JB Accepted Manuscript Posted Online 2 March 2015 J. Bacteriol. doi:10.1128/JB.02409-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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The mechanism for inhibition of Vibrio cholerae ToxT activity by the unsaturated fatty acid
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components of bile
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Sarah C. Plecha and Jeffrey H. Withey#
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Department of Immunology and Microbiology
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Wayne State University School of Medicine, Detroit, MI 48201
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# To whom correspondence should be addressed: Jeffrey H. Withey, Department of
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Immunology and Microbiology, Wayne State University, 540 E. Canfield, Detroit, MI 48201,
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USA, Tel.: (313) 577-1316; Fax: (313) 577-1155; E-mail: [email protected]
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Running Title: Mechanism for ToxT inhibition by bile
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ABSTRACT
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The gram-negative curved bacillus Vibrio cholerae causes the severe diarrheal illness
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cholera. During host infection, a complex regulatory cascade results in production of ToxT, a
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DNA-binding protein that activates the transcription of major virulence genes that encode
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cholera toxin (CT) and toxin-coregulated pilus (TCP). Previous studies have shown that bile and
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its unsaturated fatty acid (UFA) components reduce virulence gene expression and therefore
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are likely important signals upon entering the host. However, the mechanism for the bile-
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mediated reduction of TCP and CT expression has not been clearly defined. There are two likely
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hypotheses to explain this reduction: 1) UFAs decrease DNA binding by ToxT or 2) UFAs
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decrease dimerization of ToxT. The work presented here elucidates that bile/UFAs directly
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affect DNA binding by ToxT. UFAs, specifically linoleic acid, can enter V. cholerae when added
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exogenously and are present in the cytoplasm where they can then interact with ToxT.
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Electrophoretic mobility shift assays (EMSAs) with ToxT and various virulence promoters in the
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presence or absence of UFAs showed a direct reduction in ToxT binding to DNA, even in
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promoters with only one ToxT binding site. Virstatin, a synthetic ToxT inhibitor, was previously
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shown to reduce ToxT dimerization. Here we show that virstatin only affects DNA binding at
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ToxT promoters with two binding sites, unlike linoleic acid which affects ToxT binding
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promoters having either one or two ToxT binding site. This suggests a mechanism in which
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UFAs, unlike virstatin, do not affect dimerization but affect monomeric ToxT binding to DNA.
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IMPORTANCE Vibrio cholerae must produce the major virulence factors cholera toxin (CT) and toxin2
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coregulated pilus (TCP) to cause cholera. CT and TCP production depends on ToxT, the major
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virulence transcription activator. ToxT activity is negatively regulated by unsaturated fatty acids
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(UFA) present in the lumen of the upper small intestine. This study investigates the mechanism
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for inhibition of ToxT activity by UFAs and finds that UFAs directly reduce specific ToxT binding to
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DNA at virulence promoters and subsequently reduce virulence gene expression. UFAs inhibit
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ToxT monomers from binding DNA. This differs from the inhibitory mechanism of a synthetic
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ToxT inhibitor, virstatin, which inhibits ToxT dimerization. Understanding the mechanisms for
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inhibition of virulence could lead toward better cholera therapeutics. INTRODUCTION
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V. cholerae, a gram-negative curved bacillus possessing a single polar flagellum, is the
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causative agent of cholera. Cholera is a diarrheal disease contracted by consuming
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contaminated food or water and is characterized by severe diarrhea that leads to dehydration,
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and can ultimately cause death if left untreated. Each year, there are an estimated 1.4-4.3
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million cholera cases and 20,000-142,000 deaths from the disease (1, 2). To cause disease, V.
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cholerae must colonize the upper small intestine, where it expresses virulence genes including
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those that encode the two most important virulence factors: toxin co-regulated pilus (TCP) and
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cholerae toxin (CT). TCP is required for intestinal colonization, while CT is responsible for the
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massive secretion of electrolytes and water into the lumen, causing diarrhea. Transcription of
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these and other virulence genes are regulated by the major virulence transcription activator
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ToxT.
