Binding pocket alterations in dihydrofolate synthase confer resistance to para-aminosalicylic acid in clinical isolates of Mycobacterium tuberculosis

Article abstract of DOI:10.1128/AAC.01775-13

The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor paraaminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance in M. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H₂Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug. Copyright

Full text of DOI:10.1128/AAC.01775-13

Binding Pocket Alterations in Dihydrofolate Synthase Confer
Resistance to para-Aminosalicylic Acid in Clinical Isolates of
Mycobacterium tuberculosis
Fei Zhao,a,b Xu-De Wang,a Luke N. Erber,c Ming Luo,d Ai-zhen Guo,b Shan-shan Yang,a Jing Gu,a Breanna J. Turman,c Yun-rong Gao,e
Dong-fang Li,f,g Zong-qiang Cui,a Zhi-ping Zhang,a Li-jun Bi,e Anthony D. Baughn,c Xian-En Zhang,e Jiao-Yu Denga
Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Chinaa; State Key Laboratory of
Agricultural Microbiology, College of Life Science and Technology, Huazhong Agriculture University, Wuhan, Chinab; Department of Microbiology, University of
Minnesota, Minneapolis, Minnesota, USAc; Chongqing Institute of Tuberculosis Prevention and Treatment, Chongqing, Chinad; National Laboratory of
Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, Chinae; Shenzhen Key Laboratory of Unknown Pathogen Identification, Shenzhen,
Chinaf; BGI-Tianjin, Tianjin, Chinag
The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in
the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor para-
aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and dis-
rupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme
needed for the bioactivation of PAS, causes PAS resistance in M. tuberculosis strain H37Rv. Here, we demonstrate that various
missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC)
confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant
M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in
PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also
abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bio-
activation, the introduction of a wild-type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of
this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuber-
culosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.
wo billion people worldwide are latently infected with Myco- demonstrated that PAS is a poor inhibitor of DHPS activity (6).
T
bacterium tuberculosis (1). Each year, this immense reservoir of
Recently, PAS has been shown to function as an alternate substrate
for FolP1 with the formation of an analog of dihydropteroate
(H2Pte), hydroxy dihydropteroate (H2PtePAS), which can be glu-
taminated by dihydrofolate synthase (DHFS/FolC) to form the
dihydrofolate (H2Pte-Glu) analog hydroxy dihydrofolate
(H2PtePAS-Glu) (7). H2PtePAS-Glu can then inhibit the activity
of dihydrofolate reductase (DfrA), resulting in the growth arrest
of M. tuberculosis due to a limitation in its ability to synthesize
tetrahydrofolate (8). Consistent with this model for the PAS mode
of action, overexpression of an alternate enzyme with dihydrofo-
late reductase activity, RibD, confers resistance to this drug (8).
Interestingly, loss-of-function mutations in thyA, which encodes a
folate-dependent thymidylate synthase, have been associated with
PAS resistance by an undefined mechanism in both laboratory
and clinical isolates of M. tuberculosis (9, 10). As only one-third of
infected individuals yields 9 million new cases of active tubercu-
losis (TB), resulting in the transmission of tubercle bacilli to tens
of millions of naive individuals and 2 million TB-related deaths
(1). The emergent spread of multidrug-resistant (MDR) (resistant
to isoniazid and rifampin) and extensively drug-resistant (XDR)
(resistant to isoniazid, rifampin, quinolones, and any second-line
injectable drug) strains of M. tuberculosis now threatens the effi-
cacy of existing antitubercular therapies (2, 3). In high-TB-burden
countries, the reported case rates of MDR-TB doubled between
2009 and 2011, and to date, XDR-TB has been reported in 84
different countries (3). Defining the underlying molecular and
biochemical mechanisms of TB drug resistance is critical to facil-
itate the development of novel diagnostic tools and to guide tar-
get-based discovery of novel therapeutic agents.
para-Aminosalicylic acid (PAS) was first reported by Lehmann
in 1946 (4) and followed streptomycin as the second therapeutic
agent used in the treatment of TB. Despite nearly 70 years of clin-
ical use of PAS, the mechanistic basis for the susceptibility and
resistance of M. tuberculosis to this drug has not been fully defined.
Based on the structural similarity of PAS to the folate precursor
para-aminobenzoic acid (PABA) and the observation that the an-
titubercular action of PAS can be fully antagonized by exogenous
PABA (5), PAS was initially thought to interfere with folate syn-
thesis through the inhibition of dihydropteroate synthase activity
(DHPS/FolP1), similar to the action of many sulfonamides. How-
ever, subsequent enzymology with purified recombinant FolP1
Received 16 August 2013 Returned for modification 7 October 2013
Accepted 12 December 2013
Published ahead of print 23 December 2013
Address correspondence to Jiao-Yu Deng, dengjy@wh.iov.cn., or Xian-En Zhang,
x.zhang@wh.iov.cn.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.01775-13.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01775-13
March 2014 Volume 58 Number 3
Antimicrobial Agents and Chemotherapy p. 1479–1487
aac.asm.org 1479

Zhao et al.
PAS-resistant clinical isolates have been found to harbor thyA mu- RTA1.12) was used to process the raw fluorescent images and call se-
quences. An average of 520 Mb of data for each sample was generated,
representing Ͼ100-fold sequence coverage per genome. High-quality
paired-end reads were mapped to the reference H37Ra genome (GenBank
NC_009525) using SOAP2 (16) or Geneious 6.0 (Biomatters Ltd., Auck-
land, New Zealand). Single nucleotide polymorphisms were indepen-
dently verified by the sequencing of PCR amplicons of the respective loci.
