A Modified ToxT Inhibitor Reduces Vibrio cholerae Virulence in Vivo

Article abstract of DOI:10.1021/acs.biochem.8b00667

We have previously designed and synthesized small-molecule inhibitors that reduce Vibrio cholerae virulence in vitro by targeting the transcription factor ToxT. Here we report the synthesis and biological activity of derivatives of our previous bicyclic, fatty acid-like inhibitors. All of the synthesized derivatives show antivirulence activity in vitro. For the most potent compounds, a concentration of 5 μM completely inhibited ToxT-mediated tcpA expression as measured in the β-galactosidase assay. One indole compound, 3-(1-butyl-1H-indol-7-yl)propanoic acid (8), was also effective at inhibiting intestinal colonization in the infant mouse. These modified compounds may serve as good candidates for further anti-cholera drug development.

Full text of DOI:10.1021/acs.biochem.8b00667

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Article
Modified ToxT inhibitor reduces Vibrio cholerae virulence in vivo
Anne Woodbrey, Evans O. Onyango, Gabriela Kovacikova, F. Jon Kull, and Gordon W. Gribble
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00667 • Publication Date (Web): 30 Aug 2018
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Modified ToxT inhibitor reduces Vibrio cholerae virulence in vivo
Anne K. Woodbrey,a Evans O. Onyango,a Gabriela Kovacikova,b F. Jon Kulla and Gordon W. Gribble*a
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a Department of Chemistry, Dartmouth College, Hanover, New Hampshire, 03755, USA
b Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire,
03755, USA
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*Corresponding author:
Phone: (603) 646-3118
E-mail: ggribble@dartmouth.edu
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ABSTRACT
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We have previously designed and synthesized small-molecule inhibitors that reduce Vibrio cholerae virulence in
vitro by targeting the transcription factor ToxT. Here we report the synthesis and biological activity of derivatives
of our previous bicyclic, fatty acid-like inhibitors. All of the synthesized derivatives show anti-virulence activity in
vitro. For the most potent compounds, a concentration of 5 μM completely inhibited ToxT-mediated tcpA
expression as measured in the β-galactosidase assay. One indole compound, 3-(1-butyl-1H-indol-7-yl)propanoic
acid (8), was also effective at inhibiting intestinal colonization in the infant mouse. These modified compounds
may serve as good candidates for further anti-cholera drug development.
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INTRODUCTION
The cholera bacterium Vibrio cholerae is a gram-negative bacterium that naturally inhabits aquatic environments
such as brackish water and estuaries and can contaminate drinking water reservoirs. In order to cause disease, V.
cholerae must produce two primary virulence factors, the toxin co-regulated pilus (TCP) and cholera toxin (CT),
which are responsible for intestinal colonization and diarrheal symptoms, respectively.1,2 These virulence factors
are regulated at the transcriptional level by a coordinated network of proteins that respond to specific
environmental signals.3 Among these proteins is ToxT, an AraC family regulator which binds the tcpA and ctx
promoters and directly activates virulence gene expression.4–7 ToxT plays an essential role in V. cholerae virulence,
and its expression and activity are highly regulated among epidemic V. cholerae strains.8 As a result, ToxT is an
obvious target for potential cholera therapeutics.
The X-ray structure of ToxT revealed the presence of cis-palmitoleic acid at the interface between the N-
terminal and C-terminal domains of ToxT.9 This unsaturated fatty acid (UFA) was more than just a crystallographic
ligand, however, as it inhibited ToxT transcriptional activity in vitro.9 Regulation of ToxT by UFAs likely originates in
the context of the host environment. By binding ToxT and downregulating virulence gene expression, bile and
UFAs present in the intestinal lumen prevent virulence activation until V. cholerae has reached the intestinal
crypts, the appropriate location for colonization and delivery of cholera toxin.10 Based on the ToxT-bound
conformation of cis-palmitoleic acid, we recently designed and synthesized a number of compounds that
significantly reduce V. cholerae infection in vitro.11 These compounds, which we will identify by a methyl group at
the 8 position of 1,8-disubstituted naphthalene (Figure 1), reduce virulence gene expression by binding ToxT and
inhibiting ToxT-DNA binding interactions. These ‘8-methyl’ inhibitors were comparatively more potent than
virstatin (Figure 1), a small-molecule therapeutic identified via high throughput screening,12 demonstrating the
benefits of rational structure-based drug design. Virstatin presumably inhibits ToxT dimerization, a required
activity for activating virulence gene expression at the ctx promoter.13 The crystal structures of two ‘8-methyl’
compounds in complex with ToxT revealed additional opportunities for structural modifications of the small
molecules in order to further improve their inhibitory potential.11 Thus, we now report the design, synthesis, and
biological characterization of analogues of our previous ‘8-methyl’ inhibitors.
