Pyrazinamide Inhibits Trans-Translation in Mycobacterium

tuberculosis
Wanliang Shi et al.
Science 333, 1630 (2011);
DOI: 10.1126/science.1208813

This copy is for your personal, non-commercial use only.

If you wish to distribute this article to others, you can order high-quality copies for your
colleagues, clients, or customers by clicking here.

Downloaded from www.sciencemag.org on November 6, 2012

Permission to republish or repurpose articles or portions of articles can be obtained by
following the guidelines here.

The following resources related to this article are available online at

www.sciencemag.org (this information is current as of November 6, 2012 ):

Updated information and services, including high-resolution figures, can be found in the online
version of this article at:
http://www.sciencemag.org/content/333/6049/1630.full.html
Supporting Online Material can be found at:
http://www.sciencemag.org/content/suppl/2011/08/10/science.1208813.DC1.html
A list of selected additional articles on the Science Web sites related to this article can be
found at:
http://www.sciencemag.org/content/333/6049/1630.full.html#related
This article cites 33 articles, 16 of which can be accessed free:
http://www.sciencemag.org/content/333/6049/1630.full.html#ref-list-1
This article has been cited by 14 articles hosted by HighWire Press; see:
http://www.sciencemag.org/content/333/6049/1630.full.html#related-urls
This article appears in the following subject collections:
Microbiology
http://www.sciencemag.org/cgi/collection/microbio

Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the
American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright
2011 by the American Association for the Advancement of Science; all rights reserved. The title Science is a
registered trademark of AAAS.
 REPORTS
cleotidyltransferase and guanosine pentaphosphate
Pyrazinamide Inhibits Trans-Translation synthetase), a component of the RNA degrado-

in Mycobacterium tuberculosis
some involved in mRNA degradation. Because
of the known biochemical functions of RpsA, we
focused our attention first on understanding the
Wanliang Shi,1 Xuelian Zhang,2 Xin Jiang,3 Haiming Yuan,2 Jong Seok Lee,4 Clifton E. Barry 3rd,5 implications of POA binding to this protein.
Honghai Wang,2 Wenhong Zhang,6 Ying Zhang1,6* Overexpression of the wild-type RpsA in
M. tuberculosis caused a fivefold increase in
Pyrazinamide (PZA) is a first-line tuberculosis drug that plays a unique role in shortening the duration of the minimum inhibitory concentration (MIC) of
tuberculosis chemotherapy. PZA is hydrolyzed intracellularly to pyrazinoic acid (POA) by pyrazinamidase PZA (MIC = 500 mg ml1) compared with that
(PZase, encoded by pncA), an enzyme frequently lost in PZA-resistant strains, but the target of POA in of the vector control and the parental M. tuber-
Mycobacterium tuberculosis has remained elusive. Here, we identify a previously unknown target of POA as culosis strain (MIC = 100 mg ml1) at pH = 5.5.
the ribosomal protein S1 (RpsA), a vital protein involved in protein translation and the ribosome-sparing The susceptibility of the RpsA overexpressing
process of trans-translation. Three PZA-resistant clinical isolates without pncA mutation harbored RpsA M. tuberculosis strain to other drugs, including
mutations. RpsA overexpression conferred increased PZA resistance, and we confirmed that POA bound to INH, rifampin, streptomycin, kanamycin, and
RpsA (but not a clinically identified DAla mutant) and subsequently inhibited trans-translation rather than norfloxacin, was not affected.
canonical translation. Trans-translation is essential for freeing scarce ribosomes in nonreplicating Most PZA-resistant M. tuberculosis strains
organisms, and its inhibition may explain the ability of PZA to eradicate persisting organisms. have mutations in pncA that prevent conversion

Downloaded from www.sciencemag.org on November 6, 2012

of PZA to POA (1113); however, a few PZA-
yrazinamide (PZA) is a first-line tubercu- boxylate anion, where it is excreted by a weak resistant strains have been reported that do not

