Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells

Article abstract of DOI:10.1021/acsinfecdis.7b00028

The emergence of Mycobacterium tuberculosis (MTB) strains that are resistant to most or all available antibiotics has created a severe problem for treating tuberculosis and has spurred a quest for new antibiotic targets. Here, we demonstrate that trans-translation is essential for growth of MTB and is a viable target for development of antituberculosis drugs. We also show that an inhibitor of trans-translation, KKL-35, is bactericidal against MTB under both aerobic and anoxic conditions. Biochemical experiments show that this compound targets helix 89 of the 23S rRNA. In silico molecular docking predicts a binding pocket for KKL-35 adjacent to the peptidyl-transfer center in a region not targeted by conventional antibiotics. Computational solvent mapping suggests that this pocket is a druggable hot spot for small molecule binding. Collectively, our findings reveal a new target for antituberculosis drug development and provide critical insight on the mechanism of antibacterial action for KKL-35 and related 1,3,4-oxadiazole benzamides.

Full text of DOI:10.1021/acsinfecdis.7b00028

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Article
Ribosome Rescue Inhibitors Kill Actively Growing and Non-
replicating Persister Mycobacterium tuberculosis Cells
John N Alumasa, Paolo Solano Manzanillo, Nicholas D. Peterson, Tricia
Lundrigan, Anthony D. Baughn, Jeffery S. Cox, and Kenneth C. Keiler
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00028 • Publication Date (Web): 01 Aug 2017
Downloaded from http://pubs.acs.org on August 2, 2017
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Ribosome Rescue Inhibitors Kill Actively Growing and Non-
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replicating Persister Mycobacterium tuberculosis Cells
†
§
¶
§
John N. Alumasa, Paulo S. Manzanillo, Nicholas D. Peterson, Tricia Lundrigan,
¶
§
*,†
Anthony D. Baughn, Jeffery S. Cox, Kenneth C. Keiler.
†
4
01 Althouse Laboratory, Department of Biochemistry and Molecular Biology, The
Pennsylvania State University, University Park, PA 16802.
§
3
75E Li Ka Shing Center, Department of Molecular and Cell Biology, University of
California, Berkeley, #3370, Berkeley, CA 94720.
¶
Department of Microbiology and Immunology, Microbiology Research Facility, Rm4ꢀ
rd
1
15, University of Minnesota, 689 23 Ave SE, Minneapolis, MN 55455.
*
email: kkeiler@psu.edu
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The emergence of Mycobacterium tuberculosis (MTB) strains that are resistant to most
or all available antibiotics has created a severe problem for treating tuberculosis, and
has spurred a quest for new antibiotic targets. Here we demonstrate that transꢀ
translation is essential for growth of MTB and is a viable target for development of antiꢀ
tuberculosis drugs. We also show that an inhibitor of transꢀtranslation, KKLꢀ35, is
bactericidal against MTB under both aerobic and anoxic conditions. Biochemical
experiments show that this compound targets helix 89 of the 23S rRNA. In silico
molecular docking predicts a binding pocket for KKLꢀ35 adjacent to the peptidylꢀtransfer
center in a region not targeted by conventional antibiotics. Computational solvent
mapping suggests that this pocket is a druggable hot spot for small molecule binding.
Collectively, our findings reveal a new target for antiꢀtuberculosis drug development and
provide critical insight on the mechanism of antibacterial action for KKLꢀ35 and related
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,3,4ꢀoxadiazole benzamides.
KEYWORDS: Mycobacterium tuberculosis, antibiotics, 1,3,4ꢀoxadiazoles, ribosome
rescue
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Over 1.8 billion people are infected with MTB worldwide, 10% of whom are predicted to
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develop the active disease. These infections produce 1.5 million deaths annually.
Antibiotic treatment has reduced the mortality rate of MTB, but the rise of multiꢀdrug
resistant (MDRꢀTB) and extensively drug resistant (XDRꢀTB) strains has raised an
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urgent need for new antibiotics. Drugs with new chemical scaffolds and new molecular
targets are particularly desirable because they are less likely to be counteracted by
existing resistance mechanisms in clinical strains. The transꢀtranslation pathway for
rescue of nonstop ribosomes presents a potential target for antibiotics because it is
3,4
required for viability or virulence in many pathogens, and is not found in metazoans.
transꢀTranslation is used to rescue ribosomes that are trapped at the 3’ end of an
mRNA that has no inꢀframe stop codon to allow termination. During transꢀtranslation, a
specialized RNA molecule, tmRNA, and a small protein, SmpB, recognize these
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nonstop translation complexes. tmRNA acts first like a tRNA to accept the nascent
polypeptide, and then a reading frame within tmRNA is inserted into the mRNA channel.
