Inhibitors of ribosome rescue arrest growth of francisella tularensis at all stages of intracellular replication

Article abstract of DOI:10.1128/AAC.03089-15

Bacteria require at least one pathway to rescue ribosomes stalled at the ends of mRNAs. The primary pathway for ribosome rescue is trans-translation, which is conserved in >99% of sequenced bacterial genomes. Some species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient trans-translation activity. Small-molecule inhibitors of ribosome rescue have broad-spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against the tier 1 select agent Francisella tularensis to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL-10 and KKL-40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 or KKL-40 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis, addition of KKL-10 or KKL-40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis. Neither KKL-10 nor KKL-40 was cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for F. tularensis growth at all stages of its infection cycle and suggest that KKL-10 and KKL-40 are good lead compounds for antibiotic development.

Full text of DOI:10.1128/AAC.03089-15

AAC Accepted Manuscript Posted Online 7 March 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.03089-15
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Inhibitors of ribosome rescue arrest growth of Francisella tularensis at all
stages of intracellular replication.
Tyler D. P. Goralski2, Kalyan K. Dewan1, John N. Alumasa2, Victoria Avanzato1,
David E. Place1, Rachel L. Markley1, Bhuvana Katkere1, Seham M. Rabadi3,
Chandra Shekhar Bakshi3, Kenneth C. Keiler2# and Girish S. Kirimanjeswara1#.
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Department of Veterinary and Biomedical Science, The Pennsylvania State
University, University Park, PA 168021, Department of Biochemistry and
Molecular Biology, The Pennsylvania State University, University Park, PA
168022, Department of Microbiology and Immunology, New York Medical
College, Valhalla, NY 105953.
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Running Title: Ribosome rescue inhibitors against F. tularensis
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16 # Address correspondence to Girish S. Kirimanjeswara, gsk125@psu.edu and
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Kenneth C. Keiler, kkeiler@psu.edu
T.D.G. and K.K.D. contributed equally to this work.
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Abstract
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Bacteria require at least one pathway to rescue ribosomes stalled at the end of
mRNAs. The primary pathway for ribosome rescue is trans-translation, which is
conserved in >99% of sequenced bacterial genomes. Some species also have
backup systems, such as ArfA or ArfB, which can rescue ribosomes in the
absence of sufficient trans-translation activity. Small molecule inhibitors of
ribosome rescue have broad-spectrum antimicrobial activity against bacteria
28 grown in liquid culture. These compounds were tested against the Tier 1 Select
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Agent Francisella tularensis to determine if they can limit bacterial proliferation
during infection of eukaryotic cells. The inhibitors KKL-10 and KKL-40 exhibited
exceptional antimicrobial activity against both attenuated and fully virulent strains
of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 or KKL-40
to macrophage or liver cells at any time after infection by F. tularensis prevented
further bacterial proliferation. When macrophages were stimulated with the pro-
35 inflammatory cytokine interferon-γ before being infected by F. tularensis, addition
36 of KKL-10 or KKL-40 reduced intracellular bacteria by >99%, indicating that the
37 combination of cytokine induced stress and a nonfunctional ribosome rescue
38 pathway is fatal to F. tularensis. Neither KKL-10 nor KKL-40 were cytotoxic to
39 eukaryotic cells in culture. These results demonstrate that ribosome rescue is
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required for F. tularensis growth at all stages of its infection cycle, and suggest
41 that KKL-10 and KKL-40 will be good lead compounds for antibiotic development.
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Introduction
A major challenge associated with the development of effective antibiotics is the
variance observed in bacterial physiology under different growth and infection
conditions. Antibiotics capable of inhibiting bacterial replication in vitro may be
less efficacious ex vivo or in vivo. This potential difference is particularly
48 important when considering the treatment of the pathogen Francisella tularensis,
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which is shielded from the host innate immune response during infection and
replication within eukaryotic cells [1-4]. F. tularensis ssp. tularensis is a gram-
negative, facultative intracellular bacterium responsible for the vector-borne
zoonosis tularemia [2]. Human infections can occur through a number of routes,
however bites from infected insects are the most common, leading to the
ulceroglandular form of the disease [5-8]. Pneumonic tularemia, while less
common, is infectious at ≤ 10 colony-forming units (cfu) of aerosolized bacteria,
and has a 60% mortality rate if left untreated [5-8]. F. tularensis has been
classified as a Tier 1 Select Agent by the CDC due to its high infectivity and ease
of propagation [9]. Attempts to develop an effective vaccine have been
59 unsuccessful, due in part to the organism’s ability to suppress or bypass the host
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immune response early after infection [1-4]. F. tularensis strains resistant to
multiple antibiotics are a biowarfare threat [9]. In the absence of an effective
vaccine, new antibiotic targets and compounds are needed to ensure biodefense.
During bacterial translation, when the ribosome reaches the 3’ end of the
mRNA with no stop codon, the ribosome becomes stuck and is unable to
translate other proteins [10-13]. Ribosome rescue pathways that release
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ribosomes from non-stop translation complexes have been identified in most
bacteria. Ribosome rescue pathways are potential targets for new antibiotics
because they are required for virulence or viability in many pathogenic species