P losis (TB) drug used in combination with

isoniazid (INH), ethambutol, and rifampin
for the treatment of drug-susceptible TB and
frequently for multidrug-resistant TB (1). PZA
shortens TB treatment from the 9 to 12 months
required before its introduction to the current
standard of 6 months, often referred to as short
course chemotherapy (2). Despite PZAs power-
ful in vivo sterilizing activity, it has no apparent
activity for actively growing TB bacilli under nor-
mal culture conditions at neutral pH (3). It is in-
stead preferentially active against nonreplicating
persister bacteria with low metabolism at acid pH
in vitro (4) or in vivo during active inflammation
(5). Although several new drug candidates are cur-
rently in clinical development (6), they all have to
be used together with PZA for optimal efficacy in
the mouse model of TB infection (7, 8).
Despite its important role in shortening TB
therapy, the mechanism of action of PZA is
poorly understood (9). Structurally, PZA is an
analog of nicotinamide, which, like INH (10),
is a prodrug, requiring conversion into its ac-
tive form pyrazinoic acid (POA) by the bacterial
pyrazinamidase (PZase) (11). Mutation in the pncA
gene encoding the PZase (11) is the major mech-
anism for PZA resistance in M. tuberculosis
(1113). PZA is believed to enter M. tuberculosis
by passive diffusion, where it is converted to
POA by the PZase. POA is an acid with a pKa
(where Ka is the acid dissociation constant) of 2.9
and is therefore trapped within the cell as the car-
1
W. Harry Feinstone Department of Molecular Microbiology and
Immunology, Bloomberg School of Public Health, Johns Hopkins
University, Baltimore, MD 21205, USA. 2State Key Laboratory of
Genetic Engineering, School of Life Sciences, Fudan University,
Shanghai 200433, China. 3Department of Medical Microbiology
and Immunology, Shanghai University of Traditional Chinese
Medicine, Shanghai 201203, China. 4International Tuberculosis
Research Center, Changwon 475-1, Korea. 5Tuberculosis Research
Section, National Institute of Allergy and Infectious Diseases
(NIAID), National Institutes of Health, Bethesda, MD 20892,
USA. 6Department of Infectious Diseases, Huashan Hospital,
Fudan University, Shanghai 200040, China.
*To whom correspondence should be addressed. E-mail:
yzhang@jhsph.edu
efflux pump and passive diffusion (14). Small
amounts of protonated POA capable of diffusion
across the membrane have been proposed to
cause collapse of the proton gradient, reducing
membrane potential and affecting membrane
transport (15). The observations that energy in-
hibitors such as N,N-dicyclohexylcarbodiimide
(DCCD) (an F1F0 adenosine triphosphatase in-
hibitor) (15), as well as the drug candidate TMC207,
synergize with PZA in vitro (7, 16) provide some
support for this model. However, the molecular
target of PZA is unknown. Although fatty acid
synthase-I has been proposed as a target of PZA
on the basis of studies with an analog (5-Cl-PZA)
(17), a subsequent study negated this thesis (18).
Binding studies using a POA derivative, 5-
hydroxyl-2-pyrazinecarboxylic acid, and ethanol-
amine as control extracted several proteins from
M. tuberculosis cell lysates that bound to POA
(Fig. 1). In contrast, no proteins bound to the con-
trol column, indicating that the proteins bound
specifically to POA. Mass spectrometry analysis
and subsequent database searches identified a
major POA binding protein as RpsA (table S1), the
largest 30S ribosomal protein S1 (Rv1630), along
with three other proteins (Rv2731, Rv2783c, and
Rv3169) from M. tuberculosis. Two of these proteins
(Rv2731 and Rv3169) are conserved hypothetical
proteins of unknown function, whereas Rv2783c
is a bifunctional enzyme (polyribonucleotide nu-
Fig. 1. M. tuberculosis
whole-cell lysates were
loaded onto the POA-
linked and control col-
umns, and the proteins
that bound to POA (A)
and the control column
(B) were analyzed by
SDSpolyacrylamide gel
electrophoresis. Lane M,
protein ladder; lane 1,
whole-cell lysate; 2, flow-
through fraction; 3, wash fraction; and 4, elution fraction. The bands indicated by arrowheads are Rv2783c,
RpsA, Rv2731, and Rv3169, respectively.
have pncA mutations (12, 13). We previously
identified a low-level PZA-resistant M. tubercu-
losis clinical isolate DHM444 (MIC = 200 to
300 mg ml1 PZA compared with 100 mg ml1
in the sensitive control strain H37Rv) that lacked
pncA mutations (12), suggesting that its resistance
may be caused by alterations in RpsA. Sequenc-
ing the rpsA gene from this strain revealed that
it contained a 3base pair GCC deletion at the nu-
cleotide position 1314, resulting in loss of an ala-
nine at amino acid 438 (DA438) in the C terminus of
RpsA (Fig. 2), a region that is not considered to be
strictly required for protein synthesis in vivo (19).
Sequencing of an additional five PZA-resistant
M. tuberculosis clinical isolates without pncA mu-
tations identified two additional strains bearing
RpsA mutations of T5S5 (T5S) and D123A
(20) in one strain and V262M in another strain.
To determine whether the mutant RpsA from
the PZA-resistant strain DHM444 has any defect
in POA binding, we overexpressed and purified
the mutant RpsA (RpsADA438), the wild-type
M. tuberculosis RpsA, and the M. smegmatis RpsA
and measured their ability to bind to POA by using
isothermal titration calorimetry (ITC). The wild-
type M. tuberculosis RpsA was found to specifi-
cally bind to POA (Fig. 2B, VI) with a Ka = 7.53
1 6 T 2.21 106 M1, enthalpy DH = 410.9 T 8.693
10
kcalmol1, and entropy DS = 27.6 calmol1K1
(Fig. 2B, bottom graph) but did not bind the

