .05% Tween 8 0, or on s olid Middlebrook 7H11 medium (Difco) supplemented with 10% OADC or containing 10% sucrose if necessary (Table 1). Hygromycin (HYG; Roche) and ampicillin (AMP; Sigma) were added at 200 μg/ml and 100 μg/ml, respectively, to maintain vector constructs. HYG and KAN (Invitrogen) were added at 150 μg/ml and 40 μg/ml, respectively, for Msm when required. SMX and TMP were purchased from Sigma and dissolved in DMSO.

Table 1.aATCC: The American Type Culture Collection; bAMPr: ampicillin resistance gene; cKANr: kanamycin resistance gene; dHYG: hygromycin resistance gene; edif: the action site of the XerCD recombinase; fArmL: The upstream 884 bp DNA fragment of Msm folP2 gene; hArmR: the downstream 982 bp DNA fragment of Msm folP2 gene.

Vector Construction of pblMsfp2LRH

To delete the folP2 gene in M sm, the p lasmid p Bluescript II SK(+) was used to construct the vector pblMsfp2LRH. The upstream 884 bp DNA fragment (ArmL) and the downstream 982 bp DNA fragment (ArmR) of folP2 were amplified using primers Msfolp2L and Msfolp2R (Table 2), respectively. The two fragments were cloned into KpnI-EcoRI sites of pBluescript II SK(+) by 3-fragment ligation to form vector pblMSfp2LR, which was verified by restriction digestion and sequencing. The dif-HYG-dif from plasmid pTYdHm [22] was inserted into pblMSfp2LR at the HindIII site to construct pblMsfp2LRH (Fig. 3).

Table 2.DNA primers used in this study.

Fig. 3.The vector constructed in this study for deleting the folP2 gene in Msm. pUC ori: replication region in E. coli; f1(+) ori: origin of replication plasmids for making single-stranded DNA; bla: ampicillin resistance gene; Hyg: hygromycin resistance gene; dif: the recombinases XerCD action site; ArmL: The upstream 884 bp DNA fragment of Msm folP2 gene; ArmR: the downstream 982 bp DNA fragment of Msm folP2 gene.

Construction of the Msm Gene Knockout Mutants

The fragment containing the ArmL–dif-Hyg-dif–ArmR was excised from vector pblMSfp2LRH at the KpnI and EcoRI sites and transformed into induced Msm-TS53 (Msm containing pJV53Ts) competent cells as previously described [18, 22]. The folP2 gene was replaced by the Hyg gene through allelic replacement. To remove the Hyg gene, the mutants were cultured into fresh 7H9 broth w ithout HYG f or 3 d ays. T o remove v ector p JV53Ts, the mutants were cultured into fresh 7H9 broth at 42°C for 3 days, serially diluted 10-fold and plated onto 7H11 plates containing 10% sucrose, and incubated at 42°C for 72 h. The loss of the vector pJV53Ts in the mutants was subsequently confirmed by plating 100 colonies in 7H11 plates containing 10% sucrose and KAN or in 7H11 plates containing 10% sucrose at 42°C.

Successful folP2 gene deletion was confirmed by PCR using primers a, b, c, and d (Table 2) as previously described [22] and Southern blot analysis using the Ms0615–0616 gene-deleted mutant and wild-type Msm as controls. Genomic DNAs from the folP2 and Ms0615–0616 gene knockout mutant and wild-type Msm were purified, and digested with PstI and SpeI restriction enzymes. Similarly, some genomic DNAs were digested with KpnI as positive controls. DNA probes were labeled and band detection was carried out with anti-DIG alkaline phosphatase antibodies and a CDP-star substrate solution in the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). The folP2 and positive probes were generated with primers Msfp2 and Ms0615 (Table 2), respectively.

Vectors for Overexpressing folP1 and folP2 and Transformation in Msm

The E. coli/mycobacterial shuttle vector p60lux [14] and p60luxN (Table 1) carrying the strong promoter hsp60 and HYG resistance marker were used as the parental plasmids for the construction of p60fp1, p60fp2, p60Msfp1, and p60Msfp2. The plasmid p60luxN was constructed by modifying the hsp60 promoter in p60lux to remove the sequence expressing the six amino acids that were usually used to express fused proteins. An NdeI restriction sequence was introduced with the ATG sequence of NdeI as the initiation codon by primers P60-f and P60-r (Table 2).