[14,15]. Trans-translation, the primary ribosome rescue pathway, is performed
by a ribonucleoprotein complex consisting of a specialized RNA molecule called
71 transfer-messenger RNA (tmRNA), and the small protein SmpB. During trans-
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translation, tmRNA-SmpB recognizes a non-stop ribosome and releases it by
inserting a reading frame within tmRNA into the mRNA channel. Translation
resumes on tmRNA and terminates at the stop codon at the end of the reading
frame. This reaction also targets the nascent polypeptide and mRNA for
degradation [10-13]. F. tularensis mutants lacking SmpB or tmRNA are
77 attenuated for virulence in mouse models [16]. Some bacteria, such as E. coli,
78 can survive without trans-translation because they contain ArfA, a protein that
79 allows release factor 2 (RF-2) to terminate translation on ribosomes stalled at the
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3’ end of an mRNA [17-19]. Other species have ArfB, a protein that recognizes
ribosomes at the 3’ end of an mRNA and hydrolyzes the peptidyl-tRNA to rescue
82 the ribosome [20-22]. The F. tularensis genome does not contain genes
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encoding ArfA or ArfB, but it may have an unidentified backup system that
rescues ribosomes in mutants lacking SmpB or tmRNA [16].
A group of oxadiazole derivatives (Fig. 1) was identified as inhibitors of trans-
translation using a high throughput screen [23]. These molecules inhibited trans-
translation in vitro and in vivo, and inhibited growth of Shigella flexneri, Bacillus
anthracis, and Mycobacterium smegmatis in liquid culture [23]. Over-expression
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of ArfA or addition of a sub-lethal concentration of puromycin relieved growth
inhibition by KKL-35, demonstrating that KKL-35 inhibits bacterial growth by
blocking ribosome rescue [23]. ArfA over-expression also relieved growth
inhibition by KKL-10 and KKL-40, indicating that these compounds also inhibit
growth by blocking ribosome rescue (Fig. S1). Here, we describe the activity of
these oxadiazoles against F. tularensis grown in vitro and during infection of
macrophages and liver cells. The results indicate that ribosome rescue is
required for all stages of F. tularensis proliferation and suggest that molecules
similar to the oxadiazoles may be effective antibiotics against F. tularensis and
other intracellular pathogens.
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Materials and Methods
Bacterial culture
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Francisella tularensis ssp. tularensis (Schu S4) and Francisella tularensis ssp.
holartica (LVS) were grown in brain heart infusion (BHI) medium adjusted to pH
6.8 at 37 ° C and 200 rpm. Growth was monitored by performing optical density
106 (OD) readings at 600 nm. For plating assays, bacteria were diluted in 1X
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phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47
mM KH2PO4 (pH 7.2)) and grown on chocolate agar plates (Mueller Hinton agar
109 supplemented with 1% bovine hemoglobin (Remel, USA) and 1% Isovitalex X
110 Enrichment (Becton Dickinson, France)) at 37 °C in a humidified incubator with
111 5% CO2 for 48 h.
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MIC determination and in vitro F. tularensis enumeration
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For MIC assays, triplicate 2-fold serial dilutions of each compound were made in
cation-adjusted Mueller-Hinton broth (CAMHB) and added to a 96-well microtiter
plate. Stocks of each compound were prepared in 100% dimethyl sulfoxide
116 (DMSO). Overnight cultures of LVS or Schu S4 were diluted to OD600 = 0.05 in
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CAMHB to a final volume of 0.1 ml and added directly to the diluted compounds.
The microtiter plates were incubated overnight (~18 hours) at 37 °C in a
humidified incubator with 5% CO2. Bacterial growth was monitored by measuring
the optical density at 600 nm. The MIC was determined by observing the lowest
concentration at which the compound prevented a significant increase in optical
122 density. To enumerate the F. tularensis after exposure to varying concentrations
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of KKL-10 or KKL-40, the contents of the MIC assay microtiter plate were
removed and plated on chocolate agar at appropriate dilutions. After incubation
for 48 hours at 37 °C and 5% CO2, colonies were counted to calculate cfu/ml.
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BMDM Isolation
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The hind legs of euthanized C57 mice were skinned, removed at the hip joint,
and feet and excess muscle tissue were removed. Marrow was liberated by
removing the femur bones proximal to each joint and crushing them in a 70 μm
nylon mesh filter in 5 ml PBS using a sterile pestle. Marrow was added to conical
tubes and centrifuged at 500 g for 10 min at room temperature. The supernatant
was discarded, and the pellet was resuspended in Dulbecco’s Modified Eagle
Medium (DMEM) (ThermoFisher, USA). The cells were plated in 100 mm petri
134 dishes at a density of 4 x 105 cells/ml in 10 ml complete macrophage
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differentiation medium (DMEM + 20% L929 cell supernatant containing
macrophage colony-stimulating factor (M-CSF). The cells were supplemented
with and additional 5 ml of media on days 1 and 3, and BMDMs were harvested
on day 7.
Invasion assays and enumeration of intracellular F. tularensis
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RAW 264.7 macrophages (a gift from Dr. James Drake, Albany Medical College),
HepG2 human hepatic cells (a gift from Dr. Gary Perdew, The Pennsylvania
State University) or BMDMs were seeded onto 12-or 24-well cell culture plates at
143 a density of 2.5 x 105 cells/ml in RPMI-1640 medium (ThermoFisher, USA) with
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2% FBS, 1% glutamine, 1% non-essential amino acids, 1% sodium pyruvate and
1 mM HEPES. For IFN-γ prestimulation, IFN-γ was added to 50 ng/ml 12 h prior
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147 phase (OD600 = 0.5) was used to infect the cells at a multiplicity of 100:1 for
148 macrophages or 1000:1 for HepG2 cells. Infection of the cells was initiated by