Genomic DNAs of PAS-resistant strains that were not assessed by full-
genome sequencing, including genomic DNA from clinical M. tuberculo-
sis isolates (10 drug-susceptible and 85 MDR isolates obtained from the
Chongqing Institute of Tuberculosis Prevention and Treatment), were
assessed by targeted sequencing of PCR amplicons of loci associated with
PAS resistance. The primers used in the targeted sequencing are described
tations, and not all thyA mutations are associated with PAS resis-
tance (10–12), additional PAS resistance alleles have yet to be
identified. Very recently, Zheng et al. (8) described a spontaneous
PAS-resistant isolate of M. tuberculosis H37Rv harboring a substi-
tution mutation in folC that was responsible for PAS resistance,
suggesting that PAS resistance can also be mediated by alterations
in dihydrofolate synthase.
To better understand the mechanistic basis for PAS suscepti-
bility and resistance in M. tuberculosis, we characterized several
PAS-resistant laboratory and clinical isolates using genome-wide-
and targeted sequencing-based methods. Using this approach, we
identified a number of single nucleotide missense polymorphisms in Table S1 in the supplemental material.
Genetic manipulation of mycobacterial strains. FolC was amplified
clustered within the coding sequence for the H2Pte binding pocket
of FolC that were associated with PAS resistance. This association
was confirmed by complementation and gene replacement studies
in both laboratory and clinical isolates. Finally, biochemical stud-
ies were performed on purified recombinant wild-type and vari-
ant forms of FolC to assess the consequences of changes in the
H2Pte binding pocket for DHFS activity using H2Pte and
H2PtePAS as substrates. Based on these studies, a novel mecha-
nism of PAS resistance in clinical isolates of M. tuberculosis was
confirmed whereby variant forms of FolC show diminished acti-
vation of H2PtePAS to H2PtePAS-Glu. This work will be beneficial
in the further development of molecular diagnostic tools for as-
sessing PAS resistance, and it also provides new insights into stud-
ies of mechanisms of PAS action.
from wild-type M. tuberculosis H37Ra genomic DNA using PCR with the
primer pair FolCBamHIFP and FolCHindIIIRP (see Table S1 in the sup-
plemental material). The purified amplicon was digested with BamHI and
HindIII and ligated to BamHI-HindIII-cut pMV261, yielding pMV261-
FolC. PAS-resistant M. tuberculosis and M. bovis BCG strains were trans-
formed with sequence-confirmed pMV261-FolC according to the estab-
lished protocols (14) and plated on 7H10 medium containing kanamycin.
After 3 weeks of incubation at 37°C, single colonies were purified and
liquid cultures were prepared for the extraction of genomic DNA and
determination of PAS MICs. The presence of pMV261-FolC was verified
by PCR amplification using primers specific for pMV261 (JDFP and
JDRP) (see Table S1 in the supplemental material).
A modified strategy for phage-mediated allelic exchange (17) was used
to construct M. tuberculosis H37Ra derivatives containing the clinically
derived folC alleles I43A and I43T. Briefly, folC alleles were cloned into
pMV361 and used to transform M. tuberculosis H37Ra. The native copy of
folC was deleted by specialized transduction using phAE159 containing a
⌬folC allelic exchange substrate (see Fig. S1 in the supplemental material).
The deletion of the native copy of folC was verified by PCR as described in
Fig. S1.
Purification of recombinant FolP1 and FolC. FolP1 was amplified
from M. tuberculosis H37Ra genomic DNA using the primers FolPNcoIFP
and FolPHindIIIRP (see Table S1 in the supplemental material) and was
cloned into pET28a, yielding pET28a-FolP1 to introduce an N-terminal
hexahistidine tag. Following sequence verification, pET28a-FolP1 was
used to transform E. coli BL21(DE3). The cells were grown at 37°C in
Luria-Bertani broth to an OD600 of ϳ0.6, isopropyl-␤-D-thiogalactopy-
ranoside (IPTG) was added to 0.2 mM, and the cells were incubated at
16°C overnight. Following induction, the bacterial cells were harvested by
centrifugation, resuspended in 50 mM Tris-HCl, 0.5 M NaCl, 20 mM
imidazole (pH 8.0), disrupted by sonication, and clarified by centrifuga-
tion. Supernatant fluid was mixed with prewashed nickel-nitrilotriacetic
acid HisTrap HP affinity resin (GE Healthcare) at 4°C overnight. Nonspe-
cifically bound protein was removed by washing the resin with 50 mM
Tris-HCl, 0.5 M NaCl, and 60 mM imidazole (pH 8.0). Recombinant
FolP1 was eluted with 50 mM Tris-HCl, 0.5 M NaCl, and 400 mM imida-
zole (pH 8.0), and analyzed by SDS-PAGE.
Wild-type and mutant (I43T, R49W, E153A, and A183P mutations)
folC genes were amplified from genomic DNA of the respective M. tuber-
culosis strains using the primers FolCBamhIFP and FolCHindIIIRP (see
Table S1 in the supplemental material). The amplicons were digested with
BamHI and HindIII and cloned into pMAL-c2X (New England BioLabs)
to introduce a maltose binding protein (MBP) tag linked by a factor Xa
cleavage site. After sequence verification, plasmids were used to transform
E. coli BL21(DE3). The cells were grown at 37°C in Luria-Bertani broth to
an OD600 of ϳ1.0, IPTG was added to 0.1 mM, and the cells were incu-
bated at 16°C overnight. The bacterial cells were harvested by centrifuga-
tion, resuspended in column buffer (CB) (50 mM Tris·HCl, 0.5 M NaCl
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. Clinical M. tubercu-
losis strains, M. tuberculosis strain H37Ra, Mycobacterium bovis BCG, and
their derivatives were cultured in Middlebrook 7H9 broth (Difco) supple-
mented with 10% (vol/vol) oleic acid-albumin-dextrose-catalase
(OADC) (Difco), 0.5% (vol/vol) glycerol, and 0.05% (vol/vol) Tween 80
(Sigma) or on 7H10 agar medium (Difco) supplemented with 10% (vol/
vol) OADC and 0.5% (vol/vol) glycerol. Mycobacterium smegmatis strain
mc2155 was grown with Middlebrook 7H9 broth or 7H10 agar medium
(Difco) supplemented with 0.5% (vol/vol) glycerol, and 0.05% (vol/vol)