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Virulence gene expression is regulated by what is historically known as the ToxR regulon
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(3, 4). toxT transcription is activated by two pairs of inner membrane proteins, ToxR/ToxS and
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TcpP/TcpH, that bind upstream of toxT, as well as by a ToxT positive feedback loop (5-9). ToxT is
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a 32-kDA member of the AraC/XylS transcriptional regulator family, having a conserved 100-
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amino-acid DNA binding domain, consisting of two helix-turn-helix motifs, in its C-terminal
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domain (CTD).
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sequences called toxboxes that vary in configuration at individual genes (10-14). ToxT directly
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controls transcription of genes encoding not only TCP and CT, but also other accessory virulence
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genes including acfA, acfD, aldA, tagA, tarA, tarB, and tcpI (11-13, 15, 16). Except for the aldA
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promoter, in which there is a single toxbox, there are two toxboxes present at ToxT-activated
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promoters (11-14, 16). ToxT can bind to single toxboxes as a monomer but it is thought that full
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activation only occurs upon ToxT dimerization on the DNA, at least at some genes (12, 13). The
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ToxT N-terminal domain (NTD), does not share significant sequence homology with other
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proteins, but has some structural similarity to the AraC NTD, which is the domain necessary for
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AraC dimerization and arabinose binding (17). NTDs of ToxT have been shown to interact when
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separated from the CTD, although the true role of dimerization in ToxT function is not fully
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understood (16-22).
Located upstream of all ToxT-activated genes are 13-bp degenerate DNA
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There are a number of host and environmental factors that affect ToxT activity, including
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temperature, pH, bile and bicarbonate (23-27). Bile is produced by the liver and then
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subsequently stored in the gallbladder. Upon eating, bile is released into the duodenum where
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it acts to solubilize lipids. Bile itself is a complex, heterogeneous mixture that includes bile salts,
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cholesterol, bilirubin, and saturated and unsaturated fatty acids (UFAs). V. cholerae encounters
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bile early during infection and it is proposed to be a natural effector of ToxT as it is found in the 4
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same places that V. cholerae colonizes (24, 25, 27). V. cholerae has reduced virulence gene
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expression in the presence of bile and/or UFAs, with increased motility gene expression, biofilm
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formation, the induction of efflux pumps, and increased amounts of outer membrane proteins
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OmpU, OmpT and TolC (24, 25, 28-31). Bile and its UFA components have been previously
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shown to decrease CT and TCP production, but the mechanism of this effect on virulence gene
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expression levels is not fully understood (24, 25). Bile also has another role in that it is able to
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act as a bactericide for the benefit of the host, but enteric bacteria, such as V. cholerae, are not
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only adapted to live in the presence of bile, but potentially can recognize bile as a signal to
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ensure survival in the host (24, 27). Another ToxT inhibitor is a synthetic molecule called
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virstatin (21, 22). The mechanism of action for virstatin has been proposed to be through
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inhibition of ToxT dimerization (21). Whether UFAs and virstatin inhibit ToxT activity via the
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same mechanism is unknown.
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In this study we show that UFAs, specifically the unsaturated fatty acid linoleic acid, are
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able to enter the bacterial cytoplasm where ToxT is also present. We report that linoleic acid
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causes decreased transcription of ToxT-controlled virulence genes assessed. The mechanism by
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which this occurs is decreased binding affinity at ToxT-activated promoters in the presence of
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linoleic acid, regardless of the number or configuration of the toxboxes. Our results indicating
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that UFAs decrease ToxT binding to DNA even at promoters having one ToxT binding site
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suggest a mechanism in which UFAs affect the ability of monomeric ToxT to bind to DNA, in
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contrast to the results we observed using virstatin. Thus these two ToxT inhibitors exhibit
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somewhat different modes of action. These results give us a clearer understanding of the
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regulatory networks controlling virulence gene expression during human infection. 5
MATERIALS AND METHODS
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V. cholerae strains and growth conditions. All V. cholerae strains used in the study are
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derived from classical biotype O395. Strains were maintained in Luria Broth (LB) containing
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20% glycerol and stored at -70°C. All promoter::lacZ fusions used for β-galactosidase assays
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were made in plasmid pTL61T as described in previous studies (26, 32). Overnight cultures were
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grown for ~16 hours at 37°C in LB and then diluted 1:40 into LB pH 6.5 at 30°C for virulence
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inducing conditions in the presence or absence of freshly prepared .05% bile (sodium choleate),
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160µM linoleic acid, or 100µM virstatin. V. cholerae strains were grown with antibiotic
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concentration of 100µg/mL streptomycin and strains with the plasmid pTL61T were grown with
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100µg/mL ampicillin.