149 centrifugation of the culture plates at 300 g for 10 min, Cells were incubated for
150 50 min at 37 °C and 5% CO2, and extracellular bacteria were killed by removing
151 the medium, washing the wells 3 X with PBS, and incubating the cells in medium
to infection. F. tularensis LVS cultured in BHI broth to mid-exponential growth
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containing 100 g/ml gentamicin for 1 h. KKL-10 and KKL-40 was added directly
to the medium to a final concentration of 2.5 μg/ml at various times post infection
depending on the assay. Aspirating the medium, washing the wells 3X with PBS,
and lysing the cells in 100 μl 0.1% sodium deoxycholate for 5-10 min released
the intracellular bacteria. The lysate was diluted and used in plating assays for
enumerations as described above.
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Cytotoxicity assays
159 Cytotoxicity assays were performed using RAW 264.7 cells and an LDH release
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assay kit (Pierce Biochemicals, USA) following the manufacturer’s instructions.
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Results
Oxadiazole inhibitors KKL-10 and KKL-40 prevent growth of F. tularensis in
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liquid culture.
166 Broth microdilution assays were used to determine the minimum inhibitory
167 concentration (MIC) for ribosome rescue inhibitors against the attenuated live
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vaccine strain of F. tularensis ssp. holartica (LVS) and the fully virulent Schu S4
strain of F. tularensis ssp. tularensis (Table 1). The MICs of KKL-10 and KKL-40
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were lower than those of other antibiotics commonly used in the laboratory or as
therapeutics. KKL-35 also had a low MIC against LVS, but was insoluble in
growth media at concentrations of 7.95 mg/ml or higher. Therefore, In contrast,
KKL-10 and KKL-40 showed full solubility at the highest concentrations tested
174 (38 mg/ml and 35 mg/ml, respectively), so these compounds were pursued
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further. Plating assays showed that KKL-10 and KKL-40 were bacteriostatic at
their MICs against both LVS and Schu S4 (Fig. 2). Higher concentrations of KKL-
40 resulted in a >5 log decrease in cfu/ml. The bactericidal effect of KKL-10 was
less pronounced at the tested concentrations.
KKL-10 and KKL-40 arrest intracellular growth of F. tularensis during all
stages of infection.
One of the challenges of treating F. tularensis infections is that the bacteria are
able to enter host cells and proliferate without being detected [1-4]. In a typical
184 infection, F. tularensis enters naïve macrophage or dendritic cells by an
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uncharacterized phagocytic mechanism, escapes the phagosome 1-4 h post
infection, and enters the cytoplasm to proliferate rapidly. Within 24 h, the
187 bacterial numbers increase by ~100-fold. Subsequently, F. tularensis induces
188 pyroptosis to lyse the host cell and release bacteria capable of infecting new
189 macrophage or dendritic cells [24]. To determine if KKL-10 and KKL-40 arrest
190 growth of LVS inside natural host cells, RAW 264.7 mouse macrophage cells
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were infected with LVS and after 1 h extracellular bacteria were eliminated by
treatment with gentamicin. KKL-40 was added 3 h after infection and cells were
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incubated an additional 21 h before the bacteria were enumerated by plating.
KKL-40 inhibited growth of LVS in macrophages in a dose-dependent manner,
with almost no growth observed at concentrations ≥2.5 μg/ml (Fig. 3A). This
result shows that KKL-40 can cross the plasma membrane and inhibit F.
tularensis growth in the environment of a eukaryotic cell. The increased
concentration of KKL-40 required to arrest growth in macrophages compared to
growth in liquid culture was due at least in part to the presence of fetal bovine
serum, because increasing the serum concentration increased the MIC in vitro
201 and in macrophages (not shown).
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203 As described above, F. tularensis passes through multiple cellular compartments
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during its infection cycle, and the environments of these compartments can be
quite different. To determine if KKL-40 and KKL-10 could inhibit growth of F.
206 tularensis in all of these compartments, the ex vivo infection experiment was
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repeated and the inhibitor was added at different times post-infection. At all time
points, both KKL-10 and KKL-40 stopped further bacterial replication (Fig. 3B,C).
209 These data suggest that KKL-10 and KKL-40 arrest F. tularensis proliferation
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quickly, regardless of the stage of infection or the intracellular location of the
bacteria.
213 While F. tularensis proliferation primarily occurs in macrophages, the bacteria are
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also capable of infecting other cell types, such as hepatic cells, in mice and
humans [25]. To determine if KKL-10 and KKL-40 could arrest F. tularensis
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growth in different cell types, the ex vivo infection assays were repeated using
human hepatic HepG2 cells and primary bone marrow derived macrophage
(BMDM) isolated from C57 mouse femurs. For both cell types, KKL-10 and KKL-
219 40 arrested LVS growth (Fig. 4).
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KKL-10 and KKL-40 do not affect macrophage viability or function.
Macrophages play a significant role in host immunity and the innate and adaptive
immune response, so it was important to determine if KKL-10 or KKL-40 had any
negative effects on macrophage viability or function. A lactate dehydrogenase
(LDH) release assay was used to determine the cytotoxicity of KKL-10 and KKL-
40. Both molecules produced cytotoxic effects <5% at concentrations up to 17.5
μg/ml (Fig. 5A). Likewise, 24 h treatment with 2.5 μg/ml KKL-10 or KKL-40 was
cytotoxic for <5% of macrophages (Fig. 5B).
The effect of KKL-10 and KKL-40 on macrophage function was tested by pre-