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The new disubstituted naphthalene and indole derivatives (Figure 1) are prepared using similar chemistry
as we used for their 8-methyl-1-substituted naphthalene precursors, confirming that our synthetic design is
amenable to additional manipulation. Furthermore, only a handful of reactions is necessary to achieve a variety of
desired target molecules; this synthetic simplicity and robustness is an important criterion in drug design. More
importantly, the second-generation derivatives are at least as effective as the previous ‘8-methyl’ compounds. Our
newest lead compound 8 not only exhibits significantly improved activity in vitro, but protects against V. cholerae
colonization in the infant mouse. Additionally, we explore the mechanism of ToxT inhibition by synthetically
modifying the carboxylate moiety of one compound and by altering a critical lysine residue in ToxT, key factors
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involved in ToxT-inhibitor binding.
Figure 1. General structures of the ‘8-methyl’ compounds, virstatin, and newly synthesized derivatives.
MATERIALS AND METHODS
Bacterial strains and media
Classical (O395) and El Tor (C6706) strains of V. cholerae are both virulent in humans. C6706 was used for
the infant mouse colonization assay, as El Tor strains are more prevalent and more dominant in the current setting,
and thus more relevant to in vivo colonization. Additionally, our methods were based on the C6706 inoculations
reported by Hung et al. However, ToxT is more strongly expressed in O395 strains in vitro. Because we were
monitoring a ToxT-mediated effect caused by the small-molecule inhibitors, O395 strains were used for the β-
galactosidase assays.
All strains and plasmids used in this study are listed in Table 1. Strains were maintained at -80 °C in Luria-
Bertani (LB) broth containing 30% (v/v) glycerol. All strains were grown in LB at 30 °C; cultures were aerated by
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rotating culture tubes on a roller drum. Antibiotics and additives were used in LB/agar plates at the following
concentrations: kanamycin (Km), 45 μg/ml; gentamycin (Gm), 30 μg/ml; streptomycin (Sm), 100 μg/ml (for
determining CFUs) or 1 mg/ml (for allelic exchange); 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), 40
μg/ml.
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Table 1. Bacterial strains and plasmids
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Strain or plasmid
E. coli strains
S17-1 λpir
Characteristic(s)
Reference
Tpr Smr recA, thi, pro, hsdR- M+
[RP4-2-Tc::Mu:Kmr Tn7] (λpir)
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V. cholerae strains
AW40
MBN142 toxT K230A
MBN017 toxT K230A
El Tor Inaba, Smr
This work
AW46
This work
C6706 str2
KSK1184
MBN017
MBN135
MBN142
O395 Sm
Plasmids
pAKW8
Laboratory collection
C6706 str2 ꢀtoxT
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O395 Smr ꢀtoxT
O395 Smr ꢀlacZ tcpA-lacZ
MBN135 ꢀtoxT
Classical Ogawa, Smr
pKAS154 tcpF-toxT K230A-tcpJ
pKAS32 derivative, Kmr
pACYC184 Gm cassette, Gmr
This work
pKAS154
pMIN1
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Strain and plasmid construction
The O395 tcpA-lacZ fusion strain carrying toxT mutant K230A was constructed using a gBlock Gene
Fragment synthesized by IDT. The gBlock contained toxT K230A and 484 base pairs (bp) of the downstream gene
tcpJ. The gBlock was amplified using primers ToxT F Sap and TcpJ R Not (Table S1). In addition, 450 bp of the
upstream gene tcpF was amplified from O395 Sm using primers TcpF F Xba and ToxT R Sap (Table S1). The PCR
fragments were digested with appropriate restriction enzymes and ligated into pKAS154,18 which had been pre-cut
with XbaI and NotI enzymes. The resulting ligation, pAKW8, was electroporated into E. coli S17-1 λpir.14 Kmr
colonies were screened for the insert via PCR. The mutant toxT gene was then introduced into V. cholerae strain
MBN142 by allelic exchange,19 generating strain AW40. Strain MBN142 carries plasmid pMIN1, which confers Gmr
as a counter-selection for conjugation.17 The ToxT K230A mutant was confirmed by DNA sequencing.