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14
C linoleic acid uptake. V. cholerae classical biotype strain O395 was grown overnight in
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LB at 37°C, subcultured 1:40 in LB pH 6.5, and grown for two hours in the absence of linoleic
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acid. At two hours, 0.1μCi
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added for each milliliter of the subculture. Upon addition of the radiolabeled linoleic acid, 1 mL
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of the culture was immediately centrifuged, and the supernatant saved to compare to cell
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pellet cpms. The cell pellet was washed 3 times with 1 mL of PBS and centrifuged each time.
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Cell pellets were resuspended in 100μL PBS and added to 5mL Scintillation cocktail (Fisher
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Scientific). The same procedure was followed for other aliquots of the subculture at times 5, 15
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and 30 minutes. After uptake, cpm was measured for each time point using an LS6000IC liquid
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scintillation counting system (Beckman).
124 125
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C-radiolabeled linoleic acid (58.2mCi/mmol) (Perkin-Elmer) was
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C linoleic acid fractionation assay. After overnight growth, V. cholerae O395 classical
biotype was subcultured 1:40 in the absence of linoleic acid. After 2 hours, 0.1μCi
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C-
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radiolabeled linoleic acid (58.2mCi/mmol) (Perkin-Elmer) was added to one milliliter of the
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subculture and incubated at room temperature. After one hour, the bacteria were harvested by
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centrifugation and washed three times in PBS. Bacteria were then resuspended in 20mM Tris-
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HCl, pH 8.5 and 500 mM NaCl) and freeze-thawed in an ethanol and dry ice bath, then thawed
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at 37°C. This process was repeated for a total of three freeze-thaw cycles. The bacteria were
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then fractionated by centrifugation for 10 min at 15,000 x g to separate the membrane and
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cytoplasm. The cytoplasm was taken as the supernatant and the pellet was resuspended in
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PBS. Each fraction was then measured for cpm of each fraction using a LS6000IC liquid
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scintillation counting system (Beckman).
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Western blot analysis. Immunodetection of ToxT was performed using the same
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procedure as previously described (26). Briefly, bacteria were harvested after inducing
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conditions grown with DMSO only, 160µM linoleic acid, or 100µM virstatin.
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normalized at an optical density of 600nM and resuspended in 2x protein buffer. Samples were
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then boiled, separated by 14% SDS-PAGE, blotted, and blocked for 30 minutes in TBS-Tween
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buffer. After blocking, the blots were incubated overnight in a 1:2,500 dilution of rabbit
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polyclonal anti-ToxT serum, washed three times in TBS buffer, and secondary goat anti-rabbit
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IgG conjugated to alkaline phosphatase (IgG-AP) was used at a dilution of 1:5000 (Southern
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Biotech). Blots were washed again and then developed using immune-BCIP (MP Biomedicals).
Cells were
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To ensure complete fractionation, immunodetection of cytoplasmic ToxT and the outer
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membrane protein OmpU was performed. After bacteria was separated into cytoplasmic and
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membrane fractions, with whole cell lysate used as a control, a 14% SDS-PAGE gel was run,
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blotted, and blocked as described above. The membrane was then incubated overnight in anti7
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ToxT serum, to detect the cytoplasmic fraction, and 1:500 dilution of goat polyclonal anti-
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OmpU serum to detect the membrane fraction. Blots were washed three times in TBS-Tween
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buffer, and secondary rabbit anti-goat IgG-AP (to detect OmpU) followed by secondary goat
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anti-rabbit IgG-AP (to detect ToxT) were used. Blots were washed again and then developed
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using immune-BCIP.