1630 16 SEPTEMBER 2011 VOL 333 SCIENCE www.sciencemag.org


prodrug PZA (Fig. 2B, V). However, the mutant
RpsADA438 from the PZA-resistant strain DHM444
failed to bind either POA or PZA (Fig. 2B, IVand
III), and the RpsA from naturally PZA-resistant
M. smegmatis bound to POA only weakly (Fig.
2B, II). The mutant RpsADA438 did not bind to
POAwhen subjected to the affinity column described
above. Because M. tuberculosis DHM444 mu-
tant RpsADA438 and M. smegmatis RpsA showed
little or no binding to POA (Fig. 2B), it may be
inferred that POA binds to the C terminus of the
wild-type M. tuberculosis RpsA (Fig. 2A). From
the protein sequence alignment of RpsA of dif-
ferent mycobacterial species, the C-terminal region,
where the mutation occurs in the PZA-resistant
M. tuberculosis strain DHM444, is also the re-
gion that varies most between PZA-sensitive and
-resistant mycobacterial species (Fig. 2A), indi-
stress survival, virulence, and recovery from nu-
trient starvation (26). We therefore evaluated
whether the mutant RpsA has any deficiency
in tmRNA binding compared with the wild-
type M. tuberculosis RpsA and whether POA
affects the interaction between RpsA and tmRNA.
Specific binding of RpsA to tmRNA was assessed
by changes in gel mobility in the presence of
excess tmRNA. The wild-type M. tuberculosis
RpsA and the mutant RpsADA438 both bound to
the tmRNA in the absence of POA (Fig. 3A, B),
although the mutant appeared to bind more
weakly overall (Fig. 3B). However, when POA
was added to the system, it inhibited the wild-type
M. tuberculosis RpsA from binding to tmRNA at
its MIC concentration of 50 mg ml1 (Fig. 3A) but
did not inhibit binding of the mutant RpsADA438
(Fig. 3B). The prodrug PZA and the control drug
REPORTS
observed from message carrying the rare codon
cluster (Fig. 3C, lane 5). POA inhibited the trans-
translation of DHFR with the rare codon cluster at
concentrations greater than 25 mg ml1 (Fig. 3C,
lanes 1 to 3). However, POA did not affect the
translation of the wild-type DHFR gene even at
100 mg ml1 with M. tuberculosis ribosomes
(Fig. 3D), nor did it inhibit the trans-translation
of the template bearing the rare codon cluster with
either M. smegmatis or E. coli ribosomes (Fig. 3,
E and F). These observations support that POA
does not inhibit M. tuberculosis SmpB or tmRNA
function but instead directly binds to RpsA to
cause the inhibition of trans-translation. Under the
same conditions, nicotinic acid (NA) (50 mg ml1),
an analog of POA as a control compound, did
not inhibit trans-translation (Fig. 3C, lane 11).
The M. tuberculosis RpsA protein consists