The O395 strain carrying toxT K230A was constructed for TcpA immunoblotting (Figure S1) via allelic
exchange. Plasmid pAKW8 in E. coli S17-1 λpir was introduced into MBN017 (carrying plasmid pMIN1) to generate
strain AW46. The ToxT K230A mutant was confirmed by DNA sequencing.
β-galactosidase assays
Strains were grown in LB pH 6.5 with aeration for 14 hours. Small-molecule inhibitors were added at the time
cultures were inoculated. Cultures were diluted 1:10 in fresh LB 6.5 and β-galactosidase activity was determined by
the method of Miller.20 Results are the average of three or more independent (biological) experiments, each
performed in duplicate (2 technical replicates for each sample).
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Infant mouse colonization assay
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Mouse colonization assays were performed in accordance with Dartmouth College’s Institutional Animal Care and
Use Committee (IACUC). Three- to four-day-old CD1 mice (Charles River) were orogastrically inoculated with ~105
CFU bacteria (50 μl of V. cholerae C6706 cultures that had been grown overnight then diluted 1:1,000 in fresh LB).
Mice had been removed from their mothers and mixed with non-littermates 3 hours prior to inoculation. For mice
receiving the wild-type C6706 strain, DMSO or the compound was added to the inoculum. Serial dilutions of the
inocula were plated onto LB agar supplemented with 100 μg/ml Sm to determine input CFU. Infected mice were
incubated at 30 °C for 16-18 h, then sacrificed and dissected. Small intestines were removed and homogenized
with a Tissue Tearor in 4 ml sterile LB media containing 10% glycerol. Intestinal homogenates were serially diluted
and plated onto LB agar/Sm as before to enumerate the CFUs recovered. The colonization index (CI) was reported
as the ratio of output to input CFUs. Experiment was repeated.
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In vitro cytotoxicity assay
Samples were prepared identically to mice inocula: compounds were added to cultures of V. cholerae C6706 that
had been grown overnight and diluted. Cultures were then grown at 30 °C. Samples taken after one hour and
overnight growth were serially diluted and plated. The colony forming units (CFUs) of cultures grown in the
presence of compounds were compared to that of the wild-type culture. Compounds were tested at various
concentrations until in vitro growth was not at all attenuated.
Western blot
V. cholerae strains O395 Sm, O395 Sm ꢀtoxT (MBN017), and O395 Sm toxT K230A (AW46) were grown in LB pH 6.5
with aeration for 14 hours at 30 °C. Whole-cell extracts were assayed for total protein concentration using the
Pierce BCA Protein Assay Kit (Thermo Scientific). Samples were subjected to SDS-PAGE on 16% Tris-Glycine gels
(Invitrogen) and transferred to a nitrocellulose membrane using iBlot (Invitrogen). The membrane was blocked
with 3% bovine serum albumin in TBST (1x Tris-buffered saline, 0.1% Tween), incubated with anti-TcpA antibody,
and washed in TBST. After incubation with horseradish peroxidase-conjugated secondary antibody (Bio-Rad), the
membrane was washed in TBS. Blots were visualized using the Pierce ECL detection system (Thermo Scientific)
according to the manufacturer’s protocols.