Translation resumes using tmRNA as a message and terminates at a stop codon within
tmRNA, releasing the ribosome and a protein with the tmRNAꢀencoded peptide
4ꢀ6
sequence at its C terminus. Multiple proteases recognize the tmRNAꢀencoded peptide
and rapidly degrade the protein, thereby clearing both the stalled ribosome and the
7,8
incomplete polypeptide. Nonstop translation complexes occur frequently in bacteria
because they arise both from damaged mRNAs that lack a stop codon (nonstop mRNA)
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and from cleavage of mRNAs before or during translation. In some bacteria, transꢀ
translation is the only mechanism known to rescue nonstop translation complexes, and
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both tmRNA and SmpB are essential for viability. Other species have the ArfA or ArfB
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backup systems that can release ribosomes from nonstop translation complexes in the
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absence of transꢀtranslation.
The MTB genome does not encode ArfA or ArfB,
suggesting that transꢀtranslation is likely to be essential, and therefore a good candidate
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for targetꢀbased drug development. Despite a report that the antiꢀtuberculosis drug
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pyrazinamide targets transꢀtranslation, careful experiments have shown that transꢀ
translation is not inhibited by pyrazinamide or its active metabolite, pyrazinoic acid, in
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vitro or in vivo . Therefore, there are currently no antibiotics that target this pathway.
RESULTS and DISCUSSION
SmpB is essential in M. tuberculosis
To assess the importance of transꢀtranslation in MTB, we first attempted to delete the
genes encoding tmRNA (ssrA) and SmpB (smpB) from the MTB chromosome using
allelic exchange, but we could not obtain a deletion of either gene. To rigorously
determine if transꢀtranslation is essential in MTB, we engineered a strain
(TetpsmpB:rTetR) in which the expression of smpB at its chromosomal locus is
controlled by the tet repressor (TetR), such that addition of anhydrotetracycline (ATc)
shuts off SmpB production (Fig. 1a). TetpsmpB:rTetR cells grew at a similar rate to
wildꢀtype cells in the absence of ATc, but addition of ATc severely inhibited growth (Fig.
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b). Addition of ATc had no effect on growth of wildꢀtype cells or control strains lacking
tetR (Fig. 1b). These data indicate that SmpB is required for growth of MTB in culture.
This conclusion is consistent with data from saturating transposon mutagenesis screens
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that failed to recover insertions in ssrA or smpB, and with data demonstrating that the
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chromosomal copy of ssrA could only be deleted in the presence of an additional copy
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of the gene. A MTB strain deleted for smpB has been reported, but wholeꢀgenome
sequencing of this strain showed that the smpB coding sequence was present (Fig. 1c,
GenBank accession numbers: SAMN05907893 and SAMN05907849). qRTꢀPCR to
detect the SmpB mRNA in this deletion strain, ∆smpB::dif, revealed that the gene is
expressed (Fig. 1d). Taken together, these results demonstrate that transꢀtranslation is
essential for growth in MTB.
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KKL-35 kills growing and non-replicating persister cells of M. tuberculosis
KKLꢀ35 (Fig. 2a) and related 1,3,4ꢀoxadiazole benzamides, were identified by cellꢀ
based screening for inhibitors of transꢀtranslation, and were found to have broadꢀ
17,18
spectrum antibacterial activity.
To assess the ability of KKLꢀ35 to inhibit growth of
MTB, MIC and plating assays were performed. KKLꢀ35 inhibited growth of MTB cultures
with a MIC of 1.6 ꢁg/ml, and plating assays showed that 8.0 µg/ml KKLꢀ35 killed >90%
of MTB cells within seven days (Fig. 2b, c). Tuberculosis infections can be difficult to
treat in part because MTB cells can enter a nonreplicating persister state in which they
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are not sensitive to most antibiotics. We used a hypoxia persistence model to evaluate
the activity of KKLꢀ35 against nonreplicating persister bacilli. 1.6 ꢁg/ml KKLꢀ35 killed
>90 % of nonreplicating MTB cells under these conditions, demonstrating that KKLꢀ35
was equally active against nonreplicating MTB cells as it was against actively growing
cells (Fig. 2d). The observed activity against persister cells suggests that transꢀ
translation is required for survival in this state, and indicates that transꢀtranslation
inhibitors may be effective against multiple physiological states of MTB during infection.
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Despite its potency against MTB, KKLꢀ35 and its analogs displayed no cytotoxic activity
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against HeLa cells (Table 1) or HepG2 cells at concentrations >20ꢀfold MIC. The
combination of significant antibiotic activity against MTB and low cytotoxic activity for
KKLꢀ35 indicates that this compound is a promising antiꢀtubercular agent.