The folP1 and folP2 genes were amplified using primers Fp1 and Fp2 (Table 2) from Mtb H37Rv, digested with BamHI and HindIII, and then cloned into p60lux digested with the same enzymes to construct vectors p60fp1 and p60fp2, respectively, which were verified by restriction digestion and sequencing. Similarly, the folP1 and folP2 genes were amplified using primer couple Msfp1 and Msfp2 (Table 2) from Msm, digested with NdeI and HindIII, and then cloned into p60luxN cut with the same enzymes to construct vectors p60Msfp1 and p60Msfp2, which were verified by restriction digestion and sequencing. All constructs were transformed into Msm competent cells using standard electroporation at 4°C. The HYG-resistant colonies were isolated and tested individually by PCR using primers Hyg-f and Hyg-r (Table 2).

Drug Susceptibility Testing

The measurement of MICs was adapted from previous works [24, 25]. The MIC values for wild-type and recombinant Msm colonies were determined by culturing on 7H11 agar plates containing 2-fold serial dilutions of SMX (0 to 8 μg/ml) or TMP (0 to 20 μg/ml) for 72 h. The MIC values for each strain were defined as the lowest concentration of SMX or TMP needed to inhibit 99% of bacterial growth.

Growth Analysis of Msm Strains

All Msm strains were grown at 37°C with aeration in 7H9 medium ( 7H9 medium s upplemented with 1 0% O ADC, 0.05% Tween 80, and 0.2% glycerol) in the presence or absence of SMX and TMP, and wild-type Msm was used as a control. Samples were taken and measured at OD600 at 3 h intervals. All assays were performed three times.

 

Results

Construction of Recombinant Strains

To determine the role of folP1 and folP2 genes in the action of SMX and TMP against mycobacteria, we overexpressed the two genes and deleted gene folP2 in Msm. We constructed the vector pblMsfp2LRH for deleting the folP2 gene in Msm. The fragment carrying the upstream and downstream of the Msm folP2 from vector pblMsfp2LRH was transformed into competent Msm-TS53 cells. The subsequent Msm transformants were subjected to allelic exchange to disrupt the folP2 gene on their own chromosomes (Fig. 4). The folP2 gene in the recombinant strains was confirmed replaced by the HYG resistance gene by PCR analysis (Fig. 5) and further confirmation by Southern blot analysis (Fig. 6). We further found that some recombinant strains had even lost the HYG resistance genes (Fig. 5). To overexpress the folP1 and folP2 genes in Msm, we constructed the vectors p60fp1, p60fp2, p60Msfp1, and p60Msfp2 and successfully transformed them into Msm.

Fig. 4.Diagrammatic illustration of introduction of an unmarked folP2 in-frame deletion in Msm.

Fig. 5.Identification of Msm folP2 gene deletion mutants. PCR products in A and C using primers a+b, B using primers c+d, and D using primers c+b. Lane M, DNA marker; lane 1, PCR products with water as a control; lane 2, PCR products from plasmid pblMsfp2LRH, 1,453 bp and/or 353 bp band (A and C), no product (B and D); lane 3, PCR products from Msm-TS53 (Msm containing pJV53Ts); 1,136 bp (A and C), no product (B), 2,301 bp band (D); lanes 4 and 5, PCR products from MsmΔfolP2, 1,453 bp (A and C), 1,350 bp (B), and 1,519 bp (D). A 353 bp fragment was obtained with primers “a” and “b” and a 1,519 bp fragment was obtained with primers “c” and “b” in MsmΔfolP2, showing that the some cells had already lost the dif-ΩHYG-dif cassette during incubation without selection (lanes 4 and 5 in A , C, and D).

Fig. 6.Southern blot analysis of MsmΔfolP2-5H. The folP2 gene as a probe in A and an 804 bp fragment in the MSMEI_0615 gene as a probe in B. Lane 1, Msm; lane 2, MsmΔfolP2-5H; lane 3, Ms0615–0616 gene knockout mutant.

Drug Susceptibility Testing

SMX and TMP susceptibilities of the recombinant and wild-type Msm strains were tested. As shown in Table 3, the MICs of SMX and TMP in wild-type Msm were 0.5 μg/ml and 2.5 μg/ml, respectively. The MICs of SMX and TMP for Msm::p60fp1 and Msm::p60Msfp1 increased by 4- and 2-fold, respectively, compared with wild-type Msm. Interestingly, we obtained a MIC of 0.08 μg/ml for SMX in MsmΔfolP2, which was an 8-fold reduction compared with wild-type Msm, whereas there were no changes in the MICs of TMP. No observable differences were noted in the MICs of SMX for Msm::p60fp2 and Msm::p60Msfp2 compared with wild type. In addition, the MICs of TMP for Msm::p60fp2 increased 2-fold compared with wild-type Msm, whereas there were no changes for Msm::p60Msfp2.