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β-galactosidase assays. Strains to be analyzed were grown overnight in LB at 37°C and
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then subsequently subcultured 1:40 into LB pH 6.5 in the presence or absence of 160µM
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linoleic acid dissolved in DMSO. Cultures were grown under virulence inducing conditions
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(aeration at 30°C) for 3 hours and then analyzed.
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performed following the same established protocol (33).
The β-galactosidase assay was then
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qPCR. As in previous assays, after overnight growth, V. cholerae classical biotype O395
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was subcultured 1:40, either with or without 160µM linoleic acid. RNA from three biological
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samples in each condition was extracted using the RNeasy Bacteria Protect Mini Kit (Qiagen)
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and manufacturer’s protocols were followed. DNA contamination was removed using an on-
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column RNase-free DNase kit (Qiagen) and confirmed free of DNA by the absence of bands
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using logarithmic PCR. To measure the relative mRNA levels of tcpA and aldA the following
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primers were used: forward tcpA (5’-ACGCAAATGCTGCTACACAG-3’) reverse tcpA (5’-
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CCCCTACGCTTGTAACCAAA-3’), and forward aldA (5’-TTGGTGGGCATCCTAACAAT-3’) reverse
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aldA (5’-ACACCGGCACCTAAACCATA-3’). qRT-PCR was performed using one-step SYBR green
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Mastermix (Invitrogen) and the following program: cDNA synthesis for 10min. at 55° C,
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denaturing step for 5min. at 95° C, followed by 35 cycles of 95° C for 10 s, then 55° C for 30 s.
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The level of each mRNA was normalized to the level of rpoB using primers forward rpoB (5’8
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ACCTGAAGGTCCAAACATCG-3’) and reverse rpoB (5’-CAAAACCGCCTTCTTCTGTC-3’). Relative
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levels of transcript with the addition of linoleic were calculated using 2-ΔΔCT and analyzed
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comparing the ΔΔCT values as previously described (34).
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Protein Purification. Maltose binding protein-ToxT fusion (MBP-ToxT) and MBP-CRP
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purification was performed as previously described using E. coli strain BL21(DE3) with the
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plasmid pMAL-c2E carrying either MBP-ToxT or MBP-CRP (32, 35). Briefly, after MBP-ToxT or
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MBP-CRP induction by IPTG, cells were lysed by French press, run over an amylose column, and
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fractions containing MBP-ToxT or MBP-CRP were saved and dialyzed into 50mM Na2HPO4 (pH
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8.0), and 100mM NaCl and then dialyzed into the same solution containing 20% glycerol to save
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as freezer stock at -80° C.
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Electrophoretic mobility shift assays (EMSA). EMSAs were performed as previously
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described (35). Purified MBP-ToxT, or MBP-CRP with cAMP (New England Biolabs), was
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incubated with DNA probes made by PCR from the promoter sequence of interest using one
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primer radiolabeled with γ-32P (Perkin-Elmer) by T4 polynucleotide kinase (New England
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Biolabs). Binding reactions contained various amounts of MBP-ToxT with constant 10 µg/mL
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salmon sperm DNA as nonspecific competitor, 10mM Tris-acetate (pH 7.4), 1mM Potassium
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EDTA (pH 7.0), 100mM KCl, 1mM dithiothreitol (DTT), 0.3mg/mL bovine serum albumin (BSA)
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and 10% glycerol in a volume of 30µL. To each reaction, a constant concentration of the labeled
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DNA probe was added. In reactions containing linoleic acid, the final concentration was 32µM
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for each reaction, and for reactions containing virstatin, the final concentration was 50µM,
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except at the aldA promoter where a higher concentration of 100µM was used. All other
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reactions contained 3.33% (1μL in 30μL) DMSO as a solvent control. Binding reactions were 9
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incubated for 30 minutes at 37°C and then loaded into a 6% polyacrylamide gel at 4°C. Gels
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were dried for 1 hour and then analyzed by autoradiography.