Tween 80 was added to each culture. Escherichia coli strains HB101 and
BL21(DE3) were grown with Luria-Bertani medium (Difco). Plasmids
pMAL-c2X (New England BioLabs), pET-28a (Novagen), pMV361, and
pMV261 were used for the construction of expression plasmids. Antibi-
otics were added to the growth media as necessary: hygromycin at 75
␮g/ml for mycobacteria and 150 ␮g/ml for E. coli, ampicillin at 100 ␮g/ml
for E. coli, and kanamycin at 25 ␮g/ml for mycobacteria and 50 ␮g/ml for
E. coli.
IdentificationofPASresistancealleles. M. tuberculosisH37RaandM.
bovis BCG were grown in supplemented 7H9 medium to an optical den-
sity at 600 nm (OD600) of ϳ0.8, collected by centrifugation (4,000 ϫ g, 15
min, at room temperature), and resuspended in fresh medium at a con-
centration of 108 CFU/ml. Subsequently, 200 ␮l of concentrated bacterial
cells was spread onto supplemented 7H10 agar plates containing 1, 4, and
16 ␮g/ml PAS and incubated at 37°C for 3 weeks for the selection of
PAS-resistant mutants. Single colonies were purified, and liquid cultures
were grown for the extraction of genomic DNA and determination of PAS
MICs (with MIC defined as the lowest concentration of compound re-
quired to inhibit 99% of bacterial growth over 3 weeks of incubation at
37°C) according to the established protocols (13–15). PCR-free DNA li-
braries for full-genome sequencing were constructed from the genomic
DNA of selected strains using a TruSeq DNA kit (Illumina, Inc.) accord-
ing to the manufacturer’s protocol, with an average insert size of 350 bp
for each sample, and these were assayed using the Illumina MiSeq or [pH 8.0]), and disrupted by sonication. Recombinant FolC proteins were
HiSeq 2000 sequencing systems. A base-calling pipeline (version HC1.4/ first purified over an amylose resin column (product no. E8021, New
1480 aac.asm.org
Antimicrobial Agents and Chemotherapy

FolC-Mediated PAS Resistance in M. tuberculosis
England BioLabs). In brief, the column was prewashed with 10 volumes of B (acetonitrile plus 0.1% acetic acid) to 73% buffer A and 27% buffer B for
CB, crude extract was loaded, the column was washed with 10 volumes of 10 min, at a flow rate of 0.3 ml/min. Substrate consumption and product
formation were determined by HPLC-MS as described above.
CB, and recombinant MBP-tagged FolC was eluted with CB containing 10
mM maltose. To remove the MBP tag, the samples were incubated with
factor Xa at 23°C for 6 h in 20 mM HEPES (pH 8.0), 100 mM NaCl, and 2
mM CaCl2. Finally, the cleavage mixtures were dialyzed against 50 mM
phosphate buffer (PB) (pH 8.0). The samples were loaded on a HiTrap
DEAE FF column (GE Healthcare), and a step gradient from 50 mM to 1
M NaCl in PB was applied to elute FolC. The fractions were analyzed by
SDS-PAGE. Recombinant FolC was found to elute with 300 mM NaCl.
Enzymatic assays. Dihydrofolate synthase activities of wild-type FolC
and its variants using H2Pte (Schircks Laboratories) as a substrate were
measured as previously described (7). The standard reactions consisted of
1 ␮M FolC protein, 110 mM Tris-55 mM glycine (pH 9.5), 11 mM MgCl2,
5 mM dithiothreitol (DTT), 200 mM KCl, 50 mM NaCl, 10% glycerol, 1
mM L-glutamate, 5 mM ATP, and 100 ␮M H2Pte. The reactions were
carried out at 37°C for 1 h in triplicate and were stopped by the addition of
EDTA to a final concentration of 50 mM. The reaction mixtures were
injected onto a Phenomenex Luna 3-␮m C18 100-Å liquid chromatogra-
phy (LC) column (50 by 2 mm) (2) in an UltiMate 3000 ultrahigh-per-
formance liquid chromatography (UHPLC) system (Thermo Fisher Sci-
entific). The samples were eluted with a gradient from 95% buffer A (H2O
plus 0.1% acetic acid) and 5% buffer B (acetonitrile plus 0.1% acetic acid)
to 5% buffer A and 95% buffer B for 15 min, at a flow rate of 0.3 ml/min.
The peak of H2Pte-Glu was measured by UV absorbance (A284). The peak
areas of H2Pte-Glu were converted to concentrations by comparison
against an H2Pte-Glu analytical standard (Schircks Laboratories).
The dihydrofolate synthase activities of wild-type FolC and its mu-
tants using H2PtePAS as a substrate were also measured according to the
above method. H2PtePAS was enzymatically synthesized as previously
described (7). Briefly, a reaction mixture containing 1.2 ␮M FolP1, 40
mM Tris-20 mM glycine (pH 9.5), 5 mM MgCl2, 1 mM DTT, 200 mM
NaCl, appropriate amounts of 6-hydroxymethyl-7,8-pterin pyrophos-
phate (H2PtePP), and 250 ␮M PAS was incubated at 37°C for 1 h. Next,
FolP1 was removed by passing through a 10-kDa Microcon centrifugal
filter, and the mixture was subsequently used as a substrate for FolC.