RESULTS AND DISCUSSION
Synthesis and biological screening of derivatives
The crystal structures of the ‘8-methyl’ compounds in complex with ToxT revealed significant unoccupied space
above the methyl group of the compounds (Figure 2).11 We hypothesized that the ToxT-effector binding pocket
could easily accommodate an extended alkyl chain at the 8 position, and that increasing the aliphatic chain would
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exclude water from the primarily hydrophobic pocket. This is also consistent with the original crystal structure of
ToxT, which accommodated a 16-carbon monounsaturated fatty acid and showed sufficient space for an 18-carbon
UFA.9 We thus used the crystal structure to design the ‘8-alkyl’ derivatives.
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Figure 2. A cross-section of the ToxT inhibitor-binding pocket containing an ‘8-methyl’ compound (from the crystal structure,
PDB code 5SUW) (a) and an ‘8-alkyl’ derivative (predicted by AutoDock21) (b). The surface of the pocket is colored by amino acid
hydrophobicity: most hydrophilic (blue), neutral (white), most hydrophobic (orange red). Pocket calculated by CASTp.22
For 1,8-disubstituted naphthalenes 1 – 4, we employed the synthetic procedures used previously.11,23
Variation of the alkyl group at the 8 position of 8-methyl-1-substituted naphthalene was obtained by de novo
synthesis from a suitable 2-alkylfuran; Diels-Alder cycloaddition using 2-n-butyl- and 2-n-pentylfurans in place of 2-
methylfuran gave the corresponding epoxides. The reactions favored the formation of the syn-cycloadduct, the
regioselectively major product, consistent with previous findings.24 Diimide reduction and subsequent acid-
catalyzed dehydration quantitatively gave the bromonaphthalene ‘anchors’ for Heck coupling. Catalytic
hydrogenation and ester hydrolysis gave the desired final ‘8-alkyl' products.
The biological activity of compounds 1 – 4 was screened using the β-galactosidase assay.20 Cultures of
O395 V. cholerae strains harboring chromosomal tcpA-lacZ fusions were grown in the presence of the synthesized
compounds and assayed as described previously.11 Expression of tcpA was markedly decreased when treated with
the ‘8-alkyl’ compounds (Figure 3). In fact, all four compounds inhibited tcpA to a greater extent than virstatin and
all ten ‘8-methyl’ inhibitors.
As illustrated in Figure 2, the anionic carboxylate of the compound forms electrostatic interactions with
the side chains of two lysine residues of ToxT. In order to support our hypothesis that the carboxylate moiety is
required for the compounds’ inhibitory activity, we reduced the carboxylic acid of 3 to an alcohol using lithium
aluminum hydride and tested the pure product as before. The resulting alcohol 5 is essentially inactive at 5 ꢁM and
shows minimal inhibitory activity even with a 10-fold increase in concentration (Figure 3). The reduced head group
(of 5) must be responsible for this loss in activity, as the remainder of the molecule is identical to its acid
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predecessor 3. Although expected, this result nonetheless confirms our original assumption that the carboxylate is
necessary for binding and inhibiting ToxT.
Figure 3. Effect of the ‘8-alkyl’ derivatives on tcpA-lacZ expression. Activity of the untreated wild-type strain (WT) is set to
100%. Relative β-gal activities of the negative control (△ToxT) and solvent control (DMSO) are calculated as a percentage of
WT; samples containing inhibitor are calculated relative to DMSO. Final concentrations of inhibitor are 50 ꢁM (white), 5 ꢁM
(dark blue), 0.5 ꢁM (grey), and 0.05 ꢁM (light blue). Error bars represent standard deviation where n is 3. *Compound 2 is a
mixture of tetralins (see supplementary material).
A second group of derivatives – the indole-based mimics – were designed with drug metabolism in mind.
The indole moiety is ubiquitous in biology, and an indole-containing inhibitor may have advantages in vivo, such as
reduced toxicity of its degradation products. Utilization of the indole moiety in place of the naphthalene may not
necessarily improve the compounds’ ability to inhibit ToxT, but may nevertheless result in a more effective drug.