treating macrophages with one of the molecules prior to challenge with F.
tularensis. If the ability of the macrophages to phagocytose and harbor F.
tularensis was impaired by KKL-10 or KKL-40, viable counts of F. tularensis
234 should be decreased in these assays. RAW 264.7 cells were pretreated with the
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inhibitors prior to infection for the indicated times shown in Figure 5C and D. The
inhibitors were washed from the macrophages, followed by infection with LVS for
24 hours. No significant growth decrease was observed in pretreated infections
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238 compared to untreated controls, indicating that the inhibitors do not have
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profound effects on macrophage activity.
IFN-γ stimulated macrophages clear F. tularensis infections after addition
of KKL-10 or KKL-40.
243 The pro-inflammatory cytokine IFN-γ is absolutely required for host survival
244 during the course of an F. tularensis infection [26]. IFN-γ restricts bacteria to the
245 endosomal compartment, thereby preventing bacterial escape into the
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cytoplasm. Oxidative stress is induced in the endosome, which aids in the killing
of bacteria [26,27]. However, pre-stimulation of macrophages with IFN-γ has
248 been shown to have bacteriostatic effects on F. tularensis infections [28]. To
249 evaluate whether macrophages pretreated with IFN-γ were able to clear an LVS
250 infection after ribosome rescue was inhibited, BMDMs were pre-stimulated with
251 IFN-γ for 12 h, and KKL-10 or KKL-40 were added to the macrophage 3 h post-
252 infection. The combination of IFN-γ stimulation and KKL-10 or KKL-40 activity
253 resulted in a reduction of the bacterial load by >99.9% (Fig. 6). An even more
254 significant decrease was observed in IFN-γ stimulated RAW 246.7 macrophage
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after the addition of KKL-40, which killed 99.99% of bacteria.
Discussion
The data presented here demonstrate that KKL-10 and KKL-40 prevent growth of
F. tularensis in liquid culture and during ex vivo infection of eukaryotic cells.
Using the fully virulent Schu S4 strain, MIC values for KKL-40 (0.4 µg/ml) and
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KKL-10 (0.5 µg/ml) were substantially lower than antibiotics used for clinical
treatment of tularemia such as tetracycline (2.3 µg/ml) and streptomycin (4.0
263 µg/ml). KKL-40 and KKL-10 were also able to inhibit growth of F. tularensis inside
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eukaryotic cells, and were effective at all times during the infection cycle. Neither
compounds showed toxicity to macrophages nor HepG2 cells. Because the
effects of both compounds were enhanced in macrophages that were activated
by IFN-γ, their effectiveness might be increased in vivo, where there is a
complete innate and adaptive immune system. The only negative indication
observed in these experiments was the relatively high concentration of KKL-40
and KKL-10 (2.5 µg/ml) required for activity in the presence of serum, suggesting
that these molecules are likely to bind serum protein. Nevertheless, the effective
concentrations were still in line with therapeutically useful antibiotics. Overall,
these results encourage further development of KKL-40 and KKL-10 as
antibiotics for therapeutic use against F. tularensis and other intracellular
pathogens, particularly for strains that are resistant to existing drugs. Because
KKL-40 and KKL-10 have little structural resemblance to other antibiotics and
target ribosome rescue, a pathway that is not targeted by other antibiotics, cross-
resistance is unlikely to be a problem.
280 The activity of KKL-10 and KKL-40 also indicates that ribosome rescue is
281 important for F. tularensis both in vitro and during infection. Svetlanov, et al.,
282 have shown that F. tularensis mutants lacking tmRNA or SmpB are viable but
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grow slowly, are more sensitive to stress, and have attenuated virulence [16]. If
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284 these F. tularensis mutants with no trans-translation activity can survive, why is
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growth of wild-type bacteria completely stopped by KKL-10 and KKL-40, which
inhibit ribosome rescue? One possibility is that sudden removal of trans-
translation activity by addition of an inhibitor has more severe consequences
than a genetic deletion. The mutant bacteria might survive because other
physiological pathways are regulated to compensate for loss of trans-translation,
whereas after addition of an inhibitor there might not be enough time for a
291 regulatory response before growth ceases. Alternatively, F. tularensis may
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contain an unidentified factor that can rescue ribosomes in the absence of trans-
translation, and this factor is also inhibited by KKL-10 and KKL-40. In either case,
294 ribosome rescue is clearly important for F. tularensis growth in liquid culture and
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throughout the infection cycle.
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Funding Information
This work was supported by NIH (AI077917 to Girish S. Kirimanjeswara and
GM068720 to Kenneth C. Keiler) and CAS/PSU start up funds to Girish S.
Kirimanjeswara. The funders had no role in study design, data collection and
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303 interpretation, or the decision to submit the work for publication.
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Acknowledgments
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We thank Edgewood Chemical and Biological Center (ECBC) for use of facilities
and reagents. We thank Dr. James Drake from Albany Medical College for
providing RAW 264.7 macrophage. We also thank Dr. Gary Perdew from The
310 Pennsylvania State University for providing HepG2 human hepatic cells.
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378 27. Fortier AH, Polsinelli T, Green SJ, Nacy CA. 1992. Activation of
379
380
macrophages for destruction of Francisella tularensis; identification of
cytokines, effector cells, and effector molecules. Infect Immun. 60:817-825.
18