H2PtePAS and H2PtePAS-Glu were identified by UHPLC as described
above and confirmed by electrospray ionization/mass spectrometry (ESI/
MS) with an in-line LCQ Fleet ion trap mass spectrometer (Thermo
Fisher Scientific). The ESI/MS working parameters were as follows: 4 kV
capillary voltage, 300°C heat block temperature for analysis, and the ni-
trogen drying and nebulizer gases were set at 5 liter/min. All MS data were
acquired in a scan range between 100 and 1,000 m/z under the negative
ionization mode.
To compare the catalytic rate of wild-type FolC for H2PtePAS and
H2Pte, these two compounds were enzymatically synthesized as described
above. The reaction mixture contained 1.2 ␮M FolP1, 40 mM Tris-20 mM
glycine (pH 9.5), 5 mM MgCl2, 1 mM DTT, 200 mM NaCl, appropriate
amounts of H2PtePP, and 250 ␮M PAS or PABA. The reaction mixture
was incubated at 37°C until no increment of product accumulation was
detected by high-performance liquid chromatography (HPLC). FolP1
was removed by passing through a 10-kDa Microcon centrifugal filter,
and 325 ␮l of the remaining reaction mixture was used as a substrate for
FolC. The concentration of H2Pte in the FolP1 reaction mixture was esti-
mated by a comparison of the peak area of H2Pte in the mixture with that
of the standard compound, and the concentration of H2PtePAS was as-
sumed to be the same as that of H2Pte since the same amount of H2PtePP
was consumed. The FolC reaction mixture contained 0.5 ␮M FolC pro-
tein, 2.5 mM ATP, and 0.5 mM L-glutamate in 100 mM Tris-50 mM
glycine (pH 9.5), 10 mM MgCl2, 5 mM DTT, 100 mM KCl, 50 mM NaCl,
10% glycerol, and about 20 ␮M H2Pte or H2PtePAS. After incubation at
37°C for the proper times, the mixture was injected onto a Phenomenex
Luna 3-␮m C18 100-Å LC column (75 by 3 mm) (2) in an UltiMate 3000
UHPLC system linked with the LCQ Fleet. The samples were eluted with
Antagonism of PAS activity by H2Pte. M. tuberculosis H37Ra was
grown in 7H9 medium to mid-log phase (OD600, ϳ0.5), collected, and
resuspended in fresh 7H9 medium to a density of OD600 of ϳ0.1. Next,
200 ␮l of bacterial cells was aliquoted into 96-well plates. PAS (0.1 ␮g/ml)
was added (except in the no-drug controls) in combination with different
concentrations of H2Pte (0, 0.1, 0.3, 1, 3, and 10 ␮g/ml). The plates were
incubated at 37°C without shaking. An XTT [2,3-bis(2-methoxy-4-nitro-
5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] reduction as-
say was performed to determine the survival of bacterial cells at 36 h, as
previously described (18). The MICs of PAS for the wild-type M. tubercu-
losis H37Ra in the presence of different concentrations of H2Pte (3 and 10
␮g/ml) were also determined.
RESULTS
CharacterizationofmutationsassociatedwithPASresistancein
M. tuberculosis. In order to characterize novel alleles associated
with PAS resistance, M. tuberculosis H37Ra and M. bovis BCG
were plated on medium containing PAS at concentrations of 1, 4,
and 16 ␮g/ml. For M. tuberculosis H37Ra, colonies arose at a fre-
quency of 5 ϫ 10Ϫ7 on 1 ␮g/ml PAS, 2 ϫ 10Ϫ7 on 4 ␮g/ml PAS,
and 2 ϫ 10Ϫ8 on 16 ␮g/ml PAS. For M. bovis BCG, colonies arose
at a frequency of 3 ϫ 10Ϫ7 on 1 ␮g/ml PAS, 2 ϫ 10Ϫ7 on 4 ␮g/ml
PAS, and Ͻ2 ϫ 10Ϫ8 on 16 ␮g/ml PAS. Forty-three M. tuberculosis
H37Ra PAS-resistant (PASr) isolates and 23 M. bovis BCG PASr
isolates were chosen for further characterization (Table 1). Ge-
nome sequencing was performed on 16 of the spontaneous PASr
isolates of M. tuberculosis H37Ra and the parental strain. Compar-
ative genomic analysis revealed that 11 strains harbored an A-to-C
transversion within codon 153 of folC (GAG to GCG, resulting in
E153A). In addition, two other strains harbored an A-to-G tran-
sition at codon 73 of folC (AAC to AGC, resulting in N73S). Tar-
geted sequencing of folC, folP1, and dfrA, ribD, and thyA (previ-
ously linked to PAS resistance) (9–12) was performed on the
remaining 50 spontaneous PASr mutants. As shown in Table 1, an
additional 32 strains had mutations within the folC gene, and 6
strains were found to have mutations within the thyA gene. No
strains were found to harbor mutations in dfrA or ribD. Mutations
were not identified in 15 of the strains that were analyzed by tar-
geted sequencing, indicating the existence of additional PAS resis-
tance alleles (Table 1).
From whole-genome and targeted sequencing, 67% (29/43) of
the M. tuberculosis H37Ra spontaneous PASr isolates and 70%
(16/23) of the M. bovis BCG spontaneous PASr isolates had muta-
tions in folC, representing nine unique missense mutations
(R49W, N73S, S150R, S150G, F152S, F152L, E153A, E153G, and
A183P) that map to six positions that are invariant among myco-
bacterial FolC orthologs (see Fig. S2A in the supplemental mate-
rial). Alignment of the protein sequences revealed that these six
residues are also very highly conserved in FolC throughout pro-
karyotic species (see Fig. S2A and B). The crystal structures of M.
tuberculosis FolC and those from other phylogenetically distinct
species (Lactobacillus casei, Yersinia pestis, Thermotoga maritima,
and E. coli) have been determined and show a remarkable degree
of structural conservation (19–23). As shown in Fig. 1A and B, all
of the identified folC mutations, except N73, were located within
the ␣1-␣2/␣4-␣5 helix bundle of the FolC N terminus. This four-
helix bundle was mainly linked through hydrophobic interac-
a gradient from 95% buffer A (H2O plus 0.1% acetic acid) and 5% buffer tions, and two residues (A183 and F152) are involved in the link-
March 2014 Volume 58 Number 3
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Zhao et al.