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KKL-35 targets helix 89 of 23S rRNA
To determine the molecular target for KKLꢀ35, we designed and synthesized an analog,
KKLꢀ2098, incorporating a photoꢀreactive azide group and a terminal alkyne moiety
(Fig. 2a, Scheme 1). The MICs for KKLꢀ2098 against Mycobacterium smegmatis and
other bacterial species were similar to those for KKLꢀ35 (Table 1). The similarity in
activity suggests that the structural modifications in this analog did not significantly alter
antibiotic properties or target binding of the compound. We therefore used M.
smegmatis for the KKLꢀ35 target identification. Intracellular photoꢀaffinity labeling
followed by click bioconjugation was used in the molecular target identification process
20,21
(Fig. 3).
Table 1. Comparison of the antibacterial activity for KKL-35, KKL-40 and KKL-
2
098.
a
g
MIC (µg/ml)
Cytotoxicity
Compound
ID
b
c
d
e
f
HeLa
µg/ml)
B. anthracis
E. coli ∆tolC
S. flexneri
M. smegmatis
MTB
(
KKLꢀ35
KKLꢀ40
KKLꢀ2098
0.3 (0.1)
0.1 (0)
0.3 (0.1)
0.5 (0.1)
0.2 (0)
0.5 (0.1)
1.6 (0)
1.8 (0)
1.7 (0)
0.4 (0)
0.3 (0)
0.4 (0)
1.6 (0)
1.8 (0)
ND
> 31.8
> 31.8
ND
a
Data are averages from three independent assays each performed in triplicate (SD);
b
c
d
e
2
Sterne strain 34F2; Strain MG1655; Strain 2a 2457T; Strain mc 155 (ATCC
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00084); Erdman strain; From > 3 independent determinations; MICs for KKLꢀ35 and
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KKLꢀ40 for these strains have been previously reported. ; ND: not determined.
To facilitate isolation and visualization of the target we also synthesized a triꢀfunctional
probe, KKLꢀ2107 (Fig. 2a, Scheme 2), that incorporated an azide group, an affinity
conjugate and a fluorescent moiety. The target was identified by incubating KKLꢀ2098
with growing M. smegmatis cells and irradiating the culture with UV light to initiate
crossꢀlinking (Fig. 3). Following crossꢀlinking, the cells were lysed and click conjugation
was used to attach the fluorescent molecule (KKLꢀ2107) to the alkyne moiety of KKLꢀ
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098, facilitating purification and visualization of crossꢀlinked molecules. Analysis of
proteins using SDSꢀPAGE showed no fluorescent bands, indicating that KKLꢀ2098 was
not crossꢀlinked to a protein (Fig. S1). However, analysis of RNA preparations from
KKLꢀ2098–treated cells revealed a fluorescent band that coꢀmigrated with 23S rRNA on
agarose gels (Fig. 4a). Similar results were obtained when crossꢀlinking was repeated
with RNA extracts from M. tuberculosis and E. coli (Fig. S2). Primer extension assays
were used to confirm that KKLꢀ2098 was crossꢀlinked to 23S rRNA. Assays using RNA
from KKLꢀ2098–treated M. smegmatis cells reproducibly showed a prominent band that
was not present in control reactions using RNA from cells treated with KKLꢀ35 instead
of KKLꢀ2098 (KKLꢀ35 will not crossꢀlink but causes the same physiological response in
the cells) (Fig. 4b, Fig. S3). This band indicated that reverse transcriptase activity was
terminated after nucleotide 2505 (E. coli numbering), suggesting KKLꢀ2098 was crossꢀ
linked to nucleotide Ψ2504 (Fig. 4b). Primer extension on 23S rRNA from E. coli after
crossꢀlinking with KKLꢀ2098 indicated modification of nucleotide C2452 (Fig. S4), which
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base pairs with Ψ2504. Ψ2504 and C2452 are positioned at the base of H89, a
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structure that extends from the PTC to the factor binding site (Fig. 4c, d). Nucleotides
that border the PTC and those located at the base of H89 adjacent to the PTC (Fig. 5)
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are highly conserved and essential for efficient ribosome function in bacteria.
Mutation of 2504, C2452 or other nearby nucleotides, as well as conformational
changes in H89, have been shown to have moderate to severe effects on translation
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fidelity, ribosome function and/or cell growth.
Collectively, these data indicate that
the base of H89 is the target for KKLꢀ35 and related 1,3,4ꢀoxadiazole benzamides.