Additionally, the free indole NH in the 4-indole derivatives serves as a potential handle for further substitution and
provides a hydrogen-bond donor. Functionalization at this NH may provide opportunities to increase solubility.
Synthesis of the 7-indole derivatives is outlined in Scheme 1. Heck coupling of the commercially available
7-bromoindole and methyl acrylate, following the procedure employed previously,11,25 gave 6. Compound 7 was
synthesized from 6 in two steps since alkylation of 626 was always accompanied by partial hydrolysis. While the
acid and ester alkylation products were separable, we found it convenient to convert the ester to the desired acid
instead. The crude mixture from step one was subjected to saponification (step two) to yield 7, which was reduced
to 8.
Synthesis of the 4-indole derivatives was achieved starting from commercially available 4-bromoindole
(Scheme 2). Acylation of the starting material26 followed by reduction of the ketone 9 provided 10. Heck coupling
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Scheme 1. Synthesis of 7-indole derivatives.
Scheme 2. Synthesis of 4-indole derivatives.
of 10 and methyl acrylate gave the disubstituted analogue 11, which was subjected to Pd-catalyzed hydrogenation
and ester hydrolysis11 to yield final products 12 and 13. To facilitate a more facile purification, 13 was made from
its corresponding ester (the reduced product of 11) rather than directly from 12.
The expression of tcpA was decreased significantly in the presence of 5 ꢁM 7, 8, and 13 (Figure 4).
Compound 12 did not inhibit tcpA transcription at the concentrations tested, which we believe were too low for
activity. Given the effectiveness of compound 13 and its similarity to 12, we favor this explanation over the
argument that 12 is unable to bind ToxT. In fact, compound 12 was moderately effective at higher concentrations
(50 ꢁM), reducing tcpA transcript levels to 60%.
As a whole, the introduction of the indole motif was less effective in improving the inhibitory activity of
the molecules in vitro compared to the extension of the alkyl chain. If the improvement of the ‘8-alkyl’ derivatives
over the ‘8-methyl’ precursors is due to the increased number of carbons, however, any benefit provided by the
presence of the nitrogen in the indole may be counteracted by the smaller indole ring (C10 for naphthalene vs. C8
for indole). More detailed analyses are required for insights into the contributions of the presence of the N/NH,
the position of the nitrogen (the 4- vs. the 7-postion), and the increase in the angle between the alkyl chain and
the indole (which is more splayed than the alkyl chain-naphthalene angle).
In vivo activity of virstatin and compounds 4 and 8
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Table 2. CFUs of in vitro cultures grown for 1 hour and
overnight in the presence of inhibitors.
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We next tested the effectiveness of the new ToxT inhibitors to protect against V. cholerae infection in the suckling
mouse. We used a prior study identifying virstatin as an effective ToxT inhibitor in vivo as a starting point for our
experiment12 and chose compounds 4 and 8 to test alongside virstatin. In order to test our inhibitors at the
Figure 4. Effect of the indole derivatives on tcpA-lacZ expression. Relative β-gal activity is calculated as in Figure 3. Final
concentrations of inhibitor are 50 ꢁM (white), 5 ꢁM (dark blue), 0.5 ꢁM (grey), and 0.05 ꢁM (light blue). Error bars represent
standard deviation where n is 3-4.
high concentrations suggested in the previous study (upwards of 50 mM, three orders of magnitude higher than
we have tested in the β-gal assays), we first measured cytotoxicity in vitro. Samples were prepared identically to
those used for inoculating mice: untreated V. cholerae strains were grown overnight in non-inducing conditions,
cultures were diluted to the appropriate CFU, compounds were introduced, and samples were cultured overnight.
Not only was a high concentration of 8 lethal to V. cholerae, but virstatin reduced growth by 6 logs (Table 2).