TABLE 1 Mutations associated with PAS resistance in laboratory and clinical isolates of the M. tuberculosis complex
folC codon
thyA codon
(mutation)
Strain(s)
PAS resistanceb
No. of isolates
(mutation)c
M. tuberculosis H37Ra
ZF-1,a ZF-6, ZF-12, ZF-18,a ZF-26,a LNE-34,
R
R
8
WT
WT
WT
LNE-38, LNE-99
ZF-7,a ZF-8,a ZF-10,a ZF-11,a ZF-14,a ZF-15,a
18
GAG¡GCG (E153A)
ZF-16, ZF-19, ZF-20,a ZF-21,a ZF-22,a ZF-23,
ZF-24,a ZF-25,a LNE-1, LNE-7, LNE-10,
LNE-12
ZF-13, ZF-17, LNE-6
ZF-4, ZF-9
ZF-5
ZF-2
ZF-3
LNE-3,a LNE-32,a LNE-39
LNE-47, BJT-77
LNE-48, LNE-93
LNE-51
R
R
R
R
R
R
R
R
R
R
3
2
1
1
1
3
2
2
1
1
GAG¡GGG (E153G)
TTC¡TCC (F152S)
TTC¡CTC (F152L)
AGC¡AGG (S150R)
AGC¡GGC (S150G)
AAC¡AGC (N73S)
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
AAC¡AAA (N134K)
CAT¡AAT (H147N)
ACC¡ATC (T22I)
GGG¡AGG (G15R)
LNE-54
M. bovis BCG
ZF-33, ZF-42, ZF-44, ZF-46, ZF-47, ZF-48, ZF-49
ZF-27, ZF-29, ZF-30, ZF-31, ZF-35, ZF-36, ZF-37,
ZF-38, ZF-39, ZF-40, ZF-41, ZF-43, ZF-45
ZF-28, ZF-32, ZF-34
R
R
7
13
WT
WT
WT
GCC¡CCC (A183P)
R
3
CGG¡TGG (R49W)
WT
Non-MDR M. tuberculosis clinical isolates
MDR M. tuberculosis clinical isolates
Q274, 501063
Q449, 1314
Q36
Q296
504036
Q93
ND
ND
R
R
R
TBD
TBD
TBD
10
77
2
2
1
1
1
1
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
GAG¡GGG (E40G)
ATC¡GCT (I43T)
ATC¡ACC (I43A)
GGC¡AGC (G111S)
GAC¡GCC (D112A)
CGG¡TGG (R410W)
a Strains analyzed by whole-genome sequencing.
b R, resistant (MIC Ͼ 0.125 ␮g/ml PAS); ND, not determined; TBD, to be determined.
c WT, identical to wild-type gene.
age of hydrophobic networks between ␣1 and ␣5. The ␣1-␣2 loop
and the ␣4-␣5 loop form an intersection, which is locked by two
pairs of salt bridges, one pair between R49 and E153 and the other
pair between R37 and D135. The first pair of salt bridges is respon-
sible for the linkage between ␣1 and ␣5, and the second pair is
responsible for the linkage between ␣2 and ␣4.
FolC H2Pte binding pocket mutations are associated with
PAS resistance in M. tuberculosis clinical isolates. To determine
the clinical significance of folC mutations for PAS resistance, 95
recent clinical isolates of M. tuberculosis (10 drug-susceptible
strains and 85 MDR strains) were assessed for folC mutations by
targeted DNA sequencing (Table 1). As expected, all folC alleles
from the drug-susceptible strains showed full sequence conserva-
tion with H37Rv folC. Among the 85 MDR strains, 8 had missense
mutations within folC corresponding to E40G (two isolates),
I43A, I43T (two isolates), D111A, G112S, and R410W (Table 1).
Of these mutations, E40G, I43A, and I43T mapped to the H2Pte
binding pocket of FolC (Fig. 1). Interestingly, when PAS suscep-
tibility testing was performed for the 8 folC mutant strains (Q36,
Q93, Q274, Q296, Q449, 1314, 501063, and 504036), those with
the E40G, I43A, and I43T mutations (Q36, Q274, Q449, 1314, and
501063) were found to be resistant (Table 1). Thus, H2Pte binding
pocket mutations are associated with PAS resistance in clinical
FIG 1 (A) Ribbon diagram of the N-domain FolC (PDB code 2VOS). The isolates of M. tuberculosis.
Wild-type folC restores PAS susceptibility in PASr M. tuber-
culosis complex strains. To assess whether the identified folC
H2Pte binding pocket mutations were associated with PAS resis-
tance, a plasmid carrying the wild-type folC gene from M. tuber-
locations of six mutations are shown as thick colored bands (Ile43, Arg49,
Ser150, Phe152, Glu153, and Ala183), while the ADP is shown as fine bands.
(B) As shown, all the mutated residues either reside in the H2Pte binding loop
(␣1-␣2, residues 36 to 50) or are situated in the ␣4-␣5 loop. The residue Glu40
was not included in the model.
