Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

Previous studies showed that mutation of folC caused decreased expression of the dihydropteroate synthase encoding gene folP2 in Mycobacterium tuberculosis (M. tuberculosis). We speculated that mutation of folC in M. tuberculosis might affect the susceptibility to sulfamethoxazole (SMX). To prove this, 53 clinical isolates with folC mutations were selected and two folC mutants (I43A, I43T) were constructed based on M. tuberculosis H37Ra. The results showed that 42 of the 53 clinical isolates (79.2%) and the two lab-constructed folC mutants were more sensitive to SMX. To probe the mechanism by which folC mutations make M. tuberculosis more sensitive to SMX, folP2 was deleted in H37Ra, and expression levels of folP2 were compared between H37Ra and the two folC mutants. Although deletion of folP2 resulted in increased susceptibility to SMX, no difference in folP2 expression was observed. Furthermore, production levels of para-aminobenzoic acid (pABA) were compared between the folC mutants and the wild-type strain, and results showed that folC mutation resulted in decreased production of pABA. Taken together, we show that folC mutation leads to decreased production of pABA in M. tuberculosis and thus affects its susceptibility to SMX, which broadens our understanding of mechanisms of susceptibilities to antifolates in this bacterium.

. Characteristics of clinical samples used in this study. Other a : samples did not belong to the above five types; Other b : isolates were resistant to one or several MDR-or XDR-relevant drugs, but were not defined as the types above. www.nature.com/scientificreports/ www.nature.com/scientificreports/ to PAS on L-J medium was subsequently confirmed by the minimum inhibitory concentration (MIC) method on 7H10 medium (> 2 μg/ml) ( Table 3). The thyA, ribD, and folC genes of all 95 isolates were sequenced. No mutations were observed in all three genes in H37Rv, five PAS-sensitive isolates, and two PAS-resistant isolates. The remaining 87 PAS-resistant isolates harbored mutations in at least one of these three genes. Most mutations are nucleotide substitutions, and only one base insertion was found (in folC) ( Table 3) For mutations in folC, the leading mutation in this gene was at nucleotide position 128 (33 of 53 isolates). Two isolates with mutations at nucleotide position 128 switched T to G, and the other 31 isolates converted T to C (Table 3).

Most PAS-resistant clinical isolates with folC mutations were sensitive to SMX. To test SMX
susceptibilities, H37Rv, five PAS-sensitive strains, and two PAS-resistant strains with no mutation in thyA, ribD, and folC were used as control strains. SMX MICs of these eight strains were all determined to be 50 μg/ml (Table 3). PAS-resistant strains with either thyA or ribD mutations were not more sensitive to SMX. On the contrary, among the 53 PAS-resistant strains with folC mutations, 42 (79.2%) were more sensitive to SMX compared with the eight control strains (Tables 2 and 3).
M. tuberculosis H37Ra ΔfolP2, M. tuberculosis H37Ra ΔfolC pMV361::folC(I43T), and M. tuberculosis H37Ra ΔfolC pMV361::folC(I43A) were more sensitive to SMX than the parental strain. SMX susceptibilities were tested in two lab-constructed folC mutants 21 and M. tuberculosis H37Ra ΔfolP2. The results showed that, compared with their parental strain, all three mutants were more sensitive to SMX (Table 4). In addition, the results of the killing curve assay showed that the two folC mutants were more susceptible to SMX than their parental strain ( Fig. 1). www.nature.com/scientificreports/ FolC mutations did not affect the expression of folP2. Results of the quantitative real-time PCR assay showed that no difference in the folP2 expression level could be observed between M. tuberculosis H37Ra and the two folC mutants, which was confirmed by subsequent western blot analysis (Figs. 2 and 3). To determine production levels of pABA in the three different H37Ra strains, an Escherichia coli (E. coli) W3110 ΔpabB mutant was used as previously described 29 . When E. coli W3110 ΔpabB was cultured in E minimal medium plus culture filtrates from different H37Ra strains, there was obviously less growth when culture filtrates from the two folC mutants were used (Fig. 4), suggesting a decreased production of pABA.  www.nature.com/scientificreports/

