MRC/SAIMR/WITS Molecular Mycobacteriology
Research Unit, South African Institute for Medical Research,
and Department of Molecular Medicine and Haematology, University of
the Witwatersrand Medical School, Johannesburg, South Africa
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INTRODUCTION |
The major human pathogen
Mycobacterium tuberculosis is responsible for approximately
2 million deaths and 8 million new cases of tuberculosis per annum
(7). Pyrazinamide (PZA) is one of the first-line drugs used
in the recommended treatment of tuberculosis (36). This drug
plays a key role in shortening the duration of chemotherapy by virtue
of its ability to kill semidormant tubercle bacilli located in an
acidic environment which are resistant to the bactericidal effects of
other drugs (13). In a seminal study, Scorpio and Zhang
demonstrated that PZA itself is a prodrug, which requires hydrolysis by
the pncA-encoded pyrazinamidase (PZase) to its active
metabolite, pyrazinoic acid (30). The critical role played
by pncA in the mode of action of PZA is underscored by the
fact that the overwhelming majority of PZA-resistant clinical isolates
of M. tuberculosis contain mutations in this gene (4, 23). In subsequent studies on the inhibitory mechanisms of
pyrazinoic acid, Zhang et al. showed that pyrazinoic acid accumulates
under conditions of acidic external pH (39), accounting for
why PZA is active against M. tuberculosis in vitro only
under conditions of acidic pH (20). This group also showed
that mycobacterial species differed considerably in their ability to
expel pyrazinoic acid by active efflux (39). Therefore,
although M. avium has an active PZase, its resistance to PZA
is probably due to effective pyrazinoic acid efflux (34).
Similarly, the resistance of M. kansasii to PZA could be
ascribed to a combination of its low PZase and weak pyrazinoic acid
efflux activities (35), whereas that of M. smegmatis, which possesses two PZases, the highly active PzaA
(2) and PncA (11), is due to a highly active
efflux mechanism (39). In summary, the susceptibility of a
mycobacterium to PZA under acidic conditions thus appears to be
determined by the relative contributions of its PZase and pyrazinoic
acid efflux activities.
Pyrazinoic acid has been postulated to have both a specific activity
against M. tuberculosis and a nonspecific activity due to
the effect that accumulation of this membrane-permeant weak acid would
have on intracellular pH (12). We previously characterized the major PZase/nicotinamidase of M. smegmatis and showed
that overexpression of this amidase, which is structurally unrelated to
PncA, rendered M. smegmatis sensitive to PZA and
nicotinamide (2). To further explore the
relationship between PZase/nicotinamidase activity and PZA
susceptibility, we investigated the effects of PzaA expression on the
susceptibility of M. tuberculosis, M. smegmatis, and Escherichia coli to PZA and related compounds, and in
this paper we report that the PzaA protein confers hypersensitivity to
PZA, nicotinamide, and benzamide on these organisms under
acidic conditions. The results presented herein suggest that the weakly acidic hydrolysis products of these amides confer similar inhibitory effects on these organisms.
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MATERIALS AND METHODS |
Materials, bacterial strains, media, and growth conditions.
Restriction enzymes were from Roche Molecular Biochemicals or New
England Biolabs, [14C]carbonyl nicotinamide (53 mCi/mmol) was from Sigma, [
-32P]dCTP was from
Amersham, and PZA, pyrazinoic acid, nicotinamide, and benzamide
were from Sigma. The BM Chemiluminescence Western blotting kit was from
Roche Molecular Biochemicals. The bacterial strains and plasmids used
in this study are shown in Table 1. M. smegmatis was grown in Middlebrook 7H9 broth (Difco)
supplemented with 0.2% glycerol, 0.085% NaCl, 0.2% glucose, and
0.05% Tween 80 and on Middlebrook 7H10 medium supplemented with
0.085% NaCl and 0.2% glucose as the solid medium. M. tuberculosis was grown in Middlebrook 7H9 broth supplemented with
0.2% glycerol, 0.05% Tween 80, and OADC supplement (Merck) and on
Middlebrook 7H10 supplemented with OADC as the solid medium
(16). E. coli strains were grown in Luria-Bertani
broth or agar. E. coli DH5 and GM161 were used for
plasmid manipulations (29), and E. coli
BL21(DE3)/pLysS and E. coli TB-1 were used for protein
expression. Kanamycin and hygromycin were used at 50 and 200 µg/ml,
respectively, for E. coli strains and at 20 and 50 µg/ml,
respectively, for mycobacterial strains. Ampicillin and chloramphenicol
were used at 100 and 34 µg/ml, respectively, for E. coli.