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FolC-Mediated PAS Resistance in M. tuberculosis
TABLE 2 Wild-type folC confers PAS susceptibility in laboratory and
clinical PAS-resistant M. tuberculosis complex strains
MICs (␮g/ml):
Following
Following
transformation with
For PAS with pMV261 pMV261-folC
0.125
transformation
Strain(s)
M. tuberculosis H37Ra
ZF-7, ZF-8, ZF-10, LNE-1
(folC E153A)
0.125
2–4
0.125
2–4
0.125
ZF-13, LNE-6 (folC E153G)
ZF-3 (folC S150G)
ZF-4 (folC F152S)
4
8
4
4
8
4
0.125
0.125
0.125
M. bovis BCG
ZF-27, ZF-29 (folC A183P)
ZF-28, ZF-32 (folC R49W)
0.125
4
4
0.125
4
4
0.125
0.125
0.125
M. tuberculosis clinical isolates
Q5 (wild-type folC)
Q36 (folC I43T)
Q449 (folC I43A)
501063 (folC E40G)
FIG 2 DHFS activity of FolC mutants. The DHFS activities of the purified
wild-type FolC and FolC mutants were determined using H2Pte as the sub-
strate. The enzyme activity was assayed as described in Materials and Methods.
The data represent the means Ϯ the standard deviations (SD) from three
independent experiments.
0.5
4
Ͼ8
Ͼ8
0.5
4
Ͼ8
Ͼ8
0.5
1
1
1
responsible for PAS resistance in these M. tuberculosis clinical iso-
lates.
culosis H37Rv was used to transform several PASr M. tuberculosis
H37Ra, M. bovis BCG, and M. tuberculosis clinical isolates. Plas-
mid-borne expression of folC did not affect PAS susceptibility of
the parental strains H37Ra and BCG, nor that of a susceptible
clinical isolate (Table 2). In contrast, PAS susceptibility was re-
stored in 15 folC mutant PASr strains (M. tuberculosis ZF-3, ZF-4,
ZF-7, ZF-8, ZF-10, ZF-13, ZF-27, ZF-28, ZF-29, ZF-32, LNE-1,
LNE-6, Q36, Q449, and 501063) that were tested (Table 2), indi-
cating that modifications in the H2Pte binding pocket were re-
sponsible for PAS resistance and that the wild-type enzyme con-
fers PAS susceptibility.
Replacement of wild-type folC with folC I43A and I43T con-
fers PAS resistance in M. tuberculosis H37Ra. To further assess
the association between PAS resistance and the folC H2Pte binding
pocket mutations I43A and I43T in M. tuberculosis clinical isolates
Q36 and Q449, folC gene replacements were performed with these
alleles in M. tuberculosis H37Ra. Due to the essentiality of folC
(24), the gene replacements were achieved by first transforming
M. tuberculosis H37Ra with an integrative plasmid carrying either
the folC I43A or I43T allele from PASr clinical isolates and then by
subsequently deleting the native copy of folC via specialized trans-
duction (17). Deletion of the original copy of the folC gene from
the genome of M. tuberculosis H37Ra was verified by PCR (see Fig.
S1 in the supplemental material). The two resultant M. tuberculo-
sis H37Ra mutant strains were found to have the same level of PAS
resistance (MIC, 8 ␮g/ml) as strains Q36 and Q449 (Table 3),
demonstrating that the folC I43A and I43T alleles were directly
FolCH2PtebindingpocketvariantsfailtoactivateH2PtePAS
to H2PtePAS-Glu. To better understand the biochemical basis by
which FolC H2Pte binding pocket variants confer PAS resistance,
purified recombinant wild-type FolC and FolC I43T, R49W, E153A,
and A183P were assayed for their ability to catalyze the glutamination
of H2Pte using an HPLC-based method as previously described (7).
The retention times of H2Pte (substrate) and H2Pte-Glu (product)
were determined to be 3.88 min and 3.60 min (see Table S2 in the
supplemental material). As shown in Fig. 2 and in Fig. S3 in the sup-
plemental material, allFolCvariantsshoweda5-to10-fold reduction
in DHFS activity relative to the wild-type enzyme, consistent with a
defect in native substrate binding.
To assess whether alterations in the H2Pte binding pocket also
affect the efficiency of H2PtePAS glutamination, H2PtePAS was
synthesized from H2PtePP and PAS using purified recombinant
FolP1. H2PtePAS was purified (see Fig. S4 in the supplemental
material) and analyzed by HPLC-MS ([M-H]Ϫ; peak, m/z 329.21)
(Fig. 3D). DHFS activity was analyzed using H2PtePAS in lieu of
H2Pte as a substrate. Consistent with previous reports (7, 8), it was
found that wild-type FolC catalyzed the ligation of L-glutamate to
H2PtePAS, yielding H2PtePAS-Glu (Fig. 3A and B), which was
confirmed by HPLC-MS ([M-H]Ϫ; peak, m/z 458.27) (Fig. 3E). In
contrast, all four FolC variants that were tested did not catalyze the
formation of detectable H2PtePAS-Glu (Fig. 3C). The retention
times of H2PtePAS and H2PtePAS-Glu were determined to be 4.28
min and 4.21 min, respectively (see Table S2 in the supplemental
material). In addition, the catalytic rates of wild-type FolC for
H2Pte and H2PtePAS were compared. As shown in Fig. S5 in the
supplemental material, under the same conditions, the transfor-
mation from H2Pte to H2PtePAS was completed within 15 min,
whereas only about 20% of the H2PtePAS was transformed into
H2PtePAS-Glu after 15 min of incubation.
TABLE 3 folC mutations from M. tuberculosis clinical isolates confer
PAS resistance in M. tuberculosis H37Ra
M. tuberculosis strain
MIC (␮g/ml)
H37Ra ⌬folC pMV361-folC
0.125
H37Ra ⌬folC pMV361-folC I43T
H37Ra ⌬folC pMV361-folC I43A
8
8
H2Pte antagonizes the effect of PAS in M. tuberculosis
H37Ra. Since PAS acts as an alternative substrate for FolP by com-
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Zhao et al.