Docking and solvent mapping predict binding site for 1,3,4-oxadiazole
benzamides
Guided by results from the crossꢀlinking experiments, we performed in silico molecular
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docking studies using the AutodockꢀVina program. KKLꢀ35 was docked to a region of
the 50S ribosome encompassing the PTC and the fullꢀlength H89 to the edge of the
factorꢀbinding site (Fig. S5). The 6 lowest energy structures had KKLꢀ35 in the same
pocket at the base of H89 near nucleotides Ψ2504 and C2452 (Fig. 6). The
conformation of KKLꢀ35 within this pocket varied, but in all cases the main contributors
to the binding energy based on the docked conformation were the predicted polar
interactions between KKLꢀ35 and H89 originating from the carbonyl oxygen atom and
the oxadiazole core (Fig. 6). The latter contribution is in agreement with experimental
evidence showing favorable electron donor capabilities for the 1,3,4ꢀoxadiazole ring as
29
a result of a large dipole moment generated by the nitrogen atoms. Docking
experiments using KKLꢀ2098 and KKLꢀ40 localized these compounds to the same
pocket, with similar docked conformations and binding energies as KKLꢀ35 (∆G = ꢀ8.8
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kcal/mol for KKLꢀ35, ꢀ9.0 kcal/mol for KKLꢀ2098, and ꢀ9.5 kcal/mol for KKLꢀ40) (Fig.
S5). In some structures the oxadiazoleꢀamide core was oriented in a conformation that
would place the azide group of KKLꢀ2098 in position to crossꢀlink with Ψ2504 and
C2452, but in others the orientation of the oxadiazoleꢀamide was reversed (Fig. S5).
The similar docked energies of the forward and reverse conformations are a result of
partial symmetry in the molecules based on the location Hꢀdonor and acceptor atoms in
the oxadiazoleꢀamide core, and the phenylꢀrings (Fig. S5). Therefore, the docking
studies predict a binding pocket for 1,3,4ꢀoxadiazole benzamides at the base of H89,
but do not specify the conformation of the molecules within this pocket. Because the
ribosome structure bound by 1,3,4ꢀoxadiazole benzamides may be subtly different from
the available crystal and cryoꢀEM structures, we cannot exclude the possibility that
these molecules bind in the PTC where they could contact the other face of the Ψ2504ꢀ
C2452 base pair. Other antibiotics, including linezolid, bind in the PTC close to Ψ2504
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and C2452. Unlike linezolid, KKLꢀ35 and other oxadiazoles do not inhibit translation ,
so they would have to bind within the PTC in a manner that inhibited transꢀtranslation
but not translation. Ongoing structural studies should provide more insight on the
binding site for 1,3,4ꢀoxadiazole benzamides.
We used a computational solvent mapping algorithm (FTMap) to assess solvent
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accessibility and druggability of H89. The FTMap server performs solvent mapping
using standard probes with variable chemical structures to identify probable drug
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binding hot spots within a macromolecule. These hot spots, typically located within
probeꢀcluster consensus sites (CS), are regions capable of binding a variety of
chemically diverse probes and are predicted to contribute significantly to the binding
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free energy.
Probe clustering was observed at several sites on H89 (Fig. 6d), but
the base region had one major hot spot which comprised four CSs (Fig. 6d, Fig. S6).
Structural alignment of docked KKLꢀ35 with solvent mapping revealed that the predicted
binding pocket for KKLꢀ35 superimposes with the solventꢀmapped hot spot with probe
cluster coverage along the entire KKLꢀ35 structure (Fig. 6e). These mapping data
together with the docking results for KKLꢀ35 support the presence of a druggable
binding pocket for 1,3,4ꢀoxadiazole benzamides at the base of H89 adjacent to the
PTC.
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KKL-35 selectivity suggests structural changes in the ribosome during rescue
The data presented here indicate that KKLꢀ35 bind to a highly conserved region of the
ribosome, and previous results showed that KKLꢀ35 inhibits transꢀtranslation but not
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translation initiation, elongation, or termination. Binding of a drug near the PTC might
be expected to interfere with translation and mutation of nucleotides that form the KKLꢀ
3
5 binding site (for example U2460, U2492, U2493) (Fig. 5b), lead to significantly
24ꢀ27
decreased PTC activity and impaired cell growth.
However, KKLꢀ35 did not inhibit
translation of mRNAs containing an inꢀframe stop codon when tested at concentrations
17
>
100ꢀfold above the IC for inhibiting transꢀtranslation in vitro. KKLꢀ2098 crossꢀlinked
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to 23S rRNA during in vitro translation of mRNAs containing an inꢀframe stop codon as
well as translation of nonstop mRNAs (Fig. S2), indicating that the binding site may be
accessible during normal translation. No crossꢀlinking was observed in reactions that
did not contain mRNA (Fig. S2), suggesting that the inhibitors bind a structure that is
only present during translation. How could binding of KKLꢀ35 to H89 inhibit transꢀ
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translation but not translation? One possible explanation is that binding of KKLꢀ35 could
introduce polar interactions that limit flexibility of H89, preventing structural changes that
are required for ribosome rescue but not for translation. This selectivity might also
explain the ability KKLꢀ35 to inhibit ribosome rescue by ArfA and ArfB in E. coli and C.
crescentus: these alternative rescue pathways recognize nonstop translation complexes
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in the absence of transꢀtranslation.
In summary, the 1,3,4ꢀoxadiazole benzamides present a unique chemical
scaffold that is distinct both in structure and mechanism of action from existing antiꢀ
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tuberculosis drugs.