Table 3.MIC: minimum inhibitory concentration; SMX: sulfamethoxazole; TMP: trimethoprim; Msm: Mycobacterium smegmatis mc2155; MsmΔfolP2-5H: Msm deleted folP2 gene without Hyg and pJV53Ts; Msm::p60fp1: Msm containing p60fp1; Msm::p60fp2: Msm containing p60fp2; Msm::p60Msfp1: Msm containing p60Msfp1; and Msm::p60Msfp2: Msm containing p60Msfp2.

Growth Analysis of the Recombinant Msm Strains

We determined the growth curves of wild-type and mutant strains in the presence or absence of SMX and TMP. There were no substantial change on wild-type and mutant strains in the general growth conditions. However, when different concentrations of SMX and TMP were added to challenge their growth, all strains grew more slowly than their respective controls, as similarly observed in the MIC determination (Fig. 7).

Fig. 7.Growth assays for wild-type and recombinant Msm strains in response to SMX and TMP. Growth of wild-type Msm (A), MsmΔfolP2 (B), Msm::p60fp1 (C), Msm::p60fp2 (D), Msm::p60Msfp1 (E), and Msm::p60Msfp2 (F) in 7H9 medium with SMX and TMP. The solid lines with open symbols show the growth curves of strains in different concentrations of SMX; the broken lines with solid symbols show the growth curves of strains in different concentrations of TMP. The concentration is in units of μg/ml.

 

Discussion

Since 2009, several studies [6, 7, 9, 11, 13, 19] have reported that TMP and SMX could be used in the treatment of TB. Although not listed in the World Health Organization guidelines for TB treatment, TMP and SMX could be potential candidates for evaluation against Mtb. TMP-SMX has been in use for prophylactic treatment of patients with HIV in combination with other drugs, and TB co-infection with HIV has been a big challenge in the control and management of TB. Excessive use of antimicrobial drugs, including TMP/SMX, has been known to be widespread in developing countries, causing spread of serious antibiotic resistance strains that hamper the effective treatment of infectious diseases. The establishment of the mechanism of action of SMX and TMP against Mtb, which could contribute to better treatment therapy, is highly needed. Therefore, a clear understanding of the resistance mechanisms of these two drugs in mycobacteria is urgently needed.

In this study, we attempted to explore the role of the folP1 and folP2 genes in the mechanism of TMP and SMX against mycobacteria. Since the folP1 gene is essential in mycobacteria, we just deleted the folP2 gene and overexpressed both genes in Msm and examined whether overexpression of the two genes using a strong promoter in Msm would lead to increased resistance to SMX and TMP. We found that overexpression of Mtb folP1 and Msm folP1 in Msm increased MICs 4- and 2-fold compared with wild type. A similar pattern was observed in the MICs of TMP against folP1 mutants, which indicated that the folP1 gene is evidently associated with the action mechanism of TMP and SMX in mycobacteria. This probably explains why SMX is more effective against Msm than Mtb and the observed TMP resistance in Mtb. The intrinsic differences of gene folP1 in Mtb and Msm could probably be responsible for the difference in SMX and TMP susceptibilities.

We used an improved method to construct an unmarked Msm recombinant using the modified dif-ΩHYG-dif cassette, as it was easy to remove the resistance gene by the XerCD system in mycobacteria. Thus, we obtained unmarked inframe deletion gene knockout strains without polar effect, which is consistent with a recent study by Yang et al. [22]. We observed an 8-fold reduction in MICs of Msm with the deleted folP2 gene compared with wild type, hence confirming that the gene is perhaps associated with the action of SMX against mycobacteria.

We found that overexpression of Mtb folP2 in Msm increased the MICs by 2-fold, whereas no changes were observed for Msm folP2 overexpressed in Msm compared with wild type for both SMX and TMP, respectively. This imperative bacterial inhibition could perhaps explain the role of gene folP2 in SMX susceptibility in Msm and Mtb. Surprisingly, we report the same MICs for Msm deleted folP2 gene and the wild type in TMP. Previous work by Gengenbacher et al. [8] reported that folP2 does not encode a DHPS and therefore cannot act as bypass for gene folP1 in Mtb. However, our results revealed that the folP2 gene has a role in SMX efficacy against mycobacteria, although the mechanism remains unknown (Fig. 1). Computational protein-ligand docking analysis (Fig. 2) revealed the drug binding site of SMX in Mtb to perfectly fit the PABA binding pocket, with the negatively charged oxygen atoms of the sulfonyl group matching the PABA carboxyl group and their common phenyl groups engaging the same hydrophobic pocket in the substructure as similarly observed in Yersinia pestis DHPS (YpDHPS) [23].

In summary, our study provides an alternative explanation of the effects of SMX and TMP and their respective mode of action against Msm, which could be adopted in a TB treatment scheme. Moreover, we suggest the use of folP1 and folP2 as drug targets. A combination of FolP2 inhibitor and SMX (the FolP1 inhibitor) used for TB could have a better treatment outcome.