FIG 3 HPLC-MS analysis of H2PtePAS and H2PtePAS-Glu. (A) H2PtePAS and H2PtePAS-Glu coexisted in the reaction system when not all of the H2PtePAS was
consumed by wild-type FolC. (B and C) Wild-type FolC could catalyze ligation of glutamate to H2PtePAS to produce H2PtePAS-Glu in the presence of ATP and
Mg2ϩ (B); however, the FolC mutants could not catalyze this reaction (C). (D) For H2PtePAS, the calculated weight was 330.30 and found weight was ϳ329.21
([M-H]Ϫ). (E) For H2PtePAS-Glu, the calculated weight was 459.42 and found weight was ϳ458.27 ([M-H]Ϫ). All MS data were acquired in the negative mode.
peting with PABA, we wondered whether H2Pte could antagonize tested in the presence of H2Pte. In the presence of 3 ␮g/ml H2Pte,
the effect of PAS. As shown in Fig. 4, the growth inhibitory effects the MIC of PAS was determined to be to 1 ␮g/ml, and it increased
of PAS (0.1 ␮g/ml) were antagonized by H2Pte in a dose-depen- to 4 ␮g/ml in the presence of 10 ␮g/ml H2Pte. The interaction
dent manner, with full antagonism at a concentration of 10 ␮g/ml between H2Pte-Glu and PAS was also tested in M. tuberculosis
H2Pte. In addition, the MIC of PAS for M. tuberculosis H37Ra was H37Ra; however, H2Pte-Glu did not antagonize the effect of PAS.
FIG 4 H2Pte antagonized the effect of PAS on M. tuberculosis H37Ra. The survival of the bacterial cells after 36 h of treatment was determined by the XTT
reduction assay as described in Materials and Methods. H37Ra (H2Pte) indicates that bacterial cells were treated with H2Pte at different concentrations (0, 0.1,
0.3, 1, 3, and 10 ␮g/ml), while H37Ra (H2Pte ϩ PAS) indicates that bacterial cells were treated with both H2Pte (0, 0.1, 0.3, 1, 3, and 10 ␮g/ml) and PAS (0.1
␮g/ml). The data represent the means (bars) Ϯ the SD (error bars) from three independent experiments.
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FolC-Mediated PAS Resistance in M. tuberculosis
This may be explained in part by the lack of a sufficient transport PAS resistance, we assessed whether the FolC variants were altered
system for H2Pte-Glu in M. tuberculosis.
in their enzymatic activities. Consistent with previous reports (7,
8), recombinant wild-type M. tuberculosis FolP1 and FolC were
able to utilize PAS and H2PtePAS, respectively, as a substrate. Yet
DISCUSSION
Over the last several years, considerable effort has been made to the rate of DHFS activity with H2PtePAS as a substrate for FolC
understand the mechanistic basis for susceptibility and resistance was considerably lower than when H2Pte was used as a substrate.
of M. tuberculosis to PAS. Evidence for an interaction of this drug When DHFS activities in four recombinant FolC variants (I43T,
with folate metabolism first came from the observation that one- R49W, E153A, and A183P) were tested with H2Pte as a substrate,
third of PAS-resistant M. tuberculosis and M. bovis clinical isolates it was found that all four had reduced activities (from 8% to 22%)
harbored loss-of-function mutations in thyA (9). While this result relative to the wild-type FolC. Importantly, in contrast to the wild-
was corroborated by subsequent studies (11, 12), resistance mu- type enzyme, no H2PtePAS-Glu was detected from the FolC vari-
tations in the remaining two-thirds of PAS-resistant clinical iso- ant reaction mixtures, even when the reaction times were ex-
lates were not defined. Recently, Zheng et al. (8) described a spon- tended to 20 h. Consistent with these observations, we found that
taneous PAS-resistant M. tuberculosis laboratory isolate in which the addition of H2Pte into the growth medium antagonizes the
resistance was conferred by a point mutation in folC, indicating effect of PAS on M. tuberculosis H37Ra in a dose-dependent man-
that other genes of folate metabolism can contribute to PAS resis- ner. Since H2Pte is the natural substrate for FolC and is an ana-
tance in M. tuberculosis.
logue of H2PtePAS, it can compete with H2PtePAS for enzyme
To identify additional PAS resistance alleles, we genetically binding at high concentrations and block the incorporation of
characterized several spontaneously resistant mutants of both M. H2PtePAS, thus antagonizing the effect of PAS. Together, these
tuberculosis H37Ra and M. bovis BCG. Using whole-genome and data indicate that folC-linked PAS resistance is mediated by al-
targeted sequencing of 66 PAS-resistant strains, 70% of isolates tered substrate specificity that results in the failure to generate
were found to contain substitution mutations in folC. The linkage levels of H2PtePAS-Glu that are necessary to inhibit DHFR.
of these mutations to the PAS-resistant phenotype was confirmed
To understand why PAS resistance mutations in folC led to a
by folC complementation and by allelic transfer studies. Sequenc- decrease in DHFS enzymatic activity, an analysis was performed
ing of other genes (including thyA, folP1, and dfrA) in the folate using previously solved crystal structures of FolC from M. tuber-
pathway revealed that 10% of the strains had mutations in the culosis and other bacterial species (23). M. tuberculosis FolC shows
thyA gene. As 20% of the PAS-resistant strains did not have mu- a very similar three-dimensional (3D) structure to those of FolC
tations in genes associated with PAS resistance, additional resis- from L. casei, T. maritima, Y. pestis and E. coli. In general, FolC
tance alleles await identification.