KKLꢀ35 and its analogs display promising bactericidal activity
against actively growing and nonꢀreplicating persister cells of MTB while exhibiting
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minimal cytotoxicity against eukaryotic cells (Table 1). Collectively, these properties
make the 1,3,4ꢀoxadiazole benzamides good antiꢀtubercular drug candidates. In an
effort to circumvent current resistance trends, the druggable state of transꢀtranslation in
MTB presents an excellent opportunity to develop novel antiꢀtuberculosis drugs.
METHODS
Bacterial strains and growth conditions. M. tuberculosis H37Rv, ∆smpB::dif,
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∆smpB::dif::smpB and ∆ssrA::ssrA (gifts from Prof. Tanya Parish), and the Erdman
TMC 107 (ATCC 35801) strain were cultured at 37 °C in 7H9 media (Difco, Becton
Dickinson, Franklin lakes, NJ) supplemented with 10% OADC (Middlebrook), 0.5%
glycerol, and 0.05% TWEEN 80. Solid medium plates were prepared using 7H10 agar
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(
Difco) supplemented with 10% OADC (Middlebrook) and 0.5% glycerol. E. coli ∆tolC
(MG1655) was cultured in LB growth medium at 37 °C.
MIC and MBC determination for KKL-35 against M. tuberculosis. 1 ml culture was
grown in 5 ml bottles to OD600 = 0.0125 in 5 ml ink wells and KKLꢀ35 was added at
concentrations ranging from 0 to 1 mM. Cultures were incubated at 37 °C and the MIC
was recorded as the minimum concentration of drug that inhibited visible growth. The
MBC was obtained through CFUs, which were determined by plating serial dilutions of
cultures onto 7H10 agar plates. Plates were incubated for 3ꢀ4 weeks at 37 °C prior to
enumeration of CFUs.
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Assessment of growth inhibition. Autoꢀluminescent M. tuberculosis (LuxTB) was
generated by transforming the wildꢀtype Erdman strain with the pMlux plasmid,
encoding the mycobacterial MOPS promoter driving expression of a synthetic GCꢀrich
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luxCDABE operon from P. luminescens.
LuxTB was cultured in 7H9 medium to
OD600 = 0.0125. KKLꢀ35 was added and the cultures were incubated at 37 °C.
Luminescence readings were recorded every 24 h for 12 days using an Infinite M200
plate reader (Tecan Trading AG, Mannedorf, Switzerland).
M. tuberculosis SmpB depletion assays. The TetpsmpB:rTetR mutant was
constructed by replacing 500 bp upstream of the smpB ATG start site with a tet operator
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(tetO)ꢀcontaining mycobacterial promoter (Psymc). This mutation was made by
homologous recombination using a specialized mycobacterium phage system as
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previously described. After the addition of the P
promoter, the strain was
symc
transformed with a plasmid that integrates at the attB site and expresses the reverse
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repressor TetR. Repression of SmpB was achieved by incubation of cells with 300
ng/ml of anhydrotetracycline (SigmaꢀAldrich, St. Louis, MO).
M. tuberculosis hypoxia assay. For growth under hypoxia, MTB was grown in 17 ml
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glass test tubes in triplicate and gradual hypoxia was generated using the Wayne
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Model.
KKLꢀ35 was added to hypoxic cultures at various concentrations and the
cultures incubated for 1 week. These cultures were then spread on 7H10 agar plates
and incubated at 37 °C for 3ꢀ4 weeks before enumeration of the CFUs.
Genome sequencing. Full genome sequencing of strain ∆smpB::dif was performed by
generating a library from randomly sheared 350 bp genomic DNA fragments using a
TruSeq DNA Kit (Illumina Inc., San Diego, CA) following the manufacturer’s protocol.
Pairedꢀend sequencing was performed for 100 cycles using an Illumina HiSeq 2500 by
the University of Minnesota Genomics Center. Approximately 1.3 GB of data were
obtained representing >300 fold sequence coverage (NCBI BioProject accession
number PRJNA343132). Highꢀquality pairedꢀend reads were trimmed using Cutadapt
(http://cutadapt.readthedocs.io/en/stable/guide.html#trimmingꢀpairedꢀendꢀreads) and
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mapped to the H37Rv reference genome sequence using Geneious 6.0 (Biomatters
Ltd, Auckland, New Zealand). Sequence of the smpB region was independently verified
by sequencing of PCR amplicons covering open reading frames from Rv3098c ꢀ
Rv3102c.
qRT-PCR analysis of smpB expression. Midꢀexponential phase cultures of strains
H37Rv ∆smpB::dif, ∆smpB::dif::smpB and ∆ssrA::ssrA were harvested by centrifugation,
cell pellets were resuspended in buffer containing 10 mM TrisꢀHCl, 1 mM EDTA, 15
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mg/ml lysozyme and incubated at 37°C for 16 h. RNA was extracted using the
TM
E.Z.N.A. bacterial RNA kit (Omega Biotek, Norcross, GA). Residual DNA was
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removed using the TURBO DNAꢀfree kit (Life Technologies Corp., Grand Island, NY).