381 28. Kirimanjeswara G, Golden J, Bakshi C, Metzger D. 2007. Prophylactic
382
383
384
and Theraputic Use of Antibodies for Protection against Respiratory Infection
with Francisella tularensis. J Immunol. 179:532-539.
19

385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
Figure Legends
Fig 1. Chemical structures of small molecule ribosome rescue inhibitors.
KKL-10, KKL-22, KKL-35, and KKL-40 are oxadiazoles, and KKL-55 has a
related tetrazole structure.
Fig 2. KKL-40 and KKL-10 inhibit growth of F. tularensis in culture. F.
tularensis cells were enumerated after 24 h exposure to KKL-10 or KKL-40. The
cfu/ml of the initial bacterial inoculums are represented with a dotted line. (A)
LVS treated with KKL-40. (B) LVS treated with KKL-10. (C) SchuS4 treated with
KKL-40. (D) SchuS4 treated with KKL-10. Each column indicates the average of
3 biological replicates. Error bars indicate the standard deviation.
Fig 3. KKL-40 and KKL-10 inhibit F. tularensis growth at multiples stages
of the infection cycle. (A) Intracellular LVS infections were treated with varying
concentrations of KKL-40 to determine the minimum inhibitory concentration of
the compounds ex vivo. P value determined by one-way ANOVA with Tukey post
test. Inhibitory effects of (B) KKL-10 and (C) KKL-40 at various times after
intracellular LVS infection were assayed to ensure that neither compound
interfered with bacterial proliferation inside the macrophage. Arrows indicate
times of inhibitor addition, and dotted lines are growth curves after inhibitor
addition. Each point indicates the average of 3 separate infection experiments
performed on the same day, with error bars indicating the standard deviation.
406 The experiments were repeated on at least 3 different days, and data from a
407 representative day are shown.
20

