Development, optimization, and validation of a high throughput screening assay for identification of tat and type II secretion inhibitors of Pseudomonas aeruginosa

Article abstract of DOI:10.3389/fcimb.2019.00250

Antibiotics are becoming less effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa. Antimicrobial therapies based on the inhibition of specific virulence-related traits, as opposed to growth inhibitors, constitute an innovative and appealing approach to tackle the threat of P. aeruginosa infections. The twin-arginine translocation (Tat) pathway plays an important role in the pathogenesis of P. aeruginosa, and constitutes a promising target for the development of anti-pseudomonal drugs. In this study we developed and optimized a whole-cell, one-well assay, based on native phospholipase C activity, to identify compounds active against the Tat system. Statistical robustness, sensitivity and consequently suitability for high-throughput screening (HTS) were confirmed by a dry run/pre-screening test scoring a Z′ of 0.82 and a signal-to-noise ratio of 49. Using this assay, we evaluated ca. 40,000 molecules and identified 59 initial hits as possible Tat inhibitors. Since phospholipase C is exported into the periplasm by Tat, and subsequently translocated across the outer membrane by the type II secretion system (T2SS), our assay could also identify T2SS inhibitors. To validate our hits and discriminate between compounds that inhibited either Tat or T2SS, two separate counter assays were developed and optimized. Finally, three Tat inhibitors and one T2SS inhibitor were confirmed by means of dose-response analysis and additional counter and confirming assays. Although none of the identified inhibitors was suitable as a lead compound for drug development, this study validates our assay as a simple, efficient, and HTS compatible method for the identification of Tat and T2SS inhibitors.

Full text of DOI:10.3389/fcimb.2019.00250

ORIGINAL RESEARCH
published: 10 July 2019
doi: 10.3389/fcimb.2019.00250
Development, Optimization, and
Validation of a High Throughput
Screening Assay for Identification of
Tat and Type II Secretion Inhibitors of
Pseudomonas aeruginosa
Francesco Massai1,2, Michael Saleeb3, Tugrul Doruk1,2†, Mikael Elofsson3 and
Åke Forsberg1,2
*
1 Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå University, Umeå, Sweden,
2 Department of Molecular Biology, Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden, 3 Department of
Chemistry, Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden
Antibiotics are becoming less effective in treatment of infections caused by
multidrug-resistant Pseudomonas aeruginosa. Antimicrobial therapies based on the
inhibition of specific virulence-related traits, as opposed to growth inhibitors, constitute
an innovative and appealing approach to tackle the threat of P. aeruginosa infections. The
twin-arginine translocation (Tat) pathway plays an important role in the pathogenesis of P.
aeruginosa, and constitutes a promising target for the development of anti-pseudomonal
drugs. In this study we developed and optimized a whole-cell, one-well assay, based
on native phospholipase C activity, to identify compounds active against the Tat
system. Statistical robustness, sensitivity and consequently suitability for high-throughput
screening (HTS) were confirmed by a dry run/pre-screening test scoring a Z′ of 0.82 and
a signal-to-noise ratio of 49. Using this assay, we evaluated ca. 40,000 molecules and
identified 59 initial hits as possible Tat inhibitors. Since phospholipase C is exported into
the periplasm by Tat, and subsequently translocated across the outer membrane by the
type II secretion system (T2SS), our assay could also identify T2SS inhibitors. To validate
our hits and discriminate between compounds that inhibited either Tat or T2SS, two
separate counter assays were developed and optimized. Finally, three Tat inhibitors and
one T2SS inhibitor were confirmed by means of dose-response analysis and additional
counter and confirming assays. Although none of the identified inhibitors was suitable
as a lead compound for drug development, this study validates our assay as a simple,
efficient, and HTS compatible method for the identification of Tat and T2SS inhibitors.
Edited by:
Kenneth Pfarr,
Universitätsklinikum Bonn, Germany
Reviewed by:
Natalia V. Kirienko,
Rice University, United States
Giordano Rampioni,
Roma Tre University, Italy
*Correspondence:
Åke Forsberg
ake.forsberg@umu.se
†Present Address:
Tugrul Doruk,
Department of Molecular Biology and
Genetics, Ondokuz Mayis University,
Samsun, Turkey
Specialty section:
This article was submitted to
Clinical Microbiology,
a section of the journal
Frontiers in Cellular and Infection
Microbiology
Received: 02 May 2019
Accepted: 26 June 2019
Published: 10 July 2019
Keywords: Pseudomonas aeruginosa, high-throughput screening, twin arginine translocase, type II secretion,
Citation:
Massai F, Saleeb M, Doruk T,
virulence inhibitors, phospholipase C
Elofsson M and Forsberg Å (2019)
Development, Optimization, and
Validation of a High Throughput
Screening Assay for Identification of
Tat and Type II Secretion Inhibitors of
Pseudomonas aeruginosa.
INTRODUCTION
The opportunistic Gram-negative pathogen Pseudomonas aeruginosa is relentlessly rising in the
ranks as a major threat in both community- and hospital-acquired infections. P. aeruginosa is
responsible for more than 10% of all nosocomial infections (Driscoll et al., 2007), and the major
cause of morbidity and mortality among patients suffering from cystic fibrosis (CF), a genetic
Front. Cell. Infect. Microbiol. 9:250.
doi: 10.3389/fcimb.2019.00250
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HTS Assay for Tat and T2SS Inhibitors
disease that affects about 1/3,000 newborns in the USA and
Europe. This bacterium is particularly difficult to eradicate
due to its intrinsic resistance to several antibiotics, the ability
to acquire new resistance determinants via horizontal gene
transfer and the adoption of a biofilm mode of growth. As
a result of the high frequency of emergence of multidrug-
resistant (MDR) and ultimately pan-resistant strains (Page and
Heim, 2009), P. aeruginosa is recognized as one of the ESKAPE
pathogens (Rice, 2008). The World Health Organization ranks P.
aeruginosa together with Acinetobacter baumannii and various
Enterobacteriaceae as the top three pathogens for which the
necessity to develop new antimicrobials reached a critical level
(Tacconelli et al., 2018). While it is becoming evident that this
alarming rise of antibiotic resistance calls for new strategies of
treatment, a novel and appealing approach for anti-pseudomonal
drugs could come from the identification of molecules inhibiting
virulence-related traits, as opposed to growth inhibitors per se.
Selective targeting of the pathogenic potential of the bacterium
by anti-virulence drugs could hinder or inhibit the establishment
of the infection process, while granting the host immune system
a better chance of clearing the infection. This approach is also
supposed to prevent indiscriminate harm to the host commensal
flora while treating infections. The efficacy potential of targeting
bacterial virulence has been demonstrated in Vibrio cholerae with
the small molecule Virstatin, which prevents the polymerization
of the ToxT transcriptional activator, thus inhibiting expression
of cholera toxin and toxin co-regulated pilus and ultimately
resulting in reduced colonization and virulence in a mouse
model of infection (Hung et al., 2005; Shakhnovich et al., 2007).