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Binding curve analysis. Autoradiographs were analyzed using ImageJ software (NIH) as
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previously described with nonspecific binding omitted from further analysis (32). Briefly, to
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determine the Kd for samples containing either linoleic acid or virstatin and compared to
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protein bound to DNA without inhibitor, the percent of protein bound with labeled DNA was
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determined
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%Bound=Bmax*[Protein]h/(Kdh + [Protein]h) with Bmax constraint set to 100 using Graphpad Prism
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5 software. The Kd values for each condition were compared to each other using the extra sum
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of squares F test to determine if the two values were statistically different.
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for
each
lane.
This
was
then
fit
to
the
equation
RESULTS
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ToxT protein is produced in the presence of linoleic acid. Previous work has
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demonstrated that UFAs inhibit virulence gene expression by acting through ToxT (25). Oleic
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acid has been previously shown to negatively regulate TCP and CT expression levels (17). In
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preliminary experiments, we determined that linoleic acid had the strongest negative effect on
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ToxT activity of several different UFAs tested (data not shown), and previous studies have
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shown that linoleic acid makes up the largest proportion of unsaturated fatty acid content in
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human bile (36). To date, direct interaction of linoleic acid and ToxT has not been observed,
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although a palmitoleic acid molecule was visualized within the ToxT NTD in the solved crystal
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structure (17). To begin our study, we investigated whether ToxT protein production was
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affected by the addition of linoleic acid in the media. It had been previously determined that
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there are comparable levels of toxT expression in cultures grown with and without linoleic acid 10
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(25). To confirm this, we analyzed the β-galactosidase production from a toxT::lacZ reporter
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plasmid in V. cholerae grown in the presence or absence of linoleic acid (Figure 1A). When V.
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cholerae was grown under virulence-inducing conditions (LB pH=6.5, aeration) in the presence
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of linoleic acid, the amount of β-galactosidase activity indicated that there was no statistically
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significant difference (P=.799) in toxT expression whether the culture had linoleic acid or lacked
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it. This is consistent with previous data confirming that linoleic acid does not
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affect toxT transcription.
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To further examine the effect of linoleic acid on ToxT, and confirm that there were no
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effects of linoleic acid on translation, we carried out Western blot analysis to assess the ToxT
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protein levels (Figure 1B). V. cholerae was grown under virulence-inducing conditions in the
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presence or absence of linoleic acid and cell extracts were harvested. ToxT-specific polyclonal
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antibodies were used to detect protein levels. No differences in ToxT levels were observed
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regardless of the presence or absence of linoleic acid, as a ToxT-specific band was visible in
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Western blots at the same intensity under both conditions (Figure 1B, lanes 4 and 5). This band
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was not detected in the ΔtoxT control strain (Figure 1B, lane 3). Thus, ToxT was stably
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produced regardless of the presence of linoleic acid while bacteria were grown under virulence-
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inducing conditions.
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Linoleic acid enters the V. cholerae cytoplasm. As ToxT protein levels were roughly
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equivalent with or without linoleic acid, we next assessed whether a direct interaction between
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linoleic acid and ToxT would be possible. Such an interaction would require that linoleic acid be
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imported into the bacterial cytosol. To determine whether this occurs, we performed an uptake
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assay using radiolabeled linoleic acid (Fig. 2). V. cholerae was subcultured under virulence11
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inducing conditions for two hours and then 14C-labeled linoleic acid was added to the culture.
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Aliquots of the culture were taken at times 0, 5, 15, and 30 minutes after addition of
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radiolabeled linoleic acid and radioactivity was quantified by a scintillation counter. The amount
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of radioactivity found in the cell pellet was compared to the supernatant. Results from this
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assay show that linoleic acid enters the bacteria with a best fit equation for one-phase
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association of y = 24622 + 156786*(1 - e-.036x) and an R2-value of .989.