408
409 Fig 4. KKL-10 and KKL-40 inhibit intracellular growth of LVS in different cell
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
types. Inhibitors were added 3 h post infection of LVS in the indicated cells. (A)
KKL-10 treatment in BMDMs. (B) KKL-10 treatment in HepG2 cells. (C) KKL-40
treatment in BMDMs. (D) KKL-40 treatment in HepG2 cells. P values determined
by one way ANOVA with Tukey post test. Each point indicates the average of 3
separate infection experiments performed on the same day, with error bars
indicating the standard deviation. The experiments were repeated on at least 3
different days, and data from a representative day are shown.
Fig 5. KKL-10 and KKL-40 do not show cytotoxic effects on macrophages.
LDH release assays using RAW 264.7 cells pre-treated with KKL-10 or KKL-40
for 45 min. (A) or with 2.5 μg/ml of KKL-10 or KKL-40 for 24 h (B). (+) indicates
the 1X lysis buffer positive control. Plating assays after LVS infection of RAW
264.7 cells pretreated with KKL-10 (C) or KKL-40 (D) for indicated times before
the compound was washed out and LVS was added. Each point indicates the
average of 3 separate infection experiments performed on the same day, with
error bars indicating the standard deviation. The experiments were repeated on
at least 3 different days, and data from a representative day are shown.
Fig 6. Effects of KKL-10 and KKL-40 on LVS in macrophage pre-stimulated
for 12 h with IFN-γ. Plating assays after LVS infection of the indicated cells with
or without IFN-γ pre-treatment. Where indicated, 2.5 μg/ml inhibitor was added 3
h post-infection. (A) RAW 264.7 cells with KKL-40. (B) BMDMs with KKL-10. (C)
BMDMs with KKL-40. P values were determined by one-way ANOVA with Tukey
21