Following studies aimed at identifying anti-virulence compounds
have explored the possibility to disarm P. aeruginosa by targeting
various virulence mechanisms, such as quorum sensing (Hentzer
et al., 2003; Imperi et al., 2013b; Tan et al., 2013; Paczkowski et al.,
2017; D’angelo et al., 2018; Fong et al., 2018) and pyoverdine
signaling systems (Imperi et al., 2013a; Kirienko et al., 2018),
efflux pumps (Lomovskaya et al., 2001; Rampioni et al., 2017),
protein secretion pathways (Moir et al., 2011; Anantharajah
et al., 2016; Ali et al., 2019), biofilm formation (Frei et al.,
2012; Van Tilburg Bernardes et al., 2017), flagella and other cell
wall components, to mention a few (Veesenmeyer et al., 2009).
Secretion systems are promising targets, as their inhibition could
prevent the export of several virulence-related factors. Up to now,
a number of screening strategies have been developed to identify
small molecule inhibitors of the type 2 (T2SS) and type 3 (T3SS)
secretion systems, present in many Gram-negative pathogens
and mediating secretion of a variety of virulence effectors,
degradative enzymes and toxins. Another export mechanism,
the twin arginine translocase (Tat), present in a number of
Gram-negative pathogens, is involved in the translocation of
both house-keeping and virulence-related proteins across the
inner membrane of bacteria, and may be a promising target for
the development of anti-virulence drugs. Tat is an alternative
secretion system to Sec and substrates targeted by the Tat
mechanism share a conserved twin-arginine motif within the
signal peptide. Among its substrates are proteins requiring co-
factors, heterodimers, and proteins that are already folded in
the periplasm. Proteins secreted by Tat can either reside in the
periplasm or be further exported across the outer membrane
to the extracellular milieu by the T2SS. P. aeruginosa possesses
a functional Tat system, consisting of three components, TatA,
TatB, and TatC. Among its virulence-related substrates there
are potent extracellular phospholipases C (PlcH and PlcN),
initially translocated by Tat to the periplasm and subsequently
across the outer membrane by the T2SS. Tat is also required
for the export of PvdN and PvdP, both essential components
of pyoverdine biosynthesis, which are active in the periplasm
(Gimenez et al., 2018). Pyoverdine is a high-affinity siderophore
required for scavenging iron in the host environment. In
addition, it is also involved in a signaling mechanism which
controls the expression of several virulence-related products,
such as exotoxin A and PrpL endoprotease (Lamont et al.,
2002). Functional Tat is also important for bacterial survival
in high osmolarity and microaerophilic conditions, which are
relevant for the CF lung environment. In addition, Tat is also
required for biofilm formation. As a consequence of the loss of
these Tat dependent virulence traits, Tat defective mutants have
been shown to be severely attenuated for in vivo virulence in
a rat model for chronic lung infection (Ochsner et al., 2002).
This suggests that Tat substrates are not only required for the
acute infection but also for the establishment and persistence
of chronic infection (Ochsner et al., 2002). Furthermore, it has
been shown that the Tat mechanism is highly conserved and
important for virulence of a number of other bacterial pathogens,
including enterohemorragic Escherichia coli O157:H7 (Pradel
et al., 2003), Legionella pneumophila (De Buck et al., 2005),
Salmonella enterica (Mickael et al., 2010), V. cholerae (Zhang
et al., 2009), and Yersinia pseudotuberculosis (Lavander et al.,
2006). Importantly, Tat is not found in human or animal cells,
making it an attractive target for the development of novel anti-
virulence drugs and treatments. In addition, an anti-virulence
therapy targeting Tat could have an impact both on acute
and persistent/chronic infections. This would prove particularly
beneficial when treating P. aeruginosa infections, since over time
they can develop into persistent/chronic forms that are almost
inaccessible to antibiotic treatment, and many of the potentially
targetable virulence determinants described above are known
to be dispensable for the establishment and maintenance of
persistent infection (Moradali et al., 2017). Despite the potential
of an anti-Tat drug and the fact that this mechanism is well
studied and characterized in P. aeruginosa, to the best of our
knowledge there is only one previous and noteworthy attempt
to identify Tat inhibitors in P. aeruginosa, made by Vasil et al.
(2012). This screening led to the identification of two inhibitors,
N-phenyl maleimide (NPM) and Bay 11-7082. Both compounds
are known to react with cysteine and suggested to exert their
effect on Cys23 of TatC. However, these compounds are also
known to have a strong immunomodulatory and cytotoxic
activity and are therefore not suitable for development into
clinically acceptable anti-virulence drugs (Juliana et al., 2010;
Lee et al., 2012; Matuszak et al., 2012). Nevertheless, they are
still valuable and useful as biochemical tools to investigate the
Tat mechanism in P. aeruginosa. At present, due to the lack
of a promising lead compound to develop a clinically relevant
anti-virulence drug, we considered the Tat mechanism a target
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worth the effort to continue searching for potential inhibitors.
In this study we describe development and validation of a novel
screening assay for Tat inhibitors based on the measurement of
endogenously produced phospholipase C (PLC) by the wild-type
(wt) strain of P. aeruginosa PAO1. This assay was subsequently
employed in the screening of a subset of different compound
libraries and thereby we were able to identify a number of
putative Tat inhibitors. In addition, we also developed and
evaluated a number of highly useful secondary assays, based
on the measurement of other Tat-dependent, and -independent
phenotypes for validation of our hits.
Morris Hooke, 1991). Using this media we observed an up to ca.
30-fold increase in PLC activity compared to the phosphate-poor
tryptose medium (Figure S1A). This is in line with the findings of
Lonon and Morris Hooke where cultures of Burkholderia cepacia
grown in MMST showed 38-fold higher PLC activity than the
culture grown in other phosphate-poor media.
Next we assessed PLC activity directly in the culture media
of P. aeruginosa grown in the presence of the NPPC substrate.
Similarly to what we observed in the cell-free supernatant, no
PLC activity could be detected in the culture media of a Tat
mutant (Figure S1). This suggested that this method could be
used as an assay to screen for molecules targeting the Tat
system. Although the presence of bacterial cells generated a
significant background when measuring directly in the culture
(represented by the sample without added NPPC, Figure S1B),
compared to measurement of PLC in the supernatant, which
showed a negligible background, we considered that the signal-
to-background ratio (mean signal/mean background) of ca. 3 was
a suitable starting point for assay optimization. We systematically
evaluated a number of assay parameters to determine the optimal
conditions for identifying inhibitors. Different culture volumes,
NPPC concentration, initial bacterial optical density (OD) and
incubation time were all tested and evaluated in the 384-well
plate format. The screening setup that resulted in the highest
signal-to-background ratio, without reaching signal saturation
and thus having a sufficient dynamic range to detect changes
in PLC activity, was obtained when growing P. aeruginosa for
7 h at 37◦C with shaking, in 80 µl MMST with 0.5 mM NPPC,
from a starting optical density at 600 nm (OD600) of 0.02, in a
clear bottom black microplate. Finally, running the assay using
this high throughput protocol with only positive and negative
controls (i.e., wt and Tat mutant strain) yielded a signal-to-
background ratio of 3, a signal-to-noise ratio [(mean signal—
mean background)/standard deviation of background] of 49, and
a Z’ of 0.82 (Zhang et al., 1999), which ensured the statistical
robustness, sensitivity, and suitability of the assay for high
throughput screening (Figure S2).