Higher concentrations of virstatin and compound 4 were also lethal to O395 cultures either immediately or after a
few hours of growth (data not shown). Based on these results, it is unclear how additional 17.65 mM doses of
virstatin (50 ꢁg in 10 ꢁl) administered post-inoculation could be tolerated by V. cholerae,12 when in vitro growth is
severely attenuated at 1 mM (Table 2). Furthermore, the concentration of virstatin used previously was higher
than the minimal bactericidal concentration determined for V. cholerae strain C6706.12 The data in Table 2
suggests that the decrease in in vivo colonization in the presence of virstatin measured previously may be an
artificial effect caused by virstatin’s bactericidal effects on V. cholerae in the inoculum.
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1 hour
overnight
1.45 x 1010
2.00 x 104
2.37 x 1010
1.21 x 109
2.98 x 1010
0
DMSO
1 mM virstatin
0.1 mM virstatin
3.11 x 107
3.48 x 107
2.83 x 107
4.43 x 107
2.80 x 107
0
1 mM compound
0.1 mM compound
10 mM compound
1 mM compound
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5.98 x 107
1.77 x 1010
Because of the above considerations, virstatin, 4, and 8 were tested in vivo at concentrations that did not
attenuate V. cholerae growth in vitro (0.1 mM for virstatin and 4; 1 mM for 8; see Table 2) (Figure 5). Mice were
not given an additional “boost” of drug post-infection, as we were cautious to stay below lethal limits, and also
wanted to avoid any deleterious effects caused by additional handling and successive oral gavages. As shown in
Figure 5, mice treated with compound 8 exhibited a significant decrease in intestinal colonization by V. cholerae
C6706, whereas no measurable effect was observed for compound 4. Colonization was slightly inhibited for
virstatin-treated mice, but the difference was not statistically significant (p=0.1433 by Tukey-Kramer HSD means
comparison). Notably, 8, an indole derivative designed to more closely resemble bioactive natural products and
therefore possess greater therapeutic potential, was indeed less toxic to V. cholerae than 4 and virstatin. This
allowed for administration of a higher dose, which was effective at inhibiting ToxT in vivo.
0
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Figure 5. Effects of inhibitors on intestinal colonization. Mice were given the negative control strain (△ToxT), the wild-type
strain containing a solvent control (DMSO), or the wild-type strain containing an inhibitor (0.1 mM virstatin, 0.1 mM compound
4, or 1 mM compound 8). A Box-Cox transformation of the colonization index raw data (output/input CFUs) was performed to
normalize the data distribution (Figure S2). Pairwise means comparison using Tukey-Kramer HSD indicates statistically
significant differences between 2 pairs: DMSO/△ToxT (p=0.0011) and DMSO/8 (p=0.0029). Note: A decrease in CI for compound
8 was also statistically significant when the untransformed (raw) data was analyzed using the nonparametric Kruskal-Wallis test
(Figure S2).
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Figure 6. Influence of a toxT mutation on the expression of tcpA-lacZ in the presence or absence of inhibitors. β-gal activity was
assayed in the wild-type toxT background (solid) or the toxT K230A background (striped). Samples were grown alone (WT and
K230A), with a solvent control (DMSO), and in the presence of various inhibitors. Final concentrations of inhibitor are 708 ꢁM
(green), 50 ꢁM (white/grey), and 5 ꢁM (blue). Error bars represent standard deviation where n is 3. See Figure S1 for western
blot.
Activity of ToxT K230A mutant
We further explored the interactions between the small-molecule inhibitors and ToxT by modifying ToxT.
The most obvious residues for mutagenesis were Lys31 and Lys230, which interact with the carboxylate head of
the inhibitor.9,11 Mutations were introduced into a tcpA-lacZ strain via allelic exchange.19 As shown in Figure 6, the
K230A mutation caused a ~20% decrease in tcpA expression but had essentially no effect on the response of ToxT
to its inhibitors. Surprisingly, virstatin and compound 8 inhibited tcpA expression in the K230A background, while
the alcohol 5 had no effect, similar to the findings for the WT ToxT background. Oleic acid, a known ToxT inhibitor,9
also inhibited tcpA expression in the mutant background (Figure 6).