from M. tuberculosis has two domains, the amino terminal ATPase
To confirm whether folC mutations were associated with PAS domain and the C-terminal Rossmann-fold domain. The N-ter-
resistance in M. tuberculosis clinical isolates, 95 M. tuberculosis minal domain (residues 1 to 334) is composed of one ␤-sheet with
clinical isolates (including 85 MDR isolates) were collected, and six parallel strands and one anti-parallel strand in the center
the sequences of folC in these strains were analyzed. Eight of the flanked by nine ␣-helices. One central six-stranded ␤-sheet and
MDR strains were found to have five different folC mutations, five ␣-helices constitute the C-terminal domain (residues 342 to
whereas the 10 drug-susceptible strains demonstrated no such 489). Of the structure, the ␣1-␣2 loop (residues 36 to 50) of the
mutations. Five of the folC mutant strains (Q36, Q274, Q449, FolC N-terminal domain was shown to be involved in H2Pte bind-
1314, and 501063) were chosen for PAS susceptibility testing and ing (21, 23). Also, the ␣1-␣2 and the ␣4-␣5 loop form a four-helix
were confirmed to be resistant to PAS. Complementation of folC bundle through hydrophobic interactions. Since the ␣1-␣2 loop is
restored PAS susceptibility in all strains that were tested. Further, involved in H2Pte binding, the four-helix bundle may be func-
gene replacement studies in M. tuberculosis H37Ra with two mu- tionally important for interaction with H2Pte. Among the four
tant folC genes derived from two PAS-resistant clinical isolates residues tested, I43 and R49 reside within the ␣1-␣2 loop, A183 is
(Q36 and Q449) showed a direct association of these alleles with involved in the linkage of hydrophobic networks between ␣1 and
clinical PAS resistance. Thus, our data from M. tuberculosis ␣5, and E153 is closely proximal to F152, which is also involved in
H37Ra, M. bovis BCG, and MDR M. tuberculosis clinical isolates the linkage of hydrophobic networks between ␣1 and ␣5. Thus,
confirm that the folC mutation causes PAS resistance in both M. mutations in these four residues may impair H2Pte binding, lead-
bovis and M. tuberculosis.
ing to a decrease in enzymatic activity. In fact, all folC mutations
Recently, Chakraborty et al. (7) found that PAS can act as an identified, except N73, are located within the four-helix bundle, so
alternative substrate in M. tuberculosis folate metabolism. In this they might also affect H2Pte binding and lead to a decrease in
study, it was demonstrated that DHPS (FolP1) can utilize PAS in enzymatic activity. Previous analysis showed that N73 is involved
lieu of PABA in the formation of H2PtePAS. Subsequently, in nucleoside binding, so a mutation in this residue may affect
H2PtePAS can be glutaminated by DHFS (FolC) to yield ATP hydrolysis and consequently reduce the enzymatic activity as
H2PtePAS-Glu (7). These data suggest that PAS may be a prodrug well (23).
that requires activation through biotransformation by the folate
Taken together, our data demonstrate that the mutation of
biosynthesis pathway of M. tuberculosis. More recently, Zheng et FolC results in reduced DHFS activity, leading to a blockage of
al. (8) demonstrated that H2PtePAS-Glu can inhibit the enzymatic PAS bioactivation and thereby causing PAS resistance in both M.
activity of dihydrofolate reductase (DHFR), and overexpression bovis and M. tuberculosis. A confirmation of this novel mechanism
of DHFR in M. tuberculosis can confer PAS resistance. Together, of PAS resistance in mycobacteria, as summarized in Fig. 5, should
these observations indicate that PAS is a prodrug that ultimately enable a molecular diagnosis of PAS resistance in clinical isolates
targets DHFR.
and also provide a better understanding of the PAS mode of action
To better understand the mechanistic basis for FolC-linked in mycobacteria. Our data, in combination with previous reports,
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Zhao et al.
FIG 5 Schematic of FolC-mediated PAS resistance in M. bovis and M. tuberculosis. PAS is incorporated into the folate pathway by FolP1 via competition with
PABA, yielding H2PtePAS. (A) Both wild-type FolC and the FolC mutants can catalyze the formation of H2Pte-Glu. (B) In the case of H2PtePAS, the wild-type
FolC further incorporates it to produce H2PtePAS-Glu, whereas the FolC mutants do not. FolCwt, wild-type FolC; FolCmut, FolC mutants. The dotted arrow
indicates the enzymatic activities of the FolC mutants are greatly reduced compared with that of wild-type FolC, and the fork indicates that this pathway is
blocked.
lence, and mortality by country. JAMA 282:677–686. http://dx.doi.org/10
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clearly demonstrate that PAS action is closely linked with folate
metabolism in mycobacteria. Bacteria, fungi, and plants can syn-
thesize folate de novo from GTP and chorismate as precursors,
while mammals lack this pathway and must obtain folates from
their diets (25). Hence, the folate pathway represents an ideal tar-
get for designing antimicrobial drugs. For example, FolP1 is the
target of important antimicrobial agents, such as sulfonamides
and dapsone, which are structural analogs of PABA (26). A better
understanding of the mode of PAS action and the mechanisms of
PAS resistance will be very helpful for the development of new
compounds to target M. tuberculosis folate metabolism.
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ACKNOWLEDGMENTS
This work was supported by the State Key Development Program for Basic
Research of China (grant no. 2012CB518802 and 2009CB522605), the
National Natural Science Foundation of China (grant no. 31300050), and
institutional startup funds to A.D.B. from the University of Minnesota.
The Key Laboratory of Agricultural and Environmental Microbiology,
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan,
China, and the State Key Laboratory of Agricultural Microbiology, Col-
lege of Life Science and Technology, Huazhong Agriculture University,
Wuhan, China, contributed equally to this work.
We thank William R. Jacobs, Jr., for providing M. tuberculosis H37Ra,
M. bovis BCG, and M. smegmatis mc2155, Peng Gong for technical help
with structural biology analysis, and Guofeng Zhu for providing the ex-
pression plasmids pMV261 and pMV361.
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