RESULTS
HTS Assay Development
The overall aim of the study was to develop a screening
assay for Tat inhibitors based on the measurement of the
Tat-dependent, endogenously produced PLC activity directly
in the culture media of a wt strain of P. aeruginosa PAO1.
Since the proteins involved in the TAT function do not have
any measurable enzymatic activity, indirect ways have to be
used to screen for potential inhibitors. While a number of
assays that measure PLC activity are available [listed in Durban
and Bornscheuer (2007) and Stonehouse et al. (2002)], those
cited in the literature and successfully used in P. aeruginosa
(Berka et al., 1981) employ the chromogenic substrate p-
nitrophenylphosphorylcholine (NPPC), that was first described
by Kurioka and Matsuda (1976). PLC mediated cleavage of
NPPC generates p-nitrophenol, which can be readily quantified
spectrophotometrically at 405 nm. The standard protocol to
measure PLC activity of a bacterial culture involves the
centrifugation and removal of the supernatant, which is then
added to a reaction mixture containing the NPPC substrate.
In the perspective of developing an assay protocol suitable for
high throughput screening, those processing steps were deemed
inconvenient and we therefore first explored the possibility to
measure PLC activity directly in the culture media, i.e., with
bacteria grown in the presence of the different compounds from
the chemical libraries planned to be used in the screening. In
order to overcome the drawback represented by the increased
High Throughput Screening for Tat
background signal due to the presence of bacterial cells in the Inhibitors
growth media, we first decided to maximize the PLC activity
to obtain a sufficiently high signal-to-background ratio at the
initial low cell density. Previous studies have obtained high
signal intensity by overexpressing PLC from an inducible plasmid
(Ochsner et al., 2002; Vasil et al., 2012). However, to avoid the
possibility that the antibiotic required to maintain the plasmid
could interfere with the screening compounds and influence
the assay results, we decided to optimize the conditions for
native PLC expression. Since it is known that PLC production
is repressed in the presence of inorganic phosphate (Pi) and
induced in the presence of choline (Gray et al., 1981; Lucchesi
et al., 1989; Shortridge et al., 1992), we assessed the levels of PLC
activity in the supernatant of P. aeruginosa grown in phosphate-
poor media with added choline. The highest PLC activity was
obtained by growing P. aeruginosa in a modified version of
MOPS-minimal salt-tryptose medium (MMST) (Lonon and
A series of chemical libraries, grouped into diverse subsets and
together covering a wide pharmacophore space, are available
from the Chemical Biology Consortium Sweden (CBCS),
Karolinska Institute, Stockholm, Sweden. These libraries
include diversity-oriented collections (Chembridge, CBCS
internal, Asinex Elite, and Synergy), target-focused collections
(protein-protein interaction, compounds similar to kinase or
G-protein coupled receptor (GPCR) inhibitors, predicted Zn
chelators), Food and Drug Administration (FDA) approved
drugs (Prestwick), natural products inspired compounds,
macrocycles, and carboxylic acids. The complete list is detailed
in Table S1. Compounds were dissolved in dimethylsulfoxide
(DMSO) at a concentration of 10 mM and tested at a final
concentration of 10 µM for the Chembridge library and 20 µM
for the remaining libraries. Criteria used for the selection of
hit compounds were (i) ≥20% inhibition of PLC activity for
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the Chembridge library and ≥30% inhibition for the remaining
libraries, and (ii) ≤20% reduction of bacterial growth compared
to controls without compounds. The latter criterion was chosen
to avoid any unspecific effect on PLC activity due to impaired
growth. In total 39,088 molecules were screened and of these 59
compounds were able to reduce PLC activity (Figure 1) without
any significant impact on bacterial growth. All the 59 primary
hit compounds, according to the criteria above, were subjected
to verification by repeating the screening assay using 10, 20, and
40 µM of the compounds to assess whether the impact on PLC
activity showed a dose-response relationship. Of the initial 59
compounds, 13 showed a dose dependent PLC activity inhibition
with an IC50 below 30 µM (Table S2) and were selected for
further investigation.
of several virulence factors of P. aeruginosa and was shown
to play an important role in a murine lung infection model
(Filloux, 2011; Jyot et al., 2011). Although undistinguishable
in our primary screening, compounds inhibiting either Tat or
T2SS can be discriminated by secondary assays for products
relying on one of these mechanisms for their secretion or
activity. In order to assess whether Tat or T2SS was affected and
verify inhibitor selectivity, we analyzed our 13 hit compounds
with a set of purposely developed and optimized secondary
assays. A few well characterized Tat and T2SS substrates were
the basis for the development of a mutually exclusive assay
for Tat- or T2SS-selective inhibitors. Tat mediates export of
PvdN and PvdP, both essential components of pyoverdine
biosynthesis that are localized in the periplasm (Voulhoux et al.,
2006; Gimenez et al., 2018). As a consequence, a Tat mutant
is unable to produce pyoverdine. Pyoverdine can be readily
detected in the culture media by measuring the absorbance
at 405 nm, or by a fluorescence assay (em/ex. 405/460 nm).
Since PvdN and PvdP exert their function in the periplasm
and are not T2SS-dependent, a compound able to inhibit both
PLC activity and pyoverdine is likely to be a Tat inhibitor.
Additionally, in order to rule out that such compound could
be a pyoverdine inhibitor rather than a Tat inhibitor, and that
the Tat function could be dependent on pyoverdine, we verified
that PLC activity is not affected in a pyoverdine defective
background (Figure S1B). T2SS is responsible for the export
of several exoproducts of P. aeruginosa, including the LasB
elastase and the PrpL endoprotease. Elastase is translocated into
the periplasm via the Sec mechanism and is therefore Tat-
independent. Here, a standard Elastin-Congo Red assay (Ohman
et al., 1980) could be used to assess whether a compound
exerts an effect on T2SS, and as a negative counter-assay for
potential Tat inhibitors. Similarly, changes in the production
of PrpL endoprotease can be quantitatively determined with a
Secondary Assays
While the screening was aimed at identifying Tat inhibitors,
some compounds could lower the PLC activity without directly
affecting Tat function. The possible impact on PLC activity could
also be due to reduced expression, effect on enzymatic activity,
or transport by other means than via Tat, and therefore these
possibilities were investigated separately. In particular, while
Tat exports PLC to the periplasm, the subsequent translocation
across the outer membrane is mediated by the type II secretion
system (T2SS) (Voulhoux et al., 2001). Since PLC is active both
in the periplasm and in the external media, a T2SS mutant,
unable to export PLC across the outer membrane, would show
no detectable PLC activity in the supernatant, similarly to a Tat
mutant, but will display PLC activity, albeit drastically reduced,
when measured directly in the culture media (Figure S1B).