Discussion
A huge obstacle in defeating TB is the increasing resistance to scanty anti-TB drugs. This predicament reinforced our interest in exploring the usable medicines in the approved drugs treasure chest. Antifolates, due to their toxicity in folate biosynthesis, which is vital and greatly different between human cells and bacteria, are potentially important choices to treat MDR TB. In fact, after being put on the shelf for several decades, PAS was reintroduced in the 1990s for treating MDR TB 30 . However, resistance to PAS appeared in clinical M. tuberculosis isolates in the early 2000s. Although SXT has not been tried to treat TB clinically, attempts have been made to make it an option for treating drug-resistant TB [31][32][33][34] . In this context, better use of antifolates against TB has gained importance, and further in-depth studies on the mechanisms of susceptibility and resistance of antifolates are required. Very recently, researchers found that in PAS-resistant clinical isolates of M. tuberculosis with folC mutation, protein expression levels of folP2 were significantly lower than in strains with no folC mutation 28 . Though FolP2 of M. tuberculosis has been predicted to be defective in dihydropteroate synthesis activity 27 , deletion of the folP2 gene in M. smegmatis resulted in increased susceptibility to SMX 26 . We also knocked out the folP2 gene in M. tuberculosis H37Ra, and found that the resulting ΔfolP2 mutant was more sensitive to SMX. Therefore, we speculated that decreased expression of folP2 caused by folC mutation might lead to increased susceptibility to SMX.
As expected, most of the PAS-resistant isolates with mutations in the folC gene showed increased susceptibility to SMX. In addition, two lab-constructed folC mutants were also more sensitive to SMX. These data suggest that mutation of folC did lead to increased susceptibility to SMX in M. tuberculosis.
We noted that 11/53 (20.8%) PAS-resistant isolates with folC mutations were not more sensitive to SMX, including isolates with I43A or I43T mutations. All PAS-resistant isolates used in this study were MDR/XDR clinical isolates obtained from different patients, suggesting the complexity of the genetic backgrounds of those isolates. A previous study using transmission electron microscopy showed that the cell walls of MDR and XDR strains were thicker than those of the susceptible M. tuberculosis isolates 35 , indicating another possible explanation.
However, subsequent quantitative real-time PCR and western blot analysis showed that folC mutations did not affect the expression of folP2, suggesting that the increased susceptibilities of the folC mutants to SMX were not caused by decreased expression of folP2.
With respect to the mechanisms of action, SMX shares one thing with PAS: both drugs compete with pABA. As a result, deficiency in pABA biosynthesis usually leads to increased susceptibility to both drugs 29,36 . We thus speculated that mutation of folC might lead to decreased production of pABA and hence affect the susceptibility to SMX. Subsequent comparison of pABA production levels between the two lab-constructed folC mutants and their parental strain confirmed this hypothesis.
To the best of our knowledge, this is the first study on the interaction of the two antifolates in the treatment of MDR or XDR TB clinical isolates. We found that the folC mutation in M. tuberculosis leads to decreased production of pABA and hence increases sensitivity to SMX. Since our previous data showed that a small proportion (~ 9%) of the MDR strains had a mutation in folC 21 , it would be interesting to test the efficacy of SMX or SXT against those MDR strains in vivo. The regulation of folate metabolism is still obscure in M. tuberculosis, and our pioneering observations provide new evidence to guide future research. (Table 1)  www.nature.com/scientificreports/ mycobacterium species identification and (ii) drug susceptibility testing (DST) using the proportion method on L-J solid medium (Encode, Zhuhai, China), where MDR was defined as resistance to at least isoniazid and rifampicin, and XDR was defined as MDR plus resistance to any fluoroquinolones (ofloxacin, levofloxacin or moxifloxacin) and at least one injectable drug (amikacin or capreomycin). Mycobacterium species identification was performed based on (i) sequence polymorphisms in 16S rRNA, hsp65, and rpoB 38 and (ii) the results of bacterial growth on L-J solid medium containing 5 μg/ml TCH and 500 μg/ml PNB. MDR and XDR isolates were chosen for preliminary screening of PAS resistance using the same method and medium as described above. For DSTs of PAS or SMX on Middlebrook 7H10 solid medium (Difco, Becton Dickinson, Sparks, MD, USA), the two drugs (purchased from Merck, Darmstadt, Germany) were dissolved in deionized water and dimethyl sulfoxide (Merck) at concentrations of 10 mg/ml and 60 mg/ml, respectively. The drugs were frozen at − 20 °C after sterilization. The critical concentration of PAS was 1 μg/ml on L-J medium and 2 μg/ml on 7H10 medium according to policy guidelines on DST for second-line anti-TB drugs 39 . Isolates were frozen in 25% glycerol at − 70 °C until use. Our study was conducted in accordance with the Declaration of Helsinki. The institutional review board of Chongqing Public Health Medical Center approved this study and waived the requirement for written informed consent. The institutional review board waived the need for informed consent because all patients' data were analyzed in anonymity and no additional informed consent was required.

Construction of H37Ra ΔfolP2.
A modified strategy for specialized transduction was used to construct the M. tuberculosis H37Ra ΔfolP2 mutant 40 . Genomic regions flanking folP2, 824 bp upstream (a region containing MRA_1215) and 827 bp downstream (a region containing MRA_1217), were amplified by PCR. The primers used for the amplification of the upstream region of folP2 were folP2koLFP and folP2koLRP, and those for the downstream region were folP2koRFP and folP2koRRP. The recombinant plasmid p0004s-L + R was constructed by inserting the Van91I-digested PCR products into Van91I-digested plasmid p0004s. Then, p0004s-L + R was digested with PacI and ligated into the PacI-digested shuttle phasmid vector phAE159. After ligation, the recombinant cosmid phAE159-p0004s-L + R was transduced into E. coli HB101 in an in vitro λ-packaging reaction (MaxPlax Lambda Packaging Extracts, Epicentre Biotechnologies, Madison, WI, USA). The phasmid DNA prepared from confirmed selected hygromycin-resistant transductant was electroporated into M. smegmatis mc 2 155 to generate the specialized transducing phage. As described in a previous study 40 , the transducing phage at the most efficient titer was used to infect H37Ra at a multiplicity of infection of 10. Successful transduction of H37Ra was confirmed by comparing the size of the PCR-amplified products of hygromycin-resistant colonies with the wild-type H37Ra using primers folP2LYZ and folP2RYZ (Table S1).
Quantitative real-time PCR assays. An RNeasy Mini kit (Qiagen, Germany) was used to extract total RNA, and a ReverTra Ace qPCR kit (TOYOBO, Osaka, Japan) was used to synthesize cDNA. All kits were used according to the manufacturers' instructions. Gene expression levels were quantified using quantitative realtime PCR analysis on a 7900 HT Sequence Detection System (ABI, Foster City, CA, USA) with ABI Power SYBR www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.