Mycobacterium tuberculosis arylamine N-acetyltransferase acetylates and thus inactivates para-aminosalicylic acid

Article abstract of DOI:10.1128/AAC.01312-16

Mycobacterium tuberculosis arylamine N-acetyltransferase (TBNAT) is able to acetylate para-aminosalicylic acid (PAS) both in vitro and in vivo as determined by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS) techniques. The antituberculosis activity of the acetylated PAS is significantly reduced. As a result, overexpression of TBNAT in M. tuberculosis results in PAS resistance, as determined by MIC tests and drug exposure experiments. Taken together, our results suggest that TBNAT from M. tuberculosis is able to inactivate PAS by acetylating the compound.

Full text of DOI:10.1128/AAC.01312-16

AAC Accepted Manuscript Posted Online 26 September 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.01312-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Mycobacterium tuberculosis arylamine N-acetyltransferase acetylates and thus
inactivates para-aminosalicylic acid
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Xude Wanga,b, Shanshan Yanga, Jing Gua, Jiaoyu Denga*
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Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of
Virology, Chinese Academy of Sciences, Wuhan 430071, Chinaa
School of stomatology and medicine, Foshan University, Foshan 528000, Chinab
*Address correspondence to Jiaoyu Deng, dengjy@wh.iov.cn
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Running title: NAT from M. tuberculosis can inactivate PAS
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Mycobacterium tuberculosis Arylamine N-acetyltransferase (TBNAT) is able to
acetylate para-aminosalicylic acid (PAS) both in vitro and in vivo as determined
by HPLC and ESI/MS techniques. Anti-tuberculosis activity of the acetylated
PAS is significantly reduced. As a result, over-expression of TBNAT in M.
tuberculosis results in PAS resistance, as determined by MIC tests and drug
exposure experiments. Taken together, our results suggest that TBNAT from M.
tuberculosis is able to inactivate PAS by acetylating the compound.
Tuberculosis (TB) remains a major threat to global public health. Increasing
emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively
drug-resistant tuberculosis (XDR-TB) calls for urgent development of new drugs to
combat drug-resistant TB. Understanding molecular mechanisms of action and
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resistance of current drugs will provide new perspectives and approaches for drug
discovery. Para-aminosalicylic acid (PAS) used to be one of the first-line, and is now
still used as second line anti-tuberculosis agents (1). The emergence of MDR and
XDR M. tuberculosis prompted the reintroduction of PAS to protect companion
anti-tuberculosis drugs from additional acquired resistance.
Arylamine N-acetyltransferases (NATs, EC. 2.3.1.5) are cytosolic enzymes that
catalyze the transfer of the acetyl group from acetyl coenzyme A (AcCoA) to the free
amino group of arylamines, hydrazines and N-hydroxyarylamines (2-5). NATs
participate in detoxification and metabolic activation of xenobiotics and are found in a
wide variety of prokaryotic and eukaryotic species (6). The NAT enzyme was first
identified in humans in the 1960s due to its ability to inactivate the anti-tubercular
drug isoniazid (INH) (7). The human genome contains two polymorphic NAT genes,
NAT1 andNAT2 (8, 9), which play important pharmacogenetics roles in cancer
susceptibility and have the potential to contribute to personalized medicine (10, 11).
The corresponding enzymes possess distinct substrate profiles: NAT1 preferentially
N-acetylates the arylamines p-aminobenzoic acid (pABA), p-aminobenzoyl-glutamate
(pABGlu) and PAS, whereas NAT2 has higher activity towards the hydrazines
isoniazid (INH), hydralazine (HDZ) and the arylamines sulfamethazine (SMZ) (12,
13). NATs from prokaryotes had also been studied, which display unique substrate
specificity for various arylamine, hydrazide, and hydrazine substrates (14-21).
Previous studies showed that, NAT from M. tuberculosis (TBNAT) and
Mycobacterium smegmatis (MSNAT) can inactivate INH by transfer an acetyl group
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from AcCoA onto the terminal nitrogen of the drug (17, 22). Since PAS is a drug
specific for tuberculosis, and it has been shown by a colorimetric assay that PAS is a
substrate for TBNAT (23), we are wondering if NAT from M. tuberculosis could
acetylate and thus inactivate PAS. To test this hypothesis, NAT from M. tuberculosis
H37Rv (MYCTU NAT) was over-expressed in Escherichia coli BL21 (DE3) using
pCA24N vector (24) and soluble recombinant protein was obtained through Ni-NTA
column purification. Then enzymatic activity of purified recombinant TBNAT for
PAS was determined by using high-pressure liquid chromatography-electrospray
ionization mass spectrometry (HPLC-ESI/MS). The standard reaction consists of
TBNAT, AcCoA and substrates in 20 mM Tris-HCl buffer (pH 7.5). After overnight
reaction at 37°C, the TBNAT in reaction solutions was removed by passing through a
10 kDa Microcon (Millipore) centrifugal filter, and the filtered mixture was
subsequently injected into a C18 liquid chromatography column (Thermo Fisher,
Hypersil Gold aQ, 150×4.6 mm, 3 μm) in an UltiMate 3000 HPLC system (Thermo
Fisher Scientific). Samples were eluted with a gradient from 95% buffer A (H2O plus
0.1% acetic acid) and 5% buffer B (methanol plus 0.1% acetic acid) to 5% buffer A
and 95% buffer B for 30 min, at a flow rate of 0.3 ml/min. Retention times of PAS