Therefore, compounds inhibiting the T2SS could also reduce PLC
activity in our assay. Although not the main focus of this study,
the identification of T2SS inhibitors could still be a valuable
finding as this mechanism is known to contribute to the export
FIGURE 1 | Result of the screen of selected library collections for a total of ca. 40,000 compounds. Threshold for the selection of hit compounds (≥20% inhibition of
PLC activity for the Chembridge library and ≥30% inhibition for the remaining libraries) was calculated independently for each plate and is highlighted by a purple line.
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chromogenic substrate (Caballero et al., 2001). However, PrpL
endoprotease is pyoverdine-dependent and therefore also Tat-
dependent, which prevents from using such method as negative
counter-assay for Tat inhibitors. Finally, in order to rule out
false positives identified on the basis of an inhibitory effect on
PLC enzymatic activity, all compounds were tested for their
ability to affect PLC activity in the cell-free supernatant of a
P. aeruginosa PAO1 culture grown in MMST. An overview
of our whole screening strategy, with its primary assay and
mechanism-specific secondary assays, is represented in Figure 2.
The 13 selected hit compounds were tested for their ability to
inhibit pyoverdine production, elastase, or PrpL endoprotease
activity in a PAO1 wt strain. When tested for their effect on
pyoverdine production, we were able to identify 5 compounds
(Library IDs CBK067569, CBK067573, CBK297539, CBK067571,
and CBK067574, respectively renamed TAT-1, TAT-2, TAT-3,
TAT-4, and TAT-5), which caused a reduction of pyoverdine
levels comparable to their effect on PLC activity (Figure 3A).
Following the pyoverdine analysis, we tested all compounds for
their ability to reduce elastase activity. Only one compound
(library ID 7804167, renamed T2S-1) showed a strong inhibitory
effect on elastase activity (Figure 3B), which suggested that this
compound could be a T2SS inhibitor. This compound also
exerted a similar effect on PrpL endoprotease production (data
not shown). On the other hand, none of the compounds able
to inhibit pyoverdine production showed any effect on elastase
activity even at concentrations up to 100 µM (data not shown),
further supporting that they could selectively impair Tat function.
Finally, none of the 13 compounds were able to inhibit PLC
activity in the cell free supernatant (data not shown). Taken
together, these results support that TAT-1, -2, -3, -4, -5, and T2S-
1 are not exerting their effect via impact on bacterial physiology
or by impaired PLC enzymatic activity. The remaining seven
compounds, that did not show any effect either on pyoverdine
production or elastase activity, could possibly reduce PLC activity
in our screening assay by acting on PLC regulation rather
than directly on PLC enzymatic activity. Although potentially
of interest, compounds inhibiting PLC expression were not
followed up in this study. While the effect on growth exerted by
all compounds was measured simultaneously during the initial
screening assay, it was additionally verified by culturing PAO1
in LB media in the presence of increasing concentrations of the
confirmed hits, to rule out that they could have an impact on
bacterial viability in a different media than those used for the
secondary assays. As shown in Figure 3, none of the selected
compounds significantly affect growth or viability of the bacteria.
Although the putative Tat inhibitors TAT-1, -2, -3, -4, and -5
slightly impaired growth at the highest concentrations tested, the
effect is comparable to that seen in a Tat mutant and suggests that
it could be a consequence of Tat inhibition. On the other hand, no
inhibition of growth was observed in the presence of the putative
T2SS inhibitor (T2S-1) and similarly no difference compared to
wild-type was seen in a pilD mutant, which lacks functional T2SS.
Finally, TAT-1, -2, -3, -4, -5, and T2S-1 were again tested for their
ability to inhibit PLC activity on a wider concentration range and
the IC50 values were calculated (Figure 3).
Compound Characterization and
Counter-Assay
Next, we further characterized the putative inhibitors identified
in the screening. The compound that showed the strongest
activity in our primary screening, T2S-1, with an IC50 of 6.3 µM,
showed features of a putative T2SS inhibitor, rather than a Tat
inhibitor. This compound was able to inhibit elastase activity
but had no significant effect on pyoverdine production. During
the development of the screening assay we noted that complete
inhibition of the T2SS function (i.e., a T2SS defective mutant)
strongly impairs but does not completely abolish PLC activity
showing a residual activity up to 20% compared to wild type
(Figure S1B). As a consequence, our assay features a lower
signal-to-background ratio and reduced sensitivity when it comes
to identifying T2SS inhibitors. Our finding suggested that the
FIGURE 2 | Overview of the screening strategy. The Tat system is required for the export in the periplasm of PLC, and the T2SS mediates its subsequent translocation
across the outer membrane. The primary assay, based on the measurement of PLC activity, is thus able to identify both Tat and T2SS inhibitors. Compounds inhibiting
either Tat or T2SS are distinguished in secondary assays for pyoverdine (Tat-dependent) and LasB elastase (T2SS-dependent).
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FIGURE 3 | Characterization and validation of the hit compounds. (A) Structure, effect on PLC activity, pyoverdine production and growth in LB in the presence of
TAT-1, -2, -3, -4, -5. (B) Structure, effect on PLC activity, elastase and growth in LB in the presence of TSS-1. PLC activity was measured in a P. aeruginosa PAO1
culture grown in MMST with added NPPC. 80 µl of the culture was aliquoted in a 384-well microtiter plate in the presence of different concentrations of the inhibitors
◦
and grown at 37 C for 7 h, and A
was measured. Pyoverdine production was measured in a P. aeruginosa PAO1 culture grown in CAA in a 384-well microtiter
405
plate in the presence of different concentrations of the inhibitors. Bacteria were incubated at 37 C for 7 h, and fluorescence was measured at emission/excitation
◦
◦
460/405 nm. Elastase activity was measured in 100 µl of cell-free supernatants of P. aeruginosa PAO1 cultures grown at 37 C for 10 h in LB added with different
◦
concentrations of the tested compounds. Growth was measured in a P. aeruginosa PAO1 culture grown in LB at 37 C for 7 h in a 384-well microtiter plate in the
presence of different concentrations of the inhibitors. Activities have been normalized by the cell density and subtracted of background values. Bars represent
standard deviation from the mean, n = 3.
efficacy of T2S-1 was high enough to overcome the reduced
efficiency of the assay. A literature search for known T2SS
inhibitors led us to a previous study (Moir et al., 2011) where
a number of compounds targeting the T2SS of P. aeruginosa
were identified. Interestingly, two of the compounds reported
by Moir et al. (2011) (Chembridge ID 7790677 and 7801810)
show a strikingly similar chemical structure to T2S-1 with
Tanimoto coefficients of 0.90 and 0.95, respectively (Calculated
in Schrödinger software—Maestro Version 10.5.014 using linear
fingerprint where atom type scheme is all atoms equivalent and
all bonds equivalent). The two compounds are commercially
available and when tested in our assays they showed a similar
effect as T2S-1 (IC50 of 5, 6, and 6.3 µM on PLC activity, and
IC50 of 10, 15, and 11 µM on elastase activity, for compounds ID
7790677, 7801810, and T2S-1, respectively). This strengthens our
hypothesis that this compound could be a valid T2SS inhibitor
and that our screening setup is also suitable for the identification
of T2SS inhibitors.