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®
qRTꢀPCR was performed with the QuantiFast SYBR Green RTꢀPCR kit (Qiagen).
qRTꢀPCR reactions were prepared with 2X QuantiFast SYBR Green RTꢀPCR master
mix, 10 ꢂM primers, 0.1 ꢂl QuantiFast RT Mix, 1 ng RNA and were run on a
®
LightCycler 480 with following cycle conditions: 50°C for 10 min, 95°C for 5 min, 35
cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 20 s with fluorescence quantification
for each cycle. A melting curve cycle of 95°C for 15 s, 60°C for 15 s, and 95°C with 2%
ramp rate was used to determine product specificity. A no reverse transcriptase qRTꢀ
PCR control reaction was performed to test for contaminating DNA. Primers to express
41
the mature tmRNA were used for these studies.
tmRNA primer sequences: MSTSSRAꢀ5 TGCAGGCAAGAGACCACCGTA, MTSSRAꢀ6
CCGGTCACGCGAACTAGCCGAGA
Bioorthogonal photo-affinity labeling with KKL-2098. Intracellular photoꢀlabeling
was performed by adding either KKLꢀ35 or KKLꢀ2098 at the MIC to midꢀexponential
cultures of M. smegmatis, M. tuberculosis or E. coli ∆tolC. These cultures were grown
for 1 h and cells were harvested by centrifugation at 2716 g and resuspended in
phosphate buffered saline solution (1.2 g Na HPO , 0.22 g NaH PO , 8.5 g NaCl in 1L
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4
2
4
pH 7.5). This suspension was irradiated with 312 nm UV light for 10 min and cells were
recovered by centrifugation. RNA extracts were prepared using the Norgen total RNA
preparation kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the
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manufacturer’s protocols and the purity of the isolated RNA assessed by agarose gel
electrophoresis.
Primer extension assays. DNA oligonucleotides covering 23S rRNA were endꢀlabeled
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[
32]
with P using polynucleotide kinase (NEB, Ipswich, MA) according to the
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manufacturer’s instructions. Primer extension assays were performed using RNA from
KKLꢀ35 and KKLꢀ2098ꢀtreated cells with Superscript II reverse transcriptase (Thermo
Fisher Scientific, Bellefonte, PA) according to the manufacturer’s instructions. The
products were separated on an 8% polyacrylamide urea gel and visualized using a
Typhoon 9410 imager (GE Healthcare, Tyrone, PA). These experiments were repeated
using Superscript IV and Sunscript reverse transcriptase for confirmation of modified
sites.
Oligonucleotide sequences used in the primers extension assays:
M. smegmatis 23S rRNA primers
MS1 ꢀ TGTTGTAAGTTTTCGGCCGG
MS2 ꢀ CACGACGTTCTAAACCCAGC
MS3 ꢀ GCGCGTAACGAGCATCTTTA
MS4 ꢀ ACCTGTGTTGGTTTGGGGTA
MS5 ꢀ ATCAACCCGTTGTCCATCGA
MS6 ꢀ ACACGCTTAGGGGCCTTAG
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MS7 ꢀ ACACACCACTACACCACACA
MS8 ꢀ GCCATTTCCGCTAACCACAA
E. coli 23S rRNA primers
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EC 1 ꢀ GGACACGTGGTATCCTGTCTG
EC 2 ꢀ AACTGGGCGTTAAGTTGCAG
EC 3 ꢀ GGTCATCCCGACTTACCAA
EC 4 ꢀ GATGGGAAACAGGTTAATATTCCT
EC 5 ꢀ TGATCGAAGCCCCGGTAA
EC 6 ꢀ AGGTCATAGTGATCCGGTGG
EC 7 ꢀ TACGCGAGCTGGGTTTAGAA
EC 8 ꢀ GTACTAATGAACCGTGAGGCTTAA
The copper catalyzed azide-alkyne Huisgen cycloaddition (AAHC) click
conjugation assay
RNA: click conjugation reactions were performed in a 20 µl scale by combining 8 µl
acetonitrile (final 40 % v/v), KKLꢀ2107 (1 mM final), 2 µl 1M Hepes pH 7.4 (final 100
µM), 8.6 µl RNA solution, 2 µl premixed solution of CuSO /THPTA (final concentrations
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.1 mM and 1 mM respectively) and NH NH (final concentration of 0.1 mM). Samples
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were mixed and the reaction was incubated at room temperature for 15 min. 15 µl 2X
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formamide loading buffer was added and the sample was incubated at 65 °C for 10 min.