Downloaded from www.sciencemag.org on November 6, 2012

cating that changes in this region may alter PZA
susceptibility.
RpsA is essential for translation, binding
directly both to the ribosome and upstream se-
quences of mRNA (21). In addition to its func-
tion in translation, the C terminus of the RpsA is
also involved in trans-translation by specifical-
ly binding to transfer-messenger RNA (tmRNA)
(2224). A Streptomyces homolog of one of the
other proteins identified by the POA affinity
column, Rv2783c, has been identified by af-
finity chromatography on a tmRNA column and
has also been implicated in trans-translation (25).
Trans-translation is a process involved in res-
cuing ribosomes that have stalled while in the
process of decoding mRNA and involves dis-
placement of the stalled message by tmRNA fol-
lowed by translation of a short tag encoded by
tmRNA targeting the stalled protein for degrada-
tion. Trans-translation has been associated with
INH had no effect on the binding of wild-type
or mutant RpsA to the tmRNA (Fig. 3B).
In Escherichia coli, RpsA is known to bind
to the 3 terminus of the mRNA-like portion
of the tmRNA (22), and, because POA bound
to M. tuberculosis RpsA (Fig. 2B), we tested
whether POA inhibited the translation or the
trans-translation function of RpsA. POA had
no effect on conventional protein synthesis (Fig.
3D). We then tested the effects of POA on trans-
translation by using the target gene coding for
dihydrofolate reductase (DHFR) in an in vitro
cell-free translation system containing ribosomes
from M. tuberculosis, M. smegmatis, or E. coli.
In the presence of a rare codon cluster in the target
DHFR gene, ribosomes stall and translation is
blocked (27). When recombinant M. tuberculosis
SmpB and in vitro transcribed M. tuberculosis
tmRNA were added, a higher molecular weight
protein with the tmRNA-derived peptide tag was
of four imperfect repeats of the S1-like domain
thought to function directly in binding of RNA,
bridging the mRNA (or tmRNA) template and
the head of the 30S ribosome, and is terminated
at the C terminus by a 117amino acid segment.
The E. coli RpsA protein contains six repeating S1
domains, with the two N-terminal domains re-
quired for ribosome binding. The C-terminal do-
mains have been shown to be specifically involved
in trans-translation; thus the deletion of Ala438
observed in one clinical isolate is consistent with
POA exerting an effect on ribosome rescue. The
deletion occurs within a region homologous (35%
identical) to the protein Xrcc4 involved in illegit-
imate DNA recombination that forms an ex-
tensive a helix (28). Homology modeling of the
M. tuberculosis C terminus based on the Xrcc4
structure (Protein Data Bank identification code
1FU1) within the highly homologous region sug-
gests that Ala438 lies within several turns in an a

Fig. 2. RpsA alignment and ITC titration of RpsA and POA. (A) Alignment of RpsA from
M. tuberculosis H37Rv, M. tuberculosis PZA-resistant strain DHM444 and M. smegmatis.
R1 to R4 represent the four homologous RNA-binding domains in RpsA. Colored vertical
lines in gray boxes indicate sequence variations in the highly conserved RpsA sequences
compared with the wild-type M. tuberculosis RpsA sequence. The expanded region shows
the variability in amino acid sequence (20) in the C terminus of RpsA among mycobacterial
species. The red arrowhead at the position 438 amino acid residue indicates the deletion of
alanine in the C-terminal region of the mutant RpsA. (B) ITC titration of POA binding to
RpsA. ITC binding studies indicate that POA bound to the M. tuberculosis H37Rv RpsA [wild
type (WT)] (line VI), but not DHM444 RpsA (Mutant) (line IV) and only weakly with the M.
smegmatis RpsA (M. smeg) (line II). PZA did not bind to wild-type RpsA (line V) or mutant
RpsA (line III). The top graph shows raw data, and the y axis indicates the heat released per
second during RpsA and POA or PZA binding. The bottom graph shows integrated heat in
each injection of POA together with a fit, and the y axis is expressed by heat release per mole in each injection. The association constants were obtained from
fits of POA binding with M. tuberculosis RpsA (WT).