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Among the five compounds suggested to inhibit the Tat
in Scheme 2 matched exactly with the original compounds
provided in the library (Figures S3–S6). We then recorded
HSQC 1H/15N of these compounds and to our surprise the
two N-H protons are both placed at the exocyclic nitrogen
atom (Figures S7, S8). This result shows that the synthetic
procedure in Scheme 2 resulted in the regioisomers TAT-1a
and TAT-2a instead of the intended and expected products
TAT-1 and TAT-2. In fact, the structures of TAT-1a and -2a
match those of TAT-1-Library and TAT-2-Library, and do not
correspond to the structures described in the library catalog,
which are instead represented by the synthesized TAT-1 and -
2. Accordingly, we thought that this could potentially be the
reason behind the observed lack of activity of the previously
synthesized compounds outlined in Scheme 1. However, TAT-
1a and TAT-2a did not block Tat in contrast to the two original
library compounds (cf. Figure 3). At this end and despite all
chemical and biological evaluation efforts, and the confidence
about determining and synthesizing the exact compounds
received in the library, we hypothesize that an unidentified
contaminant present in the two original library compounds was
responsible for the observed dose-response specific inhibition
of Tat. However, due to the lack of additional stock materials
of these compounds from the library provider, we could not
pursue this hypothesis further and conclusively identify the Tat
inhibiting molecules.
TAT-3, -4, and -5, active in both the Tat and pyoverdine
assays, were ordered from a different batch and retested on
both PLC and pyoverdine assays, and their activity was in line
with what was observed in the screening (data not shown). This
suggests that TAT-3, -4, and -5 could have a specific effect on
the Tat function. However, in view of the weak inhibitory effect
observed in vitro on PLC activity (showing an IC50 of 22, 27, and
29 µM, respectively) and the comparable effect on pyoverdine
production, their activity was deemed not to be sufficient to
warrant further exploration. Although no compound suitable for
further development was identified during the screening, this
study validates the assay we developed as a simple, effective
and high throughput-suited method for the identification of Tat
function, TAT-1 and -2 showed the highest activity, with an IC50
for PLC activity of 14 and 18 µM, respectively. Interestingly, the
chemical structures of both of these compounds are based on
the 5-fluorocytosine scaffold. To validate the identity of the hit
compounds, they were resynthesized starting from commercially
available 2-chloro-5-fluoropyrimidine derivative 3 (Scheme 1)
(Boebel et al., 2010). An aromatic nucleophilic substitution with
3-methoxybenzyl alcohol afforded the free amino pyrimidine
derivative 4 in 73% yield. The sulphonamides TAT-1 and TAT-
2 were then obtained by reacting amino pyrimidine 4 with the
corresponding sulfonyl chlorides followed by the cleavage of
the benzyl group under acidic condition to yield the desired
sulphonamides TAT-1 and TAT-2 in 35 and 28% over two
steps, respectively (Scheme 1). To our surprise, the synthesized
compounds did not block Tat, in contrast to the original hit
compounds supplied by the library provider (data not shown).
Even at a concentration as high as 200 µM, the signal was
comparable to that of the untreated control.
We performed a comparative analysis of the structures of
synthesized TAT-1 and -2 and the remaining available aliquots
of the stock solution of the hit compounds supplied by the
library provider (henceforth renamed TAT-1-Library and TAT-
2-Library). Interestingly, the amide protons of the original hit
compounds from the library appeared at different chemical shifts
(8–9 ppm) compared to the amide protons of the synthesized
compound, which appeared at 11–12 ppm under the same
experimental conditions (Figures S3, S4). Due to the limited
quantity of the original hit compounds, we could not record
2D or 13C NMR data to investigate these compounds further.
At this point, we decided to resynthesize the hit compound via
different synthetic pathway that is based on a reported method
(Boebel et al., 2014) (Scheme 2). Starting from commercially
available 5-fluorocytosine 7, we applied in situ protection
of the cytosine amide using N,O-bis(trimethylsilyl)acetamide
followed by sulphonamide formation upon the reaction with the
corresponding sulfonyl chloride derivatives. The recorded 1H
and 19F NMR data for the compounds synthesized as outlined
SCHEME 1 | Synthetic route A for the synthesis of the target compounds TAT-1 and TAT-2.
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Massai et al.
HTS Assay for Tat and T2SS Inhibitors
SCHEME 2 | Synthetic route B for the attempted synthesis of the target compounds TAT-1a and TAT-2a.
and T2SS inhibitors, and emphasizes the importance of counter
assays and library quality control while performing a screen.
patients. The research performed on the function of the Twin
arginine transport (Tat) system in P. aeruginosa indicates that
it is important not only for acute infection but also for the
establishment of persistent/chronic infection in the CF lung. Tat
has, in fact, been shown to have a role in growth in anaerobiosis
and high osmolarity conditions, and for biofilm production.
This suggests that the Tat functionality could be significant
during chronic infections in the CF airways, where alginate and
dehydrating conditions are known to create a microaerobic and
high osmolarity environment (Ochsner et al., 2002). The recent
genome-wide identification and validation of a number of novel
Tat substrates also seem to indicate that this mechanism could
have a role in a wide range of processes involved in the adaptation
to the host environment and establishment of infection (Gimenez
et al., 2018). Despite the relevance of this mechanism during
both acute and chronic infections, to the best of our knowledge
there has been only one study published so far attempting to
identify Tat inhibitors in P. aeruginosa. Vasil et al. performed
a high throughput screening for Tat inhibitors employing a Tat
activity assay based on the colorimetric measurement of PLC
in the supernatant of a recombinant P. aeruginosa strain (Vasil
et al., 2012). This screening identified two potential inhibitors,
NPM and Bay 11-7082. Despite the promising results and
the demonstrated Tat inhibitory effect, both compounds have
a strong immunomodulatory and cytotoxic activity and are
unlikely to be suitable for development clinically acceptable anti-
virulence drugs. In addition, another study, aimed at identifying
inhibitors of the E. coli Tat system, demonstrated that neither
NPM nor Bay 11-7082 targeted the E. coli Tat mechanism,
possibly due to the lack of an uncoupled cysteine in the E. coli
TatC protein (Bageshwar et al., 2016). Consequently, this finding
suggests that NPM and Bay may not be active against the Tat
system of other Gram-negative pathogens. Nevertheless, these
compounds are still valuable as biochemical probes to investigate
the Tat function in P. aeruginosa.