Samples were analyzed by gel electrophoresis on a 1% agarose TAE gel.
Protein: KKLꢀ35 or KKLꢀ2098ꢀtreated cell pellets were resuspended in lysis buffer (100
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mM NaH PO (pH 7.5), 100 mM NaCl, 0.1% SDS, 2 mM BME), lysed by sonication, and
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clarified by centrifugation at 22,000 g for 10 min. The lysate was then subjected to click
conjugation by mixing 100 µl acetonitrile, KKLꢀ2107 (final concentration 1 mM), 172 µl
clarified lysate, a preꢀmixed solution of CuSO /THPTA (final concentrations 0.4 mM and
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mM respectively) and NH NH (final concentration of 0.1 mM). Reactions were
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incubated at room temperature for 3 h with gentle agitation and protein was precipitated
by addition of acetone. The recovered protein pellet was airꢀdried and reꢀdissolved in
binding buffer (100 mM Na PO , 100 mM NaCl, 0.1% SDS, 2 mM BME). For affinity
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4
chromatography, NeutrAvidin (Thermo Fisher Scientific, Bellefonte, PA) agarose resin
was equilibrated in binding buffer according to the manufacturer’s protocols. A mixture
of the resin and lysate was incubated for 1 h at room temperature with gentle agitation
then transferred to a column. The column was washed with 10 volumes of binding
buffer, the resin was transferred to a clean tube, and protein was eluted by addition of
1X SDS sample buffer (34.2 mM Tris pH 6.8, 13.1 mM glycerol (w/v), 1% SDS, 0.01%
bromophenol blue) and incubation at 95 °C for ~ 5 min. Samples were analyzed by SDS
PAGE.
In vitro photo-labeling and click conjugation. Assays were set up using the
PURExpress in vitro protein synthesis kit (NEB, Ipswich, MA) according to the
manufacturer’s protocols. The reactions were performed with: no DNA template, a
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nonstop DHFR template, the full length DHFR gene, full length DHFR gene with 0
bases after the stop codon or full length DHFR gene with 33 bases after the stop codon.
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KKLꢀ2098 (final concentration 1 µM) was added to a mixture of assay components
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and the samples were incubated at room temperature for 1 h. Samples were placed on
ice, irradiated with 312 nm UV light for 10 min, and used to set up click conjugation
assays in the presence of KKLꢀ2107 (final concentration 0.5 mM). After incubating for
3
0 min, an equal volume of 2X formamide loading buffer was added and the tubes were
incubated at 65 °C for 10 min. The samples were resolved on a 1% agarose gel. The
gel was first scanned for fluorescence (to visualize the conjugated probe) then stained
with ethidium bromide to visualize the RNA.
In silico molecular docking and solvent mapping. Molecular docking studies were
28
performed with the AutoDock Vina program utilizing the AutoDock tools graphical
interface. Energy minimizations for KKLꢀ35 and KKLꢀ2098 were performed using the
Open Babel module. Modeling, structural manipulation and visualization were
performed using PyMOL (Schrödinger). Receptor grid maps were generated using the
AutoGrid module and KKLꢀ35 or KKLꢀ2098 docked using the Lamarckian genetic
algorithm. Docking for KKLꢀ35 and KKLꢀ2098 to the 70S ribosome was guided by
results from the crossꢀlinking experiments with KKLꢀ2098. The dimensions of the dockꢀ
search space were adjusted to encompass the peptidylꢀtransfer center and helix 89 of
the 50S ribosomal crystal structure. These studies were performed with multiple crystal
structures: PDB ID: 4ABR, 4V69, 3DLL, 4V7T. The best binding conformations were
selected from the 10 generated from the docking event based on a criterion combining
the highest dock score and the lowest root mean square deviation values (RMSD < 1).
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Computational solvent mapping. The H89 structure was extracted from the protein
databank ribosome structure (PDB ID: 4ABR). All water molecules and ions were
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removed and mapping performed using the FTMap algorithm
(Boston University,
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MA) remotely through its servers online (http://ftmap.bu.edu). The entire surface of H89
was scanned with a mini probe library of 16 organic small molecules with variable
hydrophobic and hydrogen bonding properties. The program utilized CHARMM energy
minimized conformations for all the probes to scope from potential binding sites on H89.
The algorithm retained six bound clusters with the lowest mean interaction energies for
each probe. Probeꢀcluster consensus sites (CS) were then identified from congregated
groups of structurally diverse probe. Each of the CSs represented a potential binding
hot spot within H89 and was ranked based on the number of probe clusters it contained.
The CS with the largest number of probe clusters characterized the most probable small
molecule binding sites.
ASSOCIATED CONTENT
Supporting information
The supporting information file is available free of charge on the ACS publications
website.
Results for additional in vitro biochemical assays, in silico molecular modeling and
experimental details for the chemical synthesis (PDF)
Accession numbers
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Whole genome sequencing results for the MTB SmpB deletion strains are accessible
via GenBank using the accession numbers SAMN05907893 and SAMN05907849.