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These data, however, do not indicate whether linoleic acid is present inside the
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bacterium or simply associated with the cell surface or outer membrane. To determine whether
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linoleic acid enters the cytosol,
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grown under virulence-inducing conditions and then the bacteria were fractionated into
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membrane and cytoplasmic portions. This allowed us to see where in the bacterium linoleic
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acid was localized: the membrane, the cytoplasm, or both. Cultures were incubated for one
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hour with the radiolabeled linoleic acid. These cultures were then pelleted and washed and the
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bacteria were separated into a cytoplasm and envelope fraction. The 14C-linoleic acid in each
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fraction was then quantified using a scintillation counter. Results of this experiment show that
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most of the linoleic acid is able to enter the cell (Figure 3A). A control Western blot was
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performed to ensure complete fractionation of the bacteria and showed no ToxT in the
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membrane fraction and no OmpU in the cytoplasmic fraction (Figure 3B). Directly comparing
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the amounts of linoleic acid in the two fractions showed that about 65% of the total 14C-linoleic
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acid was present in the cytoplasmic fraction, while 35% was present in the membrane fraction.
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These data suggest that a significant fraction of extracellular linoleic acid can enter the cytosol,
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where it could then interact with ToxT.
14
C-labeled linoleic acid was added to V. cholerae cultures
12
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Linoleic acid decreases ToxT activation of virulence gene transcription. tcpA and ctxAB
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expression have been previously shown to be inhibited by the addition of oleic acid to the
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media, but the effects of linoleic acid were not assessed in those studies (17). We assessed
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whether the inhibitory effect of linoleic acid that we observed extended to a variety of ToxT-
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activated promoters with various ToxT binding site (toxbox) configurations, including tcpA,
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ctxAB, acfA, aldA, acfD, tagA, and tcpI. All of these promoters contain two toxboxes in either
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direct (tcpA,ctxAB) (13) or inverted repeat configurations (acfA, acfD, tagA, tcpI) (12-14) except
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for aldA, which contains only one toxbox (16). Plasmid-borne promoter-lacZ fusions were
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added to the wild-type V. cholerae classical strain as well as an otherwise isogenic ΔtoxT strain
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to measure transcriptional activity at these promoters with and without 160µM linoleic acid
268
(Figure 4). At each of these promoters, linoleic acid had a statistically significant negative effect
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that prevented ToxT from fully activating transcription at the promoters in wild-type O395. No
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effect of linoleic was observed in the ΔtoxT strain, confirming that linoleic acid acts through
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ToxT (Figure 4).
272
As the aldA promoter activity even in the absence of linoleic acid was much lower than
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at the other promoters, we performed quantitative real-time PCR (qPCR) to compare
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expression levels of this promoter to expression levels of the tcpA promoter upon the addition
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of linoleic acid. Bacteria were cultured using the same methods as the β-galactosidase assay
276
and then the RNA was extracted. qPCR was then performed using primers for tcpA, and aldA,
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while using rpoB as the housekeeping gene control. The qPCR results showed that both tcpA
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and aldA were downregulated upon the addition of linoleic acid to the media (Figure 5).
13
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Linoleic acid decreases ToxT-DNA binding affinity. EMSA was used to determine if
280
linoleic acid is able to directly affect ToxT binding to DNA at the ToxT-activated promoters. This
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method allows the binding pattern of ToxT to be characterized and gives a rough estimation of
282
the equilibrium dissociation constant, Kd, which represents the concentration of ToxT required
283
for binding 50% of the DNA at equilibrium. As the concentration of active ToxT varies from
284
preparation to preparation, the Kd values can only be compared per experimental condition,
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and are only an estimate. The Kd values with and without linoleic acid give a direct comparison
286
of the effect of the fatty acid on the given promoter, as a higher Kd value indicates a lower
287
binding affinity of ToxT for the DNA and a lower Kd value indicates higher binding affinity.