DISCUSSION
A major drought in antibiotic discovery calls for alternative
therapies, and recently the possibility to employ inhibitors of
single or multiple virulence traits as opposed to the growth
inhibitors per se has begun to be explored. Using in vitro
models this approach has been shown not to generate significant
selective pressure required for emergence of resistant strains
as most virulence factors do not confer direct advantage to
the cell producing them but to the whole population (Mellbye
and Schuster, 2011). Furthermore, targeting specific mechanisms
required for pathogenicity is expected to leave the commensal
flora unscathed, in contrast to the indiscriminate harm caused by
conventional antibiotics. The discovery of Virstatin, a molecule
able to block ToxT polymerization, and thus the expression
of toxin and toxin co-regulated pilus in V. cholerae, and the
observation that it could abrogate pathogenicity and mortality
in a mouse model of infection (Hung et al., 2005), paved the
way for development of similar strategies in other pathogenic
organisms and against different virulence traits. P. aeruginosa,
one of the most dreadful nosocomial pathogens, and well known
for being the main cause of mortality and morbidity in CF
patients, has also been considered for this type of antimicrobial
treatment. On account of its wide arsenal of virulence factors
and determinants, it is evident that the most efficient approach
to tackle P. aeruginosa infectious diseases would be to target
and inhibit molecular and regulatory mechanisms in charge
of the activity of more than one virulence-related phenotype
rather than aiming at suppressing single factors. Secretion
systems provide useful targets since they mediate the export
of several virulence-related factors at once. Furthermore, they
are usually not required for viability in the environment and
in many cases show a high degree of conservation between
different bacterial species, so that an inhibitor could potentially
target a broad range of pathogens. Ideally, the approach should
not only target virulence factors required for establishment of
acute infections but also those that promote chronic/persistent
infections. Possibly the most effective virulence tool and potential
drug target at disposal in P. aeruginosa and other Gram-negative
pathogens is the type three secretion system (T3SS). However,
the low overall prevalence of type III protein-secreting CF
isolates, compared to hospital-acquired infections (Jain et al.,
2004), does not make it a viable target for the treatment of CF
Due to the lack of a clinically acceptable drug targeting
Tat function, and to further explore ways to increase our
understanding and impact on virulence of this mechanism,
we decided to work out a strategy to find novel inhibitors
to the Tat system. We developed a one-well primary assay,
which was capable of direct and simultaneous monitoring of
a compound impact on both native PLC activity and bacterial
growth. Systematic testing of different experimental conditions
allowed us to optimize the assay for high-throughput applications
while retaining high sensitivity and statistical robustness, a
prerequisite for the effectiveness of the screening. In parallel we
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HTS Assay for Tat and T2SS Inhibitors
also developed additional assays for validation of potential hits
as specific Tat inhibitors. This primary assay was employed in
the screening of a subset of different compound libraries with
a total of 39,088 molecules, and was able to identify 59 hits
within our pre-set thresholds, giving an overall hit ratio of 0.15%.
Interestingly, the Prestwick library, consisting of marketed and
FDA approved drugs, yielded the highest hit ratio, where about
1.8% of the compounds showed an inhibitory effect. This was
not unexpected as many of the hits picked up by the screening
are antimicrobial compounds, which even at sub-inhibitory
concentrations are reported to potentially exert pleiotropic
effects on bacterial metabolism and physiology and normal
cell functions (Zhanel et al., 1992; Davies et al., 2006; Babic´
et al., 2010; Imperi et al., 2014). Among the non-antibiotic drug
hits we found three cardiac, one sedative, one anesthetic, one
bronchodilator, and one alcohol deterrent drug. Since all those
drugs could have significant side-effects to otherwise healthy
individuals, we did not consider them suitable candidates for the
development of anti-pseudomonal therapy and did not further
characterize these compounds/drugs. Interestingly, the alcohol
deterrent drug, disulfiram, is already known for its antibacterial
activity against P. aeruginosa, as it inhibits the betaine aldehyde
dehydrogenase which participates in the catabolism of choline
and catalyzes the irreversible oxidation of betaine aldehyde to
glycine betaine. Choline and its derivatives are abundant in
the host environment and inhibition of this enzyme causes
accumulation of the toxic betaine aldehyde intermediate inside
bacterial cells (Zaldívar-Machorro et al., 2011). Betaine aldehyde
dehydrogenase is a cytoplasmic enzyme and should therefore
not be Tat-dependent for its activity. Since we added choline
to the PLC assay culture media, we suspect that the observed
effect of disulfiram on PLC activity could be a consequence
of its effect on the choline catabolism pathway rather than a
specific Tat inhibition. When the hit distribution within the other
subset libraries was analyzed, we noticed an above average hit
ratio per compound within the arachidonic pathway inhibitors
(0.63%) and the kinase targets (0.30%) subsets. However,
none of these primary hit compounds made it through the
following confirmation experiments, suggesting that these classes
of compounds do not exert any specific effect on Tat and were
picked up in the screening due to a general effect on P. aeruginosa
physiology. We did not notice any significant difference in the hit
distribution within the other library subsets. The 59 primary hits
were tested for their effect on PLC at different concentrations in
dose response experiments and the 13 compounds that showed a
dose-response relationship and IC50 below 30 µM were selected
for further characterization. As the screening assay used for
impaired PLC activity could also identify compounds that act
on mechanisms other than Tat, like other transport mechanism,
transcription, expression, regulation, or general cell metabolism,
we established secondary assays, based on the measurement
of other Tat-dependent and/or –independent phenotypes, to
validate the hits. Since both Tat and T2SS are required for
PLC secretion, our screening assay would pick both Tat and
T2SS inhibitors, and therefore secondary assays that discriminate
between the two mechanisms were developed (Figure 2). One
assay was based on that the Tat-dependent periplasmic proteins
PvdN and PvdP are required for the synthesis of pyoverdine
and therefore any compound inhibiting Tat function would also,
as a consequence, reduce pyoverdine production. Furthermore,
pyoverdine production is not affected by the T2SS and we verified
that pyoverdine levels were not reduced in a T2SS defective
mutant (data not shown). Similarly, we confirmed that the Tat
function is not dependent on pyoverdine (Figure S1B), thus
ruling out that the identified compounds could be pyoverdine
inhibitors. Pyoverdine secretion, which can be easily detected
directly in the culture media, therefore constitutes a reliable
and effective counter assay to validate Tat-specific inhibitors. A
second assay was based on LasB elastase, whose export is T2SS
dependent but Tat-independent as secretion across the inner
membrane is mediated by the Sec-system. Elastase activity can be
measured using a standard Elastin-Congo Red assay and thereby
we could confirm whether a compound affected PLC activity
via an inhibitory effect on T2SS. This assay also served as a
negative counter-assay for potential Tat inhibitors. A compound
able to inhibit both PLC activity and pyoverdine production,
but without any impact on elastase activity, is therefore likely
to directly have impact on Tat function. Evaluation of the 13
selected hits using the pyoverdine and elastase assays resulted
in five putative Tat inhibitors that were able to inhibit both
PLC activity and pyoverdine production, but not elastase activity.