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AUTHOR INFORMATION
:
Author Contributions
J.N.A., K.C.K. and J.S.C. conceived the study. J.N.A., K.C.K, A.D.B. and J.S.C.
designed the experiments. J.N.A performed chemical synthesis and compound
characterization, in vitro biochemical analysis, target identification and in silico modeling
studies. J.N.A and K.C.K performed the primer extension assays. P.S.M. and T.L.
screened for activity against MTB, performed smpB knockꢀout experiments and carried
out the MTB hypoxia model assays. N.D.P. and A.D.B. generated, analyzed and
interpreted full genome sequencing data and qRTꢀPCR data for MTB strains H37Rv,
∆smpB::dif, ∆smpB::dif::smpB, and ∆ssrA::ssrA. J.N.A., K.C.K., J.S.C. and A.D.B.
prepared and wrote the manuscript.
Current address
Paulo S. Manzanillo: Genentech Immunology Department, San Francisco, California
USA; Nicholas D. Peterson: University of Massachusetts Medical School, Worcester,
Massachusetts USA; Tricia Lundrigan: Department of Cellular and Molecular
Pharmacology, University of California, San Francisco, California USA.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
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The authors thank Yusuke Minato for assistance with MTB MIC determinations, Teresa
Repasy for assistance with MTB studies, Tanya Parish for providing strains, and Amber
Miller for performing toxicity screens against HeLa cells. J.N.A. is grateful to Steven J.
Benkovic for use of chemical synthesis facilities. This work was supported by NIH
grants GM068720 and AI132275 to K.C.K., DP1AI124619 to J.S.C., and NIH grant
AI123146 and the Bill and Melinda Gates Foundation Grand Challenges Explorations
grant to A.D.B.
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REFERENCES
1
. World Health Organization. (2015) Global tuberculosis report. World Health
Organization Geneva, Switzerland.
http://www.who.int/tb/publications/global_report/en/.
2. Kurz, S. G., Furin, J. J., and Bark, C. M. (2016) DrugꢀResistant Tuberculosis:
Challenges and Progress. Infect. Dis. Clin. North Am. 30, 509ꢀ522. DOI:
1
0.1016/j.idc.2016.02.010
3. Keiler, K. C., and Alumasa, J. N. (2013) The potential of transꢀtranslation
inhibitors as antibiotics. Future Microbiol. 8, 1235ꢀ1237. DOI:
1
0.2217/fmb.13.110.
4
. Keiler, K. C. (2008) Biology of transꢀtranslation. Annu. Rev. Microbiol. 62, 133ꢀ
1
51. DOI: 10.1146/annurev.micro.62.081307.162948.
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a
b
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ftsX
smpB
RV 3099c
attB
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Wild-type
/
/
//
//
Wt
Wt + ATc
TetpsmpB
TetpsmpB + ATc
TetpsmpB:rTetR
TetpsmpB:rTetR + ATc
Phage mediated insertion
ftsX
ftsX
smpB
RV 3099c
attB
TetsmpB //
TetP
TetP
intergrating
rTetR plasmid
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smpB
RV 3099c
rTetR
0
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TetsmpB:rTetR //
//
Days
c
d
XhoI XhoI
smpB RV 3099c
XhoI
1
00
H37Rv
//
ftsX
ssrA
//
1,845 bp XhoI
fragment
10
1
BgIII
sites
XhoI
sites
XhoI
∆smpB::dif
predicted
/
/
ftsX
dif
RV 3099c
ssrA
//
1,445 bp XhoI
fragment
0
.1
BgIII
sites
XhoI
sites
XhoI
sites
XhoI
*
∆
smpB::dif
observed
ftsX
ftsX
dif
RV 3099c
smpB
RV 3099c
ssrA
//
0.01
/
/
H37Rv
∆
smpB::dif
observed
∆
smpB::dif
psmpB
∆ssrA::ssrA
1,445 bp XhoI
fragment
1,512 bp XhoI
fragment
Figure 1. SmpB is essential in MTB. (a) Schematic illustration for the design of the
smpB depletion constructs in MTB. (b) Growth curves for the SmpB depletion and
control strains. (c) Schematic diagram of the smpB locus in the parental H37Rv strain,
the reported ∆smpB::dif and ∆smpB::dif observed from whole genome sequencing
showing that the ∆smpB::dif strain has a copy of smpB. (d) qRT-PCR analysis showing
that both ssrA and smpB are expressed in the ∆smpB::dif strain. smpB (yellow) and
ssrA (gray) mRNA levels in mid-exponential phase MTB cells were quantified by qRT-
PCR and normalized to the housekeeping gene sigA. Mean values from 3 technical
replicates of one biological sample are shown with error bars indicating the standard
deviation.
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