www.sciencemag.org SCIENCE VOL 333 16 SEPTEMBER 2011 1631

 REPORTS
helix connecting a small globular domain to a
long helical stalk potentially involved in dimeriza-
tion. This region has been proposed to be the pri-
mary site of nucleic acid interaction in Xrcc4. There
are numerous basic residues along the helical face
and particularly in the short linker between this
helix and the globular domain potentially involved
in DNA binding, especially Arg423, Arg424, His425,
and Lys426, that may interact directly with POA
and disrupt the site of tmRNA interaction.
Trans-translation is dispensable during active
growth conditions but becomes important for
bacteria in managing stalled ribosomes or dam-
aged mRNA and proteins under stress conditions
(29, 30). It is required for stress survival and
pathogenesis in some bacteria (26). The levels
of RpsA are known to correlate with growth rate
and stress conditions that halt bacterial growth
and down-regulate RpsA in bacteria (31). When
POA binds to RpsA, it prevents binding of tmRNA,
which therefore cannot rescue stalled ribosomes.
PZA inhibition of the trans-translation process may
therefore interfere with survival under stress-
ful, nonreplicating conditions in M. tuberculosis.
The finding that POA binds to RpsA and inhibits
the trans-translation process helps to explain how
diverse stress conditions, such as starvation, acid
pH, hypoxia, and energy inhibitors and other
drugs could all potentiate PZA activity (15, 32). On
the basis of our current and previous studies, we
propose a revised model for the mode of action of
PZA that can better explain all the peculiar features
of this intriguing drug (fig. S2). Trans-translation
may provide a valuable pathway for developing
new drugs to sterilize infection more rapidly.
References and Notes
1. World Health Organization (WHO), WHO Report 2010:
Global Tuberculosis Control (2010).
2. D. A. Mitchison, Tubercle 66, 219 (1985).
3. M. S. Tarshis, W. A. Weed Jr., Am. Rev. Tuberc. 67,
391 (1953).
4. W. McDermott, R. Tompsett, Am. Rev. Tuberc. 70, 748 (1954).
5. R. M. McCune, F. M. Feldmann, H. P. Lambert,
W. McDermott, J. Exp. Med. 123, 445 (1966).
6. W. W. Yew, M. Cynamon, Y. Zhang, Expert Opin.
Emerg. Drugs 16, 1 (2011).
7. K. Andries et al., Science 307, 223 (2005);
10.1126/science.1160753.
8. E. Nuermberger et al., Antimicrob. Agents Chemother.
52, 1522 (2008).
9. Y. Zhang, D. Mitchison, Int. J. Tuberc. Lung Dis. 7, 6 (2003).
10. Y. Zhang, B. Heym, B. Allen, D. Young, S. Cole, Nature
358, 591 (1992).