Amide susceptibility testing.
The MIC of PZA is pH dependent
(27). To maximize the sensitivity of the susceptibility
tests, the lowest pH value at which visible growth of the various
strains occurred was therefore used as the test pH. Since a pH of 5.5 proved to be too low to support the growth of M. tuberculosis H37Rv, susceptibility testing was carried out at a pH
of 5.8. For M. smegmatis, a pH of 5.1 was used, under which
conditions visible colonies were formed in 5 to 6 days on solid medium
(compared with 2 to 3 days for colony formation at pH 6.6). By analogy,
a pH of 5.1 was used for susceptibility testing of E. coli
strains. PZA, nicotinamide, and benzamide susceptibility tests
for M. tuberculosis were performed in Middlebrook 7H9 broth supplemented with 0.3 g of citric acid per liter, with the pH adjusted to 5.8, and subsequently supplemented with glycerol, OADC, and
0.05% Tween 80. Amide stocks (0.1 M) were added to give the required
final concentration. Log-phase cultures of M. tuberculosis (optical density at 600 nm 
0.9) were diluted 10-fold in this medium. A 50-µl aliquot of diluted cells was then inoculated in 3 ml
of drug-containing medium, and the culture was incubated at 37°C for
2 weeks. The MIC was defined as the lowest drug concentration at which
no turbidity was visible after 2 weeks. Susceptibility tests were also
performed in unmodified Middlebrook 7H9 broth (pH 6.6) supplemented
with OADC and Tween 80, as above. Susceptibility to PZA and benzamide
was confirmed radiometrically (5) using the BACTEC PZA test
kit (Becton Dickinson). M. tuberculosis strains, subcultured
in BACTEC 12B medium as specified by the manufacturer, were grown to a
growth index of 150 for use in the inoculation of the BACTEC
susceptibility test media. Susceptibility was determined as specified
by the manufacturer but with the following minor modification: PZA and
benzamide were dissolved in reconstituting fluid (Becton Dickinson) and
filter sterilized, and 0.2 ml of each stock was added to the modified
Middlebrook 12B medium to give final concentrations of 0, 50, 100, 200, 400, and 600 µg of PZA per ml and 0, 50, 100, and 200 µg of
benzamide per ml. M. smegmatis susceptibility tests were
performed as previously described (2). The drug
susceptibility of E. coli BL21(DE3)/pLysS carrying
pET15b-derived plasmids was determined by plating on M9 minimal medium,
adjusted to pH 5.1, containing chloramphenicol, ampicillin,
isopropyl-
-D-galactopyranoside (IPTG; 1 mM), and the
appropriate amide.
Construction of a pncA::hyg
knockout mutant of M. tuberculosis.
A cosmid (Pac4)
containing pncA was identified from a library of M. tuberculosis (pYUB328::H37Rv) (1) using a
PCR-amplified M. tuberculosis pncA probe (2). A
4.67-kb Asp718 fragment containing pncA was
cloned in pGEM3Z(+)f to yield pGncA. The
hyg resistance cassette was excised from pIJ963
(19) as a BamHI-BglII
fragment and was cloned in the BclI site contained
within the pncA gene to create pGncAh. A cassette
carrying the Bacillus subtilis sacB gene under the control
of the mycobacterial hsp60 (groEL) promoter (B. G. Gordhan, S. Quan, and V. Mizrahi, unpublished
data) was cloned into the XbaI site of pGncAh to
create pGncAKO. M. tuberculosis was electroporated
with 1 µg of UV-treated pGncAKO as described by
Gordhan and Parish (10), and the cells were plated on 7H10 medium containing hygromycin and 5% sucrose. Chromosomal DNA was extracted from Hygr Sucr clones and analyzed by
Southern blotting as previously described (2, 9), using
32P-labeled probes generated using a random-prime labeling
kit (Roche Molecular Biochemicals).
Construction of PncA and PzaA expression vectors.