In addition, one putative T2SS inhibitor, able to inhibit both
PLC and elastase activity, but not pyoverdine production, was
identified. Seven compounds had no effect on neither pyoverdine
nor elastase. We hypothesize that these compounds could act
as PLC inhibitors. Of the five compounds implied to inhibit
Tat function, TAT-1 and -2 showed the highest impact on
PLC activity, with a dose-response dependent inhibition and
an IC50 below 20 µM. Both presented similar structures with
a fluorinated pyrimidine ring, which initially led us to believe
that this shared chemical group constituted the basis of their
inhibitory effect. Further characterization of these molecules and
comparison of our original results based on the compounds from
the library with our own synthesized molecules showed that
the inhibitory effect was more likely exerted by an unidentified
molecule/contaminant, present in the compound stock solution.
Although difficult to picture how two structurally similar
molecules in a library consisting of ca. 40,000 compounds could
both exert a similar effect in our assay by means of presence of
another molecule/contaminant, our thorough analysis, chemical
synthesis, and biological evaluation efforts leave no doubt that
the compounds specified in the library do not act on the Tat
system, while the unidentified molecules present both fulfill our
criteria as specific Tat inhibitors. This highlights the importance
of quality control of the library compounds and verification of
the primary hits. The hit compounds TAT-3, -4, and -5, active
in both the Tat and pyoverdine assays, could all have a selective
effect on the Tat function. However, the inhibitory effect observed
in vitro on PLC activity and pyoverdine production was relatively
weak and therefore they were not considered for further testing
in in vivo infection models or for further characterization and
drug development.
T2S-1, which inhibited both PLC and elastase activity, displays
the features of a putative T2SS inhibitor and with an IC50 of
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TABLE 1 | Strains and plasmids used in this study.
Strain/plasmid
Genotype/phenotype
Source
STRAIN
E. coli
Top10
mcrA, ꢀ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ꢀlacX74, deoR, recA1,
araD139, ꢀ(ara-leu)7697, galU, galK, rpsL(SmR), endA1, nupG
Invitrogen
+
thi pro hsdR hsdM recA RP4-2-Tc::Mu-Km::Tn7 λpir, Gm
R
S17-1(λpir)
Simon et al., 1983
P. aeruginosa
PAO1
wild type
Gift from Stephen Lory
This study
PAO1 tatC
tatC in-frame deletion mutant
pilD in-frame deletion mutant
pvdA in-frame deletion mutant
PAO1 pilD
This study
PAO1 pvdA
PLASMID
This study
R
pEX18gm
Suicide integration vector, Gm
Hoang et al., 1998
This study
pEX18Gm ꢀtatC
pEX18Gm ꢀpilD
pEX18Gm ꢀpvdA
pEX18gm derivative carrying the flanking regions of the tatC coding sequence
pEX18gm derivative carrying the flanking regions of the pilD coding sequence
pEX18gm derivative carrying the flanking regions of the pvdA coding sequence
This study
This study
6.3 µM this molecule was the strongest inhibitor identified in
our screening. This was somewhat unexpected as our screening
assay was designed to identify Tat inhibitors and not optimized
for identification of T2SS inhibitors. In fact, since in our assay
the NPPC substrate is added directly to the whole bacterial
culture, a limited yet detectable PLC activity could also occur
in the periplasm, regardless of the T2SS function, and thus
increase the background signal for T2SS inhibitors. Nevertheless,
T2S-1 was potent enough to reduce PLC activity way below
our set threshold despite the increased background, and the
effect was similarly evident in the elastase assay. T2S-1 has a
structure similar to two previously identified T2SS inhibitor in
P. aeruginosa, Chembridge ID 7790677 and 7801810 (Figure S9)
(Moir et al., 2011), and comparable activity, which suggests that
this compound is indeed a valid T2SS inhibitor and the shared
structure is likely the responsible for the inhibitory activity. For
the sake of completeness, compounds ID 7790677 and 7801810
were tested in our PLC-based assay and were found to exert
a comparable effect to T2S-1 (data not shown). However, our
identified compound, structurally similar to previously identified
T2SS, was therefore not considered for further characterization,
and development. Importantly, this finding additionally validates
our screening setup for the identification of T2SS inhibitors.
Even though our extensive screening could not identify
compounds that are suitable for further development, this study
has validated the assay we developed as a simple, efficient,
and HTS compatible method for the identification of Tat and
T2SS inhibitors. Furthermore, the limited number of molecules
obtained in our study as well as in previous attempt to identify
Tat inhibitors indicates that targeting the Tat mechanism is a
challenging task, possibly due to the difficulty for an inhibitor
to access the Tat translocon components tightly anchored in
the inner membrane, rather than dispersed in the cytoplasm
or periplasm, in order to have an impact on the Tat protein
complex. This needs to be considered in future screenings for Tat
inhibitors but we are confident that the assays we developed in
this study will be useful tools for future screenings for inhibitors
to be developed as clinical drugs or chemical probes. A better
understanding of the infection process and the identification
and development of virulence inhibitors active on different
mechanisms and targets will both contribute to broaden the
spectrum of treatment options available against P. aeruginosa.
MATERIALS AND METHODS
Bacterial Strains, Growth Media, and
Reagents
Bacterial strains and plasmids used in this study are listed in
Table 1. E. coli and P. aeruginosa cultures were grown in Luria–
Bertani broth (LB) (Sambrook et al., 1989), LB supplemented
with 1.5% agar (LA) or Pseudomonas Isolation Agar (PIA) for
general genetic procedures. Where required, antibiotics were
used at the following concentrations: kanamycin (Km, 50 µg/ml),
gentamycin (Gm, 10 µg/ml) for E. coli; Gm (200 µg/ml) for
P. aeruginosa.
Generation of Plasmids and Reporter
Strains
Standard cloning techniques (Sambrook et al., 1989) were used
to construct the plasmids listed in Table 1. In-frame deletion
of the tatC, pilD, and pvdA was performed as previously
described (Milton et al., 1996) by allelic exchange using the
suicide vectors pEX18Gm tatC, pEX18Gm pilD and pEX18Gm
pvdA. Briefly, upstream and downstream fragments of the
tatC, pilD, and pvdA genes were amplified from P. aeruginosa
PAO1 chromosomal DNA using their corresponding primers.