Downloaded from www.sciencemag.org on November 6, 2012

11. A. Scorpio, Y. Zhang, Nat. Med. 2, 662 (1996).
12. A. Scorpio et al., Antimicrob. Agents Chemother. 41,
540 (1997).
13. S. J. Cheng, L. Thibert, T. Sanchez, L. Heifets, Y. Zhang,
Antimicrob. Agents Chemother. 44, 528 (2000).
14. Y. Zhang, A. Scorpio, H. Nikaido, Z. Sun, J. Bacteriol.
181, 2044 (1999).
15. Y. Zhang, M. M. Wade, A. Scorpio, H. Zhang, Z. Sun,
J. Antimicrob. Chemother. 52, 790 (2003).
16. M. Ibrahim et al., Antimicrob. Agents Chemother. 51,
1011 (2007).
17. O. Zimhony, J. S. Cox, J. T. Welch, C. Vilchze,
W. R. Jacobs Jr., Nat. Med. 6, 1043 (2000).
18. H. I. Boshoff, V. Mizrahi, C. E. Barry 3rd, J. Bacteriol.
184, 2167 (2002).
19. I. V. Boni, V. S. Artamonova, M. Dreyfus, J. Bacteriol.
182, 5872 (2000).
20. Single-letter abbreviations for the amino acid residues
are as follows: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; K, Lys;
L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; and V, Val.
21. M. Bycroft, T. J. Hubbard, M. Proctor, S. M. Freund,
A. G. Murzin, Cell 88, 235 (1997).
22. I. K. Wower, C. W. Zwieb, S. A. Guven, J. Wower, EMBO J.
19, 6612 (2000).
23. S. Barends, A. W. Karzai, R. T. Sauer, J. Wower, B. Kraal,
J. Mol. Biol. 314, 9 (2001).
24. M. Saguy, R. Gillet, P. Skorski, S. Hermann-Le Denmat,
B. Felden, Nucleic Acids Res. 35, 2368 (2007).
25. K. Mikulk et al., Proteomics 8, 1429 (2008).
26. K. C. Keiler, Annu. Rev. Microbiol. 62, 133 (2008).
27. X. Li, R. Hirano, H. Tagami, H. Aiba, RNA 12, 248 (2006).
28. M. S. Junop et al., EMBO J. 19, 5962 (2000).
29. M. Thibonnier, J. M. Thiberge, H. De Reuse, PLoS ONE 3,
e3810 (2008).
30. A. Muto et al., Genes Cells 5, 627 (2000).
31. J. M. Lambert, G. Boileau, J. G. Howe, R. R. Traut,
J. Bacteriol. 154, 1323 (1983).
Fig. 3. (A) Concentration-dependent inhibition of tmRNA binding to WT M. tuberculosis RpsA by POA 32. Y. Zhang, S. Permar, Z. Sun, J. Med. Microbiol. 51,
(lanes 2 to 7). tmRNA from M. tuberculosis was used as the RNA-alone control (lane 1). The WT RpsA 42 (2002).
interaction with tmRNA was not affected by PZA (200 mg ml1) (lane 8) or INH (1 mg ml1) (lane 9). (B) Acknowledgments: This work was supported in part by NIH
tmRNA had impaired binding to the mutant RpsA (lane 2), and POA at different concentrations did not grant AI44063, in part by the intramural research program
of the NIAID, NIH, and by National Key Technologies
inhibit the interaction of the DHM444 mutant RpsA with tmRNA (lanes 3 to 7). The mutant RpsA interaction Research and Development Program of China
with tmRNA was not affected by PZA (200 mg ml1) (lane 8) or INH (1 mg ml1) (lane 9). (C) POA at 100, (2008ZX10003003). We thank D. E. Griffin and M. J. Klag
50, and 25 mg ml1 inhibited trans-translation of the DHFR product in a concentration-dependent manner for encouragement and support. Johns Hopkins University
in the in vitro system that contained ribosomes, tmRNA, and recombinant SmpB from M. tuberculosis and has filed a patent on detection of RpsA mutation as a marker
template pDHFR-8AGG rare codons that are required for trans-translation (lanes 1 to 3). Arrowheads for pyrazinamide resistance and use of trans-translation
indicate the trans-translation product DHFR was still present with a low concentration of POA at 12.5 mg ml1 pathway as a target for development of new antibiotics
against TB persister bacteria.
(lane 4) and in the absence of POA (lane 5). POA at different concentrations did not inhibit canonical
translation in the in vitro translation system using ribosomes from M. tuberculosis and template pDHFR with a Supporting Online Material
stop codon (lanes 6 to 10). NA, an analog of POA as a control, did not inhibit trans-translation in the above www.sciencemag.org/cgi/content/full/science.1208813/DC1
Materials and Methods
system (lane 11). POA failed to inhibit the trans-translation of DHFR using ribosomes from M. smegmatis
Figs. S1 and S2
(D) or ribosomes from E. coli (E) in the trans-translation system that contained tmRNA and recombinant Table S1
SmpB from M. tuberculosis and template pDHFR-8AGG rare codons. (F) Concentration-dependent inhibi- References
tion of trans-translation by POA with M. tuberculosis ribosomes and a DHFR template with a rare codon 24 May 2011; accepted 29 July 2011
cluster. This bar graph is from a densitometry scan of the samples in the same type of experiment as in Published online 11 August 2011;
(C), lanes 1 to 5, performed independently five times (P < 0.03, n = 5). Error bars indicate SEM. 10.1126/science.1208813

1632 16 SEPTEMBER 2011 VOL 333 SCIENCE www.sciencemag.org