288
To begin, we used the primary promoter in the tcp operon, PtcpA, as our 32P-labeled DNA
289
probe. Using purified MBP-ToxT, we added increasing amounts of protein to binding reactions
290
containing a constant concentration of labeled DNA probe. Additionally, either DMSO alone
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(Figure 6A, lanes 1-7) or 32 μM linoleic acid dissolved in DMSO were added to the reactions. A
292
representative EMSA is shown in Fig. 6A. Densitometry analysis was performed to calculate the
293
percentage of bound DNA at each concentration of MBP-ToxT and a binding curve was drawn
294
for each set of reactions to determine the percentage of bound DNA at each concentration of
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MBP-ToxT (Figure 6B). In the case of MBP-ToxT binding to PtcpA DNA, the Kd without linoleic acid
296
was 9.99 nM and the Kd with linoleic acid was 33.11 nM. These values are significantly different,
297
and indicate a lower ToxT binding affinity for PtcpA in the presence of linoleic acid. These data
298
strongly suggest there is likely a direct interaction between linoleic acid and ToxT.
299
To confirm that linoleic acid does not generally inhibit protein-DNA interactions, we
300
used cyclic AMP receptor protein (CRP) as a control, together with the region upstream of tcpP 14
301
to which CRP binds, as described above. We performed an EMSA comparing CRP binding with
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and without the addition of linoleic acid (Figure 6C) and performed densitometric analysis to
303
determine the binding curves and Kd values. The binding curve and Kd values were statistically
304
the same for CRP binding to PtcpP with or without linoleic acid (Figure 6D).
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We next determined the binding curves and Kd values at other ToxT-activated
306
promoters to determine whether different toxbox configurations may impact the effect of
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linoleic acid on ToxT binding to DNA (Figure 7). Equilibrium binding experiments on PtcpA, which
308
has two direct repeat toxboxes, showed a decrease in DNA binding upon addition of linoleic
309
acid. Other promoters have direct and inverted repeat toxbox configurations, and PaldA has only
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one toxbox (Figure 7.) If linoleic acid could decrease the binding affinity of ToxT for PaldA, it
311
could indicate the mechanism for ToxT inhibition did not involve effects on dimerization, which
312
had been previously proposed as the mechanism for decreasing virulence gene expression (18).
313
PctxAB contains two directly repeated toxboxes like PtcpA, albeit in the opposite orientation and
314
having different spacing (35). The rest of the ToxT-activated promoters previously mentioned,
315
PacfA, PacfD, PtagA, and PtcpI each contain toxboxes oriented in inverted repeat configuration as
316
well as variable spacing between the toxboxes (12-14).
317
As linoleic acid was shown above to decrease promoter activity for all of these ToxT-
318
activated promoters, we determined whether linoleic acid also affected the DNA binding
319
affinity using EMSA with each promoter region. For PctxAB, containing direct repeats, the Kd
320
without linoleic acid is 4.436 nM, and with linoleic acid is 10.05 nM, indicating decreased
321
binding affinity of ToxT (Figure 7A and 7B). As a representative of the inverted repeat toxbox
322
promoters, we chose to look at PtagA. Here, densitometry and binding curve analysis again 15
323
revealed decreased binding affinity of ToxT for the promoter upon the addition of linoleic acid,
324
with a Kd of 4.078 nM without linoleic acid, and 27.78 nM with linoleic acid (Figure 7C and 7D).
325
Lastly, we examined ToxT binding to the single toxbox promoter, PaldA. Figure 7E shows the
326
autoradiograph of ToxT binding to PaldA with and without linoleic acid. The Kd once again was
327
increased, going from 11.25 nM without linoleic acid to 48.67 nM with linoleic acid (Figure 7F).
328
These data, along with the previously described decrease in promoter activity with the addition
329
of linoleic acid as indicated by the β-galactosidase assays, suggest that linoleic acid decreases
330
binding at each promoter regardless of toxbox configuration and spacing.