A 3.39-kb
PstI fragment from Pac4 containing the M. tuberculosis
pncA gene and its promoter was cloned into pHINT to create pHncA
and into pOLYG to create pOncA. The pncA gene was also
cloned under the control of the stronger, M. smegmatis pzaA
promoter and a consensus ribosome binding site, as follows. The
pzaA promoter was excised as a 340-bp PstI
fragment from pGam (2), cloned in pOLYG, and
excised as a HindIII-XbaI fragment. This
fragment was then ligated to the XbaI-BamHI
fragment containing the pncA gene with its ribosome binding
site (9, 22) from the pET15b derivative, pTncA
(2), and cloned in
HindIII-BamHI-digested pOLYG to form pOppncA.
The integrating vector, pAINT, was derived from pHINT by excising the
hyg gene by digestion with SmaI and SacI and replacing it with the Tn903-derived
aph gene (15). The pncA-containing
4.67-kb Asp718 fragment from Pac4 was cloned in pAINT to
form pAIncA, and the pzaA-containing
HindIII-XbaI fragment from pGam was
also cloned in this vector to produce pAIam. Replicating and
integrating plasmids (100 ng) were electroporated into M. smegmatis and M. tuberculosis as previously described (9, 22). The vector pMncA, which directs the overexpression of a recombinant form of PncA fused to the maltose binding protein (MBP), was constructed as follows. The vector pTncA was digested with
NcoI (which cuts at the start codon of the pncA
gene in the plasmid) and was blunt ended before the pncA
gene was excised on a BamHI fragment. This fragment was
cloned in pMAL-c2 that had been digested with EcoRI, blunt
ended, and subsequently restricted with BamHI, to yield
pMncA, which contains an in-frame translational fusion between the
MBP-encoding malE gene and pncA. The
MBP::PncA fusion protein was induced by IPTG treatment of E. coli TB-1 cells carrying pMncA and was purified on amylose resin
as specified by the manufacturer (New England Biolabs).
Enzyme assays.
Lysates of M. smegmatis were
prepared as previously described (6). Lysates of M. tuberculosis were prepared by resuspending cell pellets in buffer
A (20 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1 mM dithiothreitol) and
homogenizing them with glass beads in a Mini bead beater (Biospec
Products, Bartlesville, Okla.) three times for 1 min each at maximum
speed, with cooling on ice between pulses. Lysates were clarified by
centrifugation at 13,000 × g for 5 min. Secreted
M. tuberculosis proteins were prepared from supernatants of
mid-log cultures by harvesting cells at 5,000 × g for
10 min and sterilization of the spent growth medium by filtration
through 0.2-µm-pore-size filters. Proteins contained in the
sterilized filtrate were concentrated either by centrifugation through
a 30-kDa Ultrafree MC filter unit (Millipore) or by precipitation with
6 volumes of ice-cold ethanol, followed by centrifugation at
17,000 × g for 1 h and resuspension in buffer A. Amidase activities were determined using the following assays. (i) The
presence of PZase activity in bacterial strains was determined using a
spectrophotometric assay following ferrous ammonium sulfate addition,
as previously described (2). (ii) PZase and nicotinamidase
specific activities were assayed by high-pressure liquid chromatography
(HPLC) using the following method, which is based on that described by
Yan and Sloan (37). Enzyme reaction mixtures were incubated
at 37°C in 20 mM PZA or nicotinamide in 100 mM sodium
phosphate buffer at pH 7.5 in a total volume of 200 µl containing 60 to 240 µg of protein from cell lysates prepared from log-phase
cultures. After incubation of the mixture for a sufficient period to
result in 0 to 10% substrate conversion, the reaction was terminated by the addition of 20 µl of 80% (wt/vol) trichloroacetic acid. Precipitates were removed by centrifugation (13,000 × g for 10 min), and 120 µl of the reaction mixture was diluted in
400 µl of 100 mM sodium phosphate buffer. Samples were filtered
through 0.45-µm-pore-size filters, and 20-µl aliquots were
separated on a Phenomenex Luna C18 column (150 by 4.6 mm)
with 10% methanol elution buffer using a SpectraSystem P4000 pump
(Thermo Separation Products). Substrates and products were detected
using a SpectraSystem UV3000 detector set at 254 and 280 nm. At a flow
rate of 1 ml/min, pyrazinoic acid eluted at 3.31 min, PZA eluted at
4.15 min, nicotinic acid eluted at 2.68 min, and nicotinamide
eluted at 2.95 min. Peaks were integrated using the PC1000 system