431
post test. Each point indicates the average of 3 separate infection experiments
432 performed on the same day, with error bars indicating the standard deviation.
433
434
435
The experiments were repeated on at least 3 different days, and data from a
representative day are shown.
22

436
Table 1
Antimicrobial activity of ribosome rescue inhibitors and various antibiotics.
MIC (μg/ml)*
LVS
MIC (μg/ml)*
SchuS4
Compound
KKL-40
KKL-10
KKL-35
KKL-22
KKL-55
0.12
0.12
0.10
0.25
0.80
0.82
0.90
0.22
1.46
>94
0.44
0.48
ND
ND
ND
tetracycline
2.30
2.08
1.25
4.0
kanamycin
gentamicin
streptomycin
ampicillin
>94
ND, not determined.
* Mean values from at least three broth microdilution assays (μg/ml).
23

Table 1
Antimicrobial activity of ribosome rescue inhibitors and various antibiotics.
MIC (μg/ml)*
LVS
MIC (μg/ml)*
SchuS4
Compound
KKL-40
KKL-10
0.12
0.44
0.12
0.10
0.25
0.80
0.82
0.90
0.22
1.46
>94
0.48
ND
KKL-35
KKL-22
ND
KKL-55
ND
tetracycline
kanamycin
gentamicin
streptomycin
2.30
2.08
1.25
4.0
ampicillin
>94
ND, not determined.
* Mean values from at least three broth microdilution assays (μg/ml) .

Fig 1. Chemical structures of small molecule ribosome rescue inhibitors. KKL-10, KKL-22, KKL-35, and KKL-40 are oxadiazoles,
and KKL-55 has a related tetrazole structure.

D
D
10
10
8
8
6
6
4
4
2
2
0
0
0
0
8
5
0
0
2
2
.
.
5
0
0
.
.
8
5
5
.
.
0
1
0
1
.
.
9
9
9
1
.
.
9
0
0
1
1
0
0
1
[KKL-10] (mg/ml)
[KKL-10] (mg/ml)
Fig 2. KKL-40 and KKL-10 inhibit growth of F. tularensis in culture. F. tularensis cells were enumerated
after 24 h exposure to KKL-10 or KKL-40. The cfu/ml of the initial bacterial inoculums are represented with
a dotted line. (A) LVS treated with KKL-40. (B) LVS treated with KKL-10. (C) SchuS4 treated with KKL-40. (D)
SchuS4 treated with KKL-10. Each column indicates the average of 3 biological replicates. Error bars
indicate the standard deviation.