We could not successfully obtain the K31A mutant or the K31A/K230A double mutant, however; V.
cholerae would acquire the mutant alleles under antibiotic pressure, but strains containing toxT K31A were not
recoverable under the conditions used for growth. It is possible that V. cholerae selects against toxT K31A and
K31A/K230A by consistently excising the mutant copy along with the plasmid. An alternative explanation is that
allelic exchange was successful, but cells containing the mutant toxT were non-viable and no colonies containing
toxT K31A or K31A/K230A could be observed. While there is precedence for functional ToxT K31A, the previous
construct was MBP-toxT K31A expressed from a plasmid.27
CONCLUSIONS
Structural modifications to our previously identified ToxT inhibitors were conducted in order to improve anti-
virulence efficacy and drug-like properties. Substituting an indole ring for naphthalene decreased cytotoxicity, and
one indole derivative significantly reduced V. cholerae colonization in vivo. We further explored protein-ligand
binding interactions by modifying the carboxylate head group of the compound and a key residue in the effector-
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binding pocket of ToxT. Compound 5, the alcohol derivative, showed no inhibitory activity in vitro, confirming the
carboxylate moiety is essential for inhibiting ToxT. Therefore, it seems logical that amino acids interacting with the
carboxylate – lysines 31 and 230 – are also critical for ToxT-inhibitor binding. Surprisingly, mutating lysine 230 had
no effect; ToxT K230A behaved like wild-type in the presence of inhibitors. This suggests that, if K230 is not
required for inhibition, K31 must be important. We were unable to confirm the importance of lysine 31, however,
as we could not produce a V. cholerae strain carrying toxT K31A. One explanation for this result is that the K31A
mutant renders ToxT insoluble and prone to aggregation, causing impaired cell growth and/or toxicity.
Alternatively, V. cholerae may exert strong negative selection on toxT K31A if the mutant reduces fitness or is
otherwise deleterious. Regardless of the explanation, it seems that lysine 31 must be conserved. Given our
hypothesis that this residue is critical for inhibitor binding, it is therefore unlikely that V. cholerae will develop
resistance to our compounds. Finally, the finding that K230 is not required for ToxT activity highlights the
importance of future studies aimed at refining the structural model for ToxT activation/inhibition, which thus far
has involved both lysine residues.9,11
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ASSOCIATED CONTENT
Supporting Information
TcpA western blot (Figure S1); Oligonucleotides used in this study (Table S1); Colonization Index (CI) data (Table
S2); Histograms and boxplot of the CI data (Figure S2); Experimental methods for synthesis of compounds;
Experimental spectra of synthesized compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: (603) 646-3118; E-mail: ggribble@dartmouth.edu.
Author Contributions
A.K.W. performed nearly all experiments; E.O.O. contributed to organic synthesis; G.K. contributed to microbiology
assays. A.K.W., F.J.K., and G.W.G. designed the study. The manuscript was written by A.K.W., with minor edits
made by F.J.K. and G.W.G.
Funding
Research was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the National
Institutes of Health (NIH) under award number F31AI124549 (to A.K.W.). This work was also supported by Donors
of the Petroleum Research Fund administered by the American Chemical Society (to G.W.G.), and NIH grants
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AI072661 (to F.J.K.), AI025096 (to Karen Skorupski in the Department of Microbiology and Immunology, Geisel
School of Medicine), and AI120068 (to Karen Skorupski and F.J.K.).
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Notes
8
A patent based on molecules included in this manuscript has been awarded to AKW, EOO, FJK, and GWG.
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Acknowledgements
We thank Karen Skorupski for providing laboratory resources for this study, as well as feedback on the manuscript.
We gratefully acknowledge Carol S. Ringelberg of the Genomics and Molecular Biology Shared Resources (GMBSR),
Geisel School of Medicine, for statistical analysis.
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Modified ToxT inhibitor reduces Vibrio cholerae virulence in vivo
Anne K. Woodbrey,a Evans O. Onyango,a Gabriela Kovacikova,b F. Jon Kulla and Gordon W. Gribble*a
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O
OH
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