software for LC (version 3.5; Thermo Separation Products). Each
mycobacterial strain was tested in at least three independent
experiments, using a minimum of five time points during the enzyme
reaction. (iii) Nicotinamidase activity was additionally determined
using the following radiochemical assay. Either 10 µl of cell lysate
(containing 4 mg of protein per ml) or 20 µl of concentrated culture
filtrate in buffer A was added to 2 µl of a solution containing 5 mM
[14C]nicotinamide at a specific activity of 53 mCi/mmol. The reaction mixtures were incubated at 37°C for 0, 10, 75, or 140 min before the reactions were quenched by the addition of 2 µl
of acetic acid (2 M). The total assay mixture was then spotted
onto a silica gel thin-layer chromatography (TLC) plate (Aldrich)
containing unlabeled nicotinic acid and nicotinamide as
internal standards. The plates were developed in a mixture of
1-butanol, NH3, and water (8:1:1), and spots corresponding
to the internal standards were visualized under UV light, excised, and
counted in toluene-based liquid scintillation fluid in a Beckman
LS6000IS scintillation counter. (iv) An analogous, nonradioactive assay
was used to monitor the benzamidase activities of whole-cell
suspensions or cell lysates in 10- to 20-µl assay mixtures containing
10 mM benzamide. The reaction products were spotted on TLC plates,
which were developed and visualized for benzamide or benzoic acid under
UV light, as described above. Protein concentrations were determined by
a Bradford assay (Bio-Rad assay kit II).
Intracellular localization assays.
PzaA-specific antiserum
was prepared by purifying PzaA from inclusion bodies in IPTG-induced
E. coli BL21(DE3)/pLysS carrying pTam (2) by a
combination of sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) of the inclusion bodies and electroelution of the protein from gel slices using a BioTrap BT1000 device
(Schleicher & Schuell). A New Zealand White rabbit was immunized
subcutaneously with 200 µg of protein in 1 ml of phosphate-buffered
saline at weekly intervals over a 6-week period before antiserum was
prepared. Purified MBP::PncA was used for the production of PncA
antiserum by immunization of a New Zealand White rabbit, as described
above. M. tuberculosis expresses very low levels of PncA, in
contrast to the higher levels detected in M. smegmatis
recombinants carrying the various PncA expression vectors. To enrich
for PncA-specific antibodies, serum was incubated with strips of
polyvinylidene difluoride membrane with bound PncA [obtained from
IPTG-induced BL21(DE3)/pLysS(pTncA) cell lysates that had been
fractionated by SDS-PAGE and transferred to polyvinylidene
difluoride], which had previously been treated with blocking solution
(Western blotting kit). The strips were washed extensively in
Tris-buffered saline prior to elution of antibody by incubation in 0.2 M glycine (pH 2.8)-1 mM EDTA for 20 min. Eluted antibody was
neutralized with 1 M Tris-HCl and stabilized in 1% blocking solution,
and precipitates were removed by centrifugation. Cell lysates and
culture supernatants were prepared as described above, precipitated in
80% acetone, and denatured in SDS-PAGE loading buffer. For cell
lysates, approximately 20 µg of total protein was loaded per lane of
the gel. Western blotting was performed with a Western blotting
chemiluminescence kit as specified by the manufacturer.
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RESULTS |
Construction and characterization of a
pncA::hyg knockout mutant of M. tuberculosis.
To investigate the effects of M. smegmatis pzaA expression on the PZA susceptibility
of M. tuberculosis, we constructed a PZase-deficient mutant of M. tuberculosis by targeted
knockout of its PZase/nicotinamidase-encoding
pncA gene (30), for use as a host strain. The
insertionally inactivated pncA::hyg
allele, in which the hyg marker was flanked on either
side by 1,733 bp (5') and 2,493 bp (3') of homologous DNA, was cloned
in a suicide plasmid carrying a sacB counterselection
marker. Plasmid DNA, pretreated by UV irradiation (10,
14), was electroporated into M. tuberculosis,
which was directly plated on medium containing hygromycin and
sucrose. Of 19 colonies genotypically screened by Southern blot
analysis, 8 corresponded to allelic exchange mutants, 8 were
site-specific single-crossover recombinants that were spontaneously
Sucr, and 3 were spontaneous Hygr mutants (Fig.