B
7
6
5
4
3
time of KKL-10 addition
Untreated
15 h
12 h
9 h
6 h
0
3
6
9
12 15 18 21 24
Hours (post infection)
C
7
6
5
4
3
time of KKL-40 addition
Untreated
15 h
12 h
9 h
6 h
0
3
6
9
12 15 18 21 24
Hours (post infection)
Fig 3. KKL-40 and KKL-10 inhibit F. tularensis growth at multiples stages of the infection cycle. (A) Intracellular
LVS infections were treated with varying concentrations of KKL-40 to determine the minimum inhibitory
concentration of the compounds ex vivo. P value determined by one-way ANOVA with Tukey post test. Inhibitory
effects of (B) KKL-10 and (C) KKL-40 at various times after intracellular LVS infection were assayed to ensure that
neither compound interfered with bacterial proliferation inside the macrophage. Arrows indicate times of inhibitor
addition, and dotted lines are growth curves after inhibitor addition. Each point indicates the average of 3 separate
infection experiments performed on the same day, with error bars indicating the standard deviation. The
experiments were repeated on at least 3 different days, and data from a representative day are shown.

p <0.001
A
***
8
7
6
5
4
3
KKL-10
—
—
+
3 h
24 h
BMDM
Fig 4. KKL-10 and KKL-40 inhibit intracellular growth of LVS in different cell types. Inhibitors were added
3 h post infection of LVS in the indicated cells. (A) KKL-10 treatment in BMDMs. (B) KKL-10 treatment in
HepG2 cells. (C) KKL-40 treatment in BMDMs. (D) KKL-40 treatment in HepG2 cells. P values determined
by one way ANOVA with Tukey post test. Each point indicates the average of 3 separate infection
experiments performed on the same day, with error bars indicating the standard deviation. The
experiments were repeated on at least 3 different days, and data from a representative day are shown.

A
A
C
8
6
4
2
0
100
100
50
50
0
0
50 25 3.125 (+) 50 25 3.125
19 9.5 1.19 (+)
—
—
—
19 9.5 1.19
[KKL-10]
+
+
+
(mM)
(mM)
(mg/ml)
(mg/ml)
3 h
9 h
24 h
[KKL-10]
[KKL-10]
[KKL-40]
[KKL-40]
D
B
8
100
6
4
2
0
50
0
—
—
—
[KKL-10]
(+)
[KKL-40]
[KKL-40]
+
+
+
3 h
9 h
24 h
Fig 5. KKL-10 and KKL-40 do not show cytotoxic effects on macrophages. LDH release assays using
RAW 264.7 cells pre-treated with KKL-10 or KKL-40 for 45 min. (A) or with 2.5 μg/ml of KKL-10 or KKL-
40 for 24 h (B). (+) indicates the 1X lysis buffer positive control. Plating assays after LVS infection of
RAW 264.7 cells pretreated with KKL-10 (C) or KKL-40 (D) for indicated times before the compound was
washed out and LVS was added. Each point indicates the average of 3 separate infection experiments
performed on the same day, with error bars indicating the standard deviation. The experiments were
repeated on at least 3 different days, and data from a representative day are shown.

Fig 6. Effects of KKL-10 and KKL-40 on LVS in macrophage pre-stimulated for 12 h with
IFN-γ. Plating assays after LVS infection of the indicated cells with or without IFN-γ pre-
treatment. Where indicated, 2.5 μg/ml inhibitor was added 3 h post-infection. (A) RAW
264.7 cells with KKL-40. (B) BMDMs with KKL-10. (C) BMDMs with KKL-40. P values were
determined by one-way ANOVA with Tukey post test. Each point indicates the average of 3
separate infection experiments performed on the same day, with error bars indicating the
standard deviation. The experiments were repeated on at least 3 different days, and data
from a representative day are shown.