Both PCR fragments for each gene, carrying complementary 3′
ends obtained through PCR primer design, were then used as
templates in an overlapping PCR to generate a recombinant
fragment that encompasses each gene flanking regions and
containing the desired deletion. These PCR fragments were then
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HTS Assay for Tat and T2SS Inhibitors
cloned into the suicide vector pEX18gm using the BamHI and
HindIII restriction sites, to generate the plasmids pEX18Gm tatC,
pEX18Gm pilD, and pEX18Gm pvdA. Allelic exchange in P.
aeruginosa PAO1 following conjugal mating with E. coli S17-1
λpir donor strains and sucrose counter-selection was performed
as described (Milton et al., 1996). The resulting PAO1 tatC,
pilD, and pvdA mutant strains were confirmed by PCR and
sequence analysis.
presence or absence of different concentrations of the tested
compounds (100 µl final volume). At 16 h of growth, following
measurement of OD600, 20 µl supernatant were collected
from the centrifuged culture and incubated with 20 µl of the
chromogenic substrate Chromozym PL (Sigma) (O’callaghan
et al., 1996; Caballero et al., 2001) in 70 µl phosphate buffer
(pH 7.0) in a 96-well, black, clear bottom microplate (Greiner
655090). The absorbance at 410 nm (A410) was measured at 2 min
intervals for 30 min, and the change in absorbance per minute
was determined. PAO1 culture grown in the absence of any added
compound was used as negative control, while PAO1 pvdA and
PAO1 tatC cultures were both used as positive controls.
High-Throughput PLC Assay
P. aeruginosa PAO1 was grown overnight at 37◦C on LB
agar plates. Bacteria were scraped from the plate surfaces and
diluted in 0.02 in MOPS Minimal Salt Medium (MMST) (3 mM
KCl, 12 mM [NH4]2SO4, 3.2 mM MgSO4, 3 mM NaCl, 20 mM Elastase Assay
glucose, 0.1% tryptose [Becton Dickinson], 50 mM MOPS pH 6.8,
0.05% choline chloride, 20 µM FeCl3) (modified from Lonon and
Morris Hooke, 1991) to an optical density of 600 nm (OD600).
Where required, NPPC was added to the culture media to the
final concentration of 0.5 mM. 80 µl of the culture was aliquoted
Elastase activity was measured in 100 µl of cell-free supernatants
of P. aeruginosa cultures grown at 37◦C for 10 h in LB added with
different concentrations of the tested compounds by the elastin-
Congo red method as described previously (Ohman et al., 1980).
with a Matrix WellMate liquid dispenser (Thermo Scientific) Growth Assay
in each well of a 384-well, black, clear bottom microtiter plate
(Greiner 781096) in the presence of the screening compounds
(final concentrations of 10 µM for the Chembridge library,
20 µM for the other libraries). Microplates were incubated at
Overnight cultures of P. aeruginosa PAO1 and PAO1 tatC were
diluted to OD600 = 0.01 in LB and grown at 37◦C in 384-
well, black, clear bottom microtiter plates (Greiner 781096) in
the presence or absence of different concentrations of the tested
compounds. OD600 was measured at 7 h of growth.
37◦C with shaking. The OD600 and absorbance of 405 nm (A405
)
were measured at 0 h and after 7 h of growth in a Synergy H4
Hybrid Microplate Reader (BioTek). In each microtiter plate,
16 wells containing the same culture grown in the absence of
any added compound were used as negative controls, while 16
wells containing the same culture without added NPPC were
used as positive controls (background). DMSO was added to the
control groups at the same final concentration as the screening
compounds. Absorbance values were normalized by the cell
density and subtracted of positive control values.
General Chemistry
All reactions were carried out under inert nitrogen gas
atmosphere. Chemicals and reagents were purchased from Sigma
Aldrich with exception of 4-amino-5-fluoropyrimidin-2-ol that
was purchased from Matrix Scientific. Organic solvents were
dried using the dry solvent system (Glass Contour Solvent
Systems, SG Water USA) except CH3CN, which was distilled
from CaH2. Microwave reactions were performed in Biotage
Initiator and Flash chromatography was performed on and
Biotage Isolera One using appropriate SNAP Cartridge KP-
Sil and UV absorbance at 254 nm. Thin layer chromatography
(TLC) was performed on Silica gel 60 F254 (Merck) with
detection by UV light unless a staining solution is mentioned.
Preparative high performance liquid chromatography (HPLC)
was performed on Gilson System HPLC, using a YMC-Actus
Triart C18, 12 nm, S-5 µm, 250 × 20.0 mm, with a flow rate 18
or 20 mL/min, detection at 254 nm and the eluent system: A:
H2O and B: CH3CN over 25 or 30 min. LC-MS were recorded by
detecting positive/negative ion (electrospray ionization, ESI) on
Agilent 1,290 infinity II−6,130 Quadrupole using H2O/CH3CN
(0.1% HCOOH) as the eluent system or on Agilent 1,290
infinity−6,150 Quadrupole using YMC Triart C18 (1.9 ꢁm, 20
× 50 mm column) and H2O/CH3CN (0.1% HCOOH) as the
eluent system. The NMR spectra were recorded at 298 K on
Bruker-DRX 400 MHz and 600 MHz using the residual peak
of the solvent DMSO-d6 (δH 2.50 ppm) or CDCl3 (δH 7.26
ppm) as internal standard for 1H, and DMSO-d6 (δc 39.50
ppm) and CDCl3 (δc 77.16 ppm) as internal standard for 13C.
All final compounds were >95% pure according to (HPLC)
UV-trace, 1H and 13C NMR. Synthesis method and spectra
for compounds TAT-1, TAT-2, TAT-1a and TAT2a are detailed
in the Supplementary Material.
Pyoverdine Assay
Overnight cultures of P. aeruginosa PAO1 and PAO1 tatC
were diluted to OD600 = 0.01 in the iron-limiting casamino
acids medium (CAA) (5 g/l casamino acids (Becton Dickinson),
5 mM K2HPO4, 1 mM MgSO4), and grown at 37◦C in 384-
well, black, clear bottom microtiter plates (Greiner 781096)
in the presence or absence of different concentrations of the
tested compounds (80 µl final volume). OD600 and pyoverdine
fluorescence (emission at 460 nm, EM460, after excitation at
405 nm, EX405) were assessed at 0 h and after 16 h of growth
in an Infinite M200 plate reader (Tecan). PAO1 culture grown
in the absence of any added compound was used as negative
control, while PAO1 culture added with 20 µM FeCl3 and PAO1
tatC culture were both used as positive controls (background).
DMSO was added to the control groups at the same final
concentration as for the screening compounds. Absorbance
values were normalized by cell density and subtraction of positive
control values.
PrpL Endoprotease Assay
Overnight cultures of P. aeruginosa PAO1 and PAO1 tatC were
diluted to OD600 = 0.01 in CAA and grown at 37◦C in 384-well,
black, clear bottom microtiter plates (Greiner 781096) in the
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HTS Assay for Tat and T2SS Inhibitors
DATA AVAILABILITY
and analyzed the screening data. MS performed the chemistry
experiments. FM wrote the manuscript with support from ÅF,
ME, and MS. All authors contributed to manuscript revision,
read and approved the submitted version.
The raw data supporting the conclusions of this manuscript will
be made available by the authors, without undue reservation, to
any qualified researcher.
SUPPLEMENTARY MATERIAL
AUTHOR CONTRIBUTIONS
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fcimb.
2019.00250/full#supplementary-material
ÅF, ME, and FM contributed to the conception and design of
the study. FM and TD performed the biological experiments
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Conflict of Interest Statement: The authors declare that the research was
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