1). As predicted from the
well-established role of the pncA gene in M. tuberculosis (30), the
pncA::hyg mutant was highly resistant to PZA
(MIC, >2,000 µg/ml [Table 2]), and it lacked PncA protein (Fig. 2A, lane 2, and data not shown) and PZase activity in both cell lysate and culture
supernatant fractions (Table 2 and data not shown).
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FIG. 1.
Construction and genotypic characterization of the
M. tuberculosis pncA::hyg mutant and its
pncA-complemented derivative. The restriction map of the
pncA locus, illustrating the
pncA::hyg allele carried on the
Asp718 fragment of the allelic exchange substrate,
pGncAKO, is shown beneath the Southern blot. Genomic DNA was
digested with PstI, and Southern blots were probed with a
PCR-generated pncA probe (2). The expected sizes
of the wild-type and inactivated PstI alleles are 3.39 and
2.65 kb, respectively. Lanes: 1, pncA::hyg
mutant complemented with pAIncA (showing the mutant allele and the
additional 4.7-kb allele arising from the integrated copy of
pncA); 2, 5, and 6, independent isolates of the
pncA::hyg mutant; 3, product of homologous
recombination by single crossover (showing both wild-type and
mutant alleles); 4, wild-type H37Rv.
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TABLE 2.
Correlation between amidase activity and amide drug
susceptibility of strains of M. smegmatis, M. tuberculosis, and E. coli
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FIG. 2.
Determination of the cellular localization of PncA and
PzaA in mycobacteria by Western blot analysis. Western blotting was
carried out as described in Materials and Methods. The locations of
PncA and PzaA are indicated by arrowheads, and the sizes are given in
kilodaltons. (A) Detection of PncA in M. tuberculosis
strains. Lanes: 1, cell lysate from wild-type H37Rv; 2, cell lysate
from the pncA::hyg mutant; 3, cell lysate
from the pncA-complemented mutant,
pncA::hyg::pAIncA; 4, culture filtrate
protein precipitate from wild-type H37Rv; 5, culture filtrate
protein precipitate from
pncA::hyg::pAIncA. (B) Detection of PncA
in M. smegmatis strains. Lanes: 1, cell lysate from
mc2155(pOncA); 2, cell lysate from wild-type
mc2155; 3, cell lysate from
mc2155::pHncA; 4, cell lysate from
mc2155(pOppncA); 5, culture filtrate protein
precipitate from mc2155::pHncA. (C) Detection of PzaA
in M. tuberculosis and M. smegmatis strains.
Lanes: 1, cell lysate from wild-type mc2155; 2, cell lysate
from the pzaA::aph mutant; 3, cell lysate
from wild-type H37Rv; 4 and 5, cell lysate from the
pzaA-complemented mutant,
pncA::hyg::pAIam; 6, culture filtrate
protein precipitate from the
pncA::hyg::pAIam mutant; 7, culture
filtrate protein precipitate from wild-type mc2155.
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PZase and nicotinamidase specific activities were determined by
HPLC analysis of enzyme reaction products. The PZase specific activities determined by the HPLC-based method were quantitatively different from those determined by the previously described
spectrophotometric assay (2). The difference was most
pronounced for M. tuberculosis, which expresses a very low
PZase activity, whose absolute value appears to have been overestimated
using the spectrophotometric assay (0.02 U/mg [2]).
The sensitivity of pyrazinoic acid detection by this assay is limited
by the high background caused by the gradual time- and buffer-dependent
oxidation of the ferrous metal ion, which occurs following addition of
the ferrous ammonium sulfate. In contrast, the HPLC assay provided a
sensitive alternative method for specific amidase activity
determination because it allowed the direct quantitation of separated
substrate and product. HPLC analysis revealed very low but detectable
PZase and nicotinamidase activities in the parental M. tuberculosis H37Rv strain, whereas no pyrazinoic or nicotinic acid
conversion could be detected in cell lysates prepared from the
pncA::hyg mutant strain over a 120-min
incubation with 240 µg of total protein. These results were
independently corroborated by determination of nicotinamidase activity
analysis using [14C]nicotinamide as a substrate,
which revealed that there was no detectable conversion to nicotinic
acid by a cell lysate prepared from the mutant strain under conditions
resulting in 
90% hydrolysis by a lysate prepared from the wild-type
strain (75-min incubation with 40 µg of total protein). As expected,
integration of a functional copy of pncA at the
attB locus of the mutant (Fig. 1, lane 1) restored the
phenotype of the mutant to that of the wild type in terms of PZA
susceptibility, level and cellular localization of PncA protein (Fig.
2A, lanes 3 and 5, and data not shown), and PZase and nicotinamidase
activity (Table 2).
Expression of M. smegmatis pzaA in M. tuberculosis confers hypersensitivity to PZA and related aromatic
amides.
Integration of a single copy of the M. smegmatis
pzaA gene in the pncA::hyg mutant
resulted in an increase in both the specific activity of PZase and the
PZA sensitivity to levels higher than those conferred by integration of
the M. tuberculosis pncA gene at the same (attB)
locus (Table 2). This result is in agreement with the recently
demonstrated ability of PzaA to confer PZA susceptibility on M. bovis BCG (11). An inverse correlation was observed
between the PZase activity and the MIC of PZA. Furthermore, analysis of the cellular localization of the PzaA protein in M. tuberculosis by Western blotting confirmed that, like PncA, this
protein localized exclusively in the cytoplasm (Fig. 2C, lanes 4 to 6).
The pzaA-complemented mutant was also highly sensitive to
both nicotinamide and benzamide in a pH-dependent manner: under
acidic (pH 5.8) conditions, the MICs of PZA and benzamide for the
pzaA-complemented mutant were 
10 and 20 µg/ml,
respectively, whereas at the normal pH of the growth medium (pH 6.6),
the MIC of both amides was 100 µg/ml. In contrast, although
pncA complementation restored nicotinamide sensitivity to the pncA::hyg mutant, the
complemented strain, like the parental wild type, was completely
insensitive to benzamide.
The biochemical basis for this observation was investigated by
performing an amidase activity analysis on cell lysates from various
strains with benzamide as a substrate (Fig.
3). Whereas strains of E. coli
and M. smegmatis expressing PzaA hydrolyzed all three amides
(Fig. 3A, lanes 2 to 4 and 10 to 12), those overexpressing PncA did not
(lanes 13 to 15), suggesting that PzaA has a broader substrate range
than PncA. The pncA-complemented
pncA::hyg mutant of M. tuberculosis and the M. smegmatis
pzaA::aph mutant did not hydrolyze benzamide (Fig.
3A, lanes 5 to 7; Fig. 3B, lanes 1 and 2). In contrast, wild-type
M. smegmatis and the pzaA-complemented pncA::hyg mutant of M. tuberculosis were proficient in benzamidase activity (Fig. 3A,
lanes 2 to 4; Fig. 3B, lanes 4 and 5). Although the M. smegmatis
pzaA::aph mutant retained residual PZase activity (Fig. 3A, lane 18), which was recently attributed to the presence of a
functional PncA in this organism (11), this mutant exhibited no benzamidase activity (lanes 5 to 7). In conjunction, these observations confirmed the restricted substrate specificity of PncA
relative to PzaA.
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FIG. 3.
Benzamidase and PZase activities of cell lysates
expressing PncA or PzaA. (A) Cell lysates from M. smegmatis
(lanes 2 to 9, 17 and 18) or IPTG-induced E. coli carrying
either pTam or pTncA (lanes 10 to 15) were incubated with the
respective amides, and the reaction products were analyzed by TLC, as
described in Materials and Methods. Lanes: 1, benzamide (BZA) and
benzoic acid (BOA) standards; 2, mc2155 incubated with
benzamide for 0 min; 3, mc2155 incubated with benzamide for
10 min; 4, mc2155 incubated with benzamide for 40 min; 5, pzaA::aph incubated with benzamide for 0 min;
6, pzaA::aph incubated with benzamide for 10 min; 7, pzaA::aph incubated with benzamide
for 40 min; 8, boiled mc2155 lysate incubated with
benzamide for 0 min; 9, boiled mc2155 lysate incubated with
benzamide for 40 min; 10, E. coli(pTam) incubated with
benzamide for 0 min; 11, E. coli(pTam) incubated with
benzamide for 7 min; 12, E. coli(pTam) incubated with
benzamide for 20 min; 13, E. coli(pTncA) incubated with
benzamide for 0 min; 14, E. coli(pTncA) incubated with
benzamide for 7 min; 15, E. coli(pTncA) incubated with
benzamide for 20 min; 16, benzoic acid standard; 17, mc2155
incubated with PZA for 20 min; 18, pzaA::aph
incubated with PZA for 20 min; 19, pyrazinoic acid (POA) standard; 20, PZA plus pyrazinoic acid standards. (B) Benzamidase activity in cell
lysates of M. tuberculosis strains. Lanes: 1, pncA::hyg::pAIncA incubated with
benzamide for 30 min; 2, pncA::hyg::pAIncA incubated with
benzamide for 150 min; 3, benzamide plus benzoic acid standard; 4, pncA::hyg::pAIam incubated with benzamide
for 30 min; 5, pncA::hyg::pAIam incubated
with benzamide for 150 min.
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The phenotypic effects of heterologous expression of PzaA in M. tuberculosis were significantly exacerbated by overexpression of
this amidase from the multicopy plasmid pOam. A transformant carrying
this plasmid displayed marked pH sensitivity, as evidenced by its poor
growth at pH 5.8 and by the fact that at pH 6.6, the MIC of PZA was 5 µg/ml whereas that of BZA was <20 µg/ml. This observation is
consistent with the notion that the PZA susceptibility of M. tuberculosis is limited by its relatively weak PZase and pyrazinoic acid efflux activities.
Overexpression of M. smegmatis PzaA also confers
aromatic amide sensitivity on M. smegmatis and E. coli.
We have previously shown that the natural resistance of
M. smegmatis to PZA and nicotinamide can be overcome
by overexpression of PzaA (2). This initial observation was
extended by analyzing the effects of PzaA overexpression from pOam and
PncA overexpression from pOncA and pOppncA on the susceptibility of
M. smegmatis to these drugs. Western blot analysis was
used to monitor the heterologous expression of M. tuberculosis PncA from these various pncA-based plasmids in M. smegmatis (Fig. 2B). This analysis confirmed
the intracellular localization of PncA, expressed heterologously
from the single-copy vector pHncA (Fig. 2B, lane 3) and from the
multicopy vectors pOncA and pOppncA (Fig. 2B, lanes 1 and 4). However,
in spite of the obvious overexpression of PncA revealed by
Western blot analysis, neither multicopy vector conferred amide
susceptibility on M. smegmatis, in accordance with the
relatively low specific activity of PZase in strains carrying these
plasmids (Table 2). In contrast, the PzaA-overexpressing plasmid pOam
conferred a 30-fold increase in the specific activities of PZase and
nicotinamidase in M. smegmatis, which resulted in a
concomitant increase in the sensitivity of this organism to benzamide,
in addition to its previously observed hypersensitivity to PZA and
nicotinamide (2). The amide susceptibility study
also revealed that M. smegmatis is naturally more
susceptible to benzamide than to PZA (Table 2). However, comparison of
the benzamide susceptibilities of the wild type and the PZase-deficient
pzaA::aph mutant suggested that the
susceptibility to benzamide at concentrations above 500 µg/ml was
hydrolysis independent.
High-level overexpression of PncA resulted in a marked increase in the
susceptibility of E. coli to PZA and nicotinamide
but not to benzamide. The sensitivity of E. coli to
overproduction of PZase was such that BL21(DE3)/pLysS cells carrying
the PzaA-overexpressing plasmid pTam were unable to form colonies at
low pH on minimal medium, even in the absence of exogenously added
amides. The few colonies that did grow all carried rearranged plasmids,
confirming the toxicity at low pH of PzaA overproduction. In
conjunction, these observations underscored the close link between
aromatic amide susceptibility at an acidic pH and the ability of the
organism to hydrolyze the amide to its corresponding acid.
| |
DISCUSSION |
In this study, we constructed a PZase-defective pncA
mutant of M. tuberculosis as a tool to investigate the
effect of heterologous expression of a PZase/nicotinamidase enzyme of
higher specific activity and broader substrate specificity than PncA on
the sensitivity of M. tuberculosis to aromatic amides, which
are structurally related to PZA. The observation of an exclusively
intracellular localization for the PncA protein in M. tuberculosis is in agreement with the observations of others
(25, 39), but our observation of the lack of nicotinamidase
activity in both the intracellular and extracellular fractions of the
pncA::hyg mutant differs from that of Raynaud
et al. (24, 25), who have reported the existence of
extracellular nicotinamidase activity in M. tuberculosis.
However, this discrepancy might be due to differences in growth medium, since this factor could affect the expression and secretion of enzymes
(38).
M. tuberculosis, M. smegmatis, and E. coli are all rendered hypersensitive to inhibition by benzamide by
the heterologous expression of PzaA, which was shown to catalyze the
hydrolysis of this amide. The MIC of benzamide for the
pzaA-complemented pncA::hyg mutant
(20 µg/ml) was comparable to that of PZA (10 µg/ml). As neutral
molecules, PZA and benzamide are expected to equilibrate rapidly
across the membrane of M. tuberculosis. Intracellular PzaA would then hydrolyze the amides to their corresponding, weak acids. Since the pKa values of benzoic acid and
pyrazinoic acid are 4.1 and 2.9, respectively, the Henderson-Hasselbach
equation predicts that both acids would be 99.9% deprotonated in the
intracellular environment, which has a pH of 7 (39). The
similarity in the MICs of PZA and benzamide implies a similar
inhibitory mechanism for these two drugs and suggests that, as
previously observed for pyrazinoic acid (39), benzoic acid
accumulates in M. tuberculosis under conditions of an acidic
external pH.
Inhibition by the intracellular accumulation of pyrazinoic acid,
nicotinic acid, and benzoic acid, three aromatic acids with very
different structures, makes it unlikely that pyrazinamide (or, more
correctly, pyrazinoic acid) acts by binding and inhibiting a specific
macromolecular target. The simplest assumption compatible with our
results would be that the accumulation of these aromatic anions exerts
a nonspecific inhibitory effect on cellular metabolism (27,
29). However, the bacteriostatic effect of these compounds may
also involve an additional mechanism. Thus, when PzaA and PncA
hydrolyze the parent amides in the cell, ammonia will be produced in
addition to the aromatic acids. The ammonia can be assimilated in
anabolic metabolism or can diffuse out of the mycobacterial cell, which
will result in acidification of the cytoplasm. Most bacterial cells
have a mechanism to reverse such cytosolic acidification; for example,
in E. coli, intracellular pH homeostasis is maintained at an
acidic external pH by its potassium transport systems (31), and in Streptococcus faecalis, the activity of
H+-ATPase is critical in the maintenance of neutral
cytosolic pH (17). During adaptation to acidic external pH,
which tends to acidify the cytoplasm, several proteins are induced,
some of which assist in regulating intracellular pH homeostasis
(18). These mechanisms may be sufficient to keep the
cytosolic pH neutral, even when ammonia is rapidly depleted and/or lost
from the cytoplasm. However, there are two possible complications that
could hinder the pH homeostasis in M. tuberculosis treated
with PZA and other aromatic acid amides. First, even in the absence of
these amides, the machinery that reverses cytosolic acidification may
already be working close to its capacity in the acidic medium used,
because under these conditions the efflux of protons will become
difficult and a spontaneous influx of protons from the medium could
possibly occur. Second, M. tuberculosis displays a
pronounced sensitivity to acidic pH compared to that of other
mycobacteria such as M. avium, both in vitro (3, 13,
33) and within the environment provided by the host cell vacuole
(8), suggesting that M. tuberculosis may not be
very competent in correcting the acidification of the cytoplasm. We
therefore think that in addition to the accumulation of aromatic
anions, acidification of the cytoplasm, or at least the metabolic
consequences of reversing such acidification, may contribute to the
inhibitory effects of these aromatic amides. The intracellular pH of
M. tuberculosis was shown to remain neutral following a 2-h
incubation at pH 5.0 with PZA at 50 µg/ml (39), but the
acid response machinery of this organism may be functioning at maximum
capacity under such conditions. It will therefore be of interest to
examine the intracellular pH of M. tuberculosis treated with
higher concentrations of PZA or that of M. tuberculosis expressing the pzaA gene under such conditions.
This work was supported by the Glaxo Wellcome Action TB
Initiative. V.M. was also supported by the South African Medical
Research Council, the National Research Foundation and the South
African Institute for Medical Research.
We thank Jo Michael and Chris van der Westhuizen for assistance with
the HPLC assays, Bhavna Gordhan for assistance with targeted gene
knockout, André Trollip for assistance with the BACTEC tests, Bill Jacobs for providing the pYUB328::H37Rv library, Ken Duncan and Ruth McAdam for advice and encouragement, and anonymous reviewers for constructive comments.
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Abstract
Introduction