(substrate) and acetylated PAS (4-acetamidosalicylic acid, AcPAS) (product) were
determined to be 16.53 min and 21.15 min according to the pure standard (Fig.1A).
We observed that PAS can be almost completely converted into AcPAS after
overnight reaction, resulting a peak of m/z at 196.05 ([M+H]+) representing the
AcPAS detected by ESI/MS (Thermo Fisher Scientific, LCQ Fleet) in the positive
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ionization mode (Fig.1B). This result showed that, TBNAT can transfer the acetyl
group from AcCoA to the N4 position (the amino group) of PAS and yield AcPAS in
vitro.
To verify if TBNAT could also acetylate PAS in vivo, the vector pMV261-NATTB
was constructed and electroporated into M. tuberculosis H37Ra to over-express
TBNAT, then levels of AcPAS in both the wild type strain and the TBNAT
over-expressed strain were analyzed by HPLC after PAS treatment. 5 μM PAS was
added into the culture media when M. tuberculosis cells were grown to mid-log phase
(OD600 of 0.5–1.0) and incubated under 37°C for 12 hours. Then, cells were
collected and disrupted by glass beads, and proteins in cell extracts were removed by
using a 3 kDa Microcon centrifugal filter, and the level of AcPAS in the extract was
analysis by HPLC as described above. As shown in Figure 2, an obvious
accumulation of AcPAS could be observed (Fig.2A, indicated by square frame) in the
TBNAT over-expressed strain after PAS treatment, which was not observed in the
wild type strain (Fig.2B). Meanwhile, when the TBNAT over-expressed and wild type
strain was not treated with PAS, AcPAS could not be detected (Fig.2C and D). These
results showed that, TBNAT is also able to acetylate PAS in vivo.
To further investigate acetylation of PAS could affect the anti-tuberculosis
activity of the compound, MICs of PAS and AcPAS for M. tuberculosis H37Ra were
tested. The MIC of PAS was determined to be 0.02 μg/ml (0.1 μM), whereas that for
AcPAS (From Santa Cruz Biotechnology, Catalog: sc-207963) was determined to be
10 μg/ml (50 μM). Thus, the anti-tuberculosis activity of acetylated PAS was
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significantly reduced. Meanwhile, in order to investigate whether TBNAT could affect
the anti-tuberculosis activity of PAS in vivo, PAS susceptibilities were determined for
both the wild type strain and the TBNAT over-expressed strain. We found that,
over-expression of TBNAT leads to a two times increase of PAS MIC. Besides, drug
exposure experiments were also performed, and the results were shown in Figure3.
For the TBNAT over-expressed strain, the killing effect of PAS peaked after 7 days of
treatment, and then bacteria started to grow again. However, for the wild type strain,
continuous killing by PAS could be observed even after 21 days of treatment. In
addition, after 7days of PAS treatment, the survival rate of the TBNAT over-expressed
strain is much higher than that of the wild type strain. These results demonstrated that,
over-expression of TBNAT in M. tuberculosis greatly enhances the tolerance to PAS
treatment.
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We found that, though acetylation greatly reduces the anti-tuberculosis activity of
PAS, AcPAS still has weak anti-tuberculosis activity. Previous studies showed that,
PAS acts as a prodrug, it needs to be incorporated into the folate pathway through
dihydropteroate synthase (DHPS) and dihydrofolate synthase (DHFS) and thus forms
a hydroxyl dihydrofolate antimetabolite, which in turn inhibits dihydrofolate
reductase (DHFR) (25, 26). We speculated that the AcPAS might also be incorporated
into the folate biosynthesis pathway, though with much lower efficiency or could
compete with pABA as they are structural analogues, thus affect folate biosynthesis
(25). In conclusion, our results presented here demonstrate that NAT from M.
tuberculosis can acetylate PAS and thus inactivate the anti-tuberculosis drug both in
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vitro and in vivo.
ACKNOWLEDGMENT
This study was supported by the National Natural Science Foundation of China (grant
no. 31300050).
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Figure Legends
Figure 1. HPLC-ESI/MS analysis of TBNAT activity for PAS. TBNAT reaction
mixture was separated by HPLC. PAS (retention time 16.53 min) (A) could be
acetylated by TBNAT, yielding acetylated PAS (retention time 21.15 min) (A).
Acetylated products were then detected by ESI/MS (m/z =196.05) ([M+H]+) which
were indicated by arrows and chemical structures were shown alongside (B). MS data
was acquired in the positive mode.
Figure 2. HPLC analysis of AcPAS accumulation in M. tuberculosis. The TBNAT
over-expressed (A and B) and wild type (C and D) M. tuberculosis were grown to
mid-log phase (OD600 of 0.5–1.0), and treated with (A and C) or without (B and D) 5
μM PAS for 12 hours, then cell extracts were prepared and filtered to remove protein
before HPLC analysis.
Figure 3.Killing curve of PAS against M. tuberculosis. M. tuberculosis was grown to
mid-log phase (OD600 of 0.5–1.0) and diluted to about 107 CFU/ml in fresh medium.
PAS was then added and aliquots were taken at regular intervals. Serial dilutions were
performed before plating. M. tuberculosis H37Ra (■) and M. tuberculosis H37Ra
over-expressing TBNAT (▲) with no drug added; M. tuberculosis H37Ra (□) and M.
tuberculosis H37Ra over-expressing TBNAT (△) with 0.5 μg/ml PAS added. The data
represent the mean ± SD (standard deviations) of three independent experiments.
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