331
Another negative ToxT effector, virstatin, acts differently than linoleic acid. The results
332
above suggest a direct negative effect of linoleic acid on ToxT binding to DNA. We next
333
examined whether this was also true for another negative ToxT regulator, virstatin. Previous
334
studies suggested that virstatin negatively affects the ability of ToxT to dimerize (21). To begin,
335
we looked at ToxT levels using Western blot analysis to compare V. cholerae grown with or
336
without virstatin (Figure 1B, lanes 6 and 7). The ToxT levels were unchanged, confirming that
337
ToxT production is not affected by virstatin. We then performed β-galactosidase assays on the
338
previously described ToxT-regulated genes to assess the effects of virstatin (Figure 8). These
339
data show decreased ToxT activity at all promoters except for aldA, where virstatin had no
340
significant effect. A previous study suggested that the aldA promoter was affected by virstatin
341
(21). We then performed EMSAs to determine whether virstatin affected the DNA binding
342
ability of ToxT at tcpA, ctxAB, and aldA, as representatives of the different toxbox
343
configurations (Figure 9). Virstatin decreased binding at tcpA and ctxAB promoters, as indicated
344
by analysis of the binding curve and Kd, (Figure 9A-D) but did not decrease ToxT binding at the 16
345
aldA promoter even at a higher concentration of virstatin (Figure 9E). In fact, the Kd significantly
346
decreased from 21.83nM to 11.79nM with the addition of virstatin, indicating a higher binding
347
affinity of ToxT for the aldA promoter when virstatin is present (Figure 9F).
348
349
350
DISCUSSION
351
The activation of virulence gene expression relies on the major virulence transcription
352
activator, ToxT. Previous studies have looked at the involvement of bile, UFAs, and virstatin on
353
V. cholerae virulence gene expression and ToxT dimerization (17, 21, 22, 24, 25, 27). Most of
354
this work with negative effectors has been done only on tcpA and ctxAB; here, in addition to
355
these genes, we also examined the effects of negative effectors on ToxT activity at aldA, acfA,
356
acfD, tagA, and tcpI. All ToxT-controlled promoters showed a decrease in expression in the
357
presence of linoleic acid. Prior to entry into the mucus layer of epithelial cells and colonization,
358
V. cholerae must pass through the lumen of the small intestine where high concentrations of
359
bile and UFAs are present. Linoleic acid needs to be taken up by V. cholerae and enter the
360
cytoplasm in order to directly interact with ToxT. Here, we show that linoleic acid is able to
361
enter the cell and that the majority of it enters the cytoplasm. One caveat is that there may be
362
periplasmic components present in the cytoplasmic portion in our fractionation studies.
363
However, the observation that a majority of linoleic acid was in this fraction, combined with our
364
other observations that linoleic acid directly impacts DNA binding by ToxT in vitro and ToxT
17
365
activity in vivo, strongly suggest that at least some linoleic acid is present in the bacterial
366
cytosol where it directly interacts with ToxT.
367
Previous work has described the effect of negative ToxT effectors on virulence gene
368
expression, but their direct effect on ToxT DNA binding affinity had not been previously
369
assessed. It has been proposed that the mechanism of action for virstatin and UFAs is inhibition
370
of ToxT dimerization (18, 21). The binding sites for ToxT, toxboxes, and their configurations
371
have been extensively studied (12,13,16,35), but whether toxbox configuration factors into the
372
inhibition of ToxT activity by linoleic acid had not been examined. Here we show that the
373
decrease in binding affinity in response to linoleic acid can be seen at each ToxT-activated
374
promoter that we examined, regardless of toxbox configuration. This suggests that linoleic acid
375
has a general effect on DNA binding by ToxT monomers. Previous work strongly suggested that
376
ToxT binds to DNA as a monomer (12, 13, 16) and that ToxT does not behave as a typical
377
obligatorily dimeric activator, such as AraC.
378
We also looked at virstatin and its method of action on ToxT. A previous study examined
379
virulence gene promoter activity in the presence of virstatin; however, unlike this earlier study,
380
the effect of virstatin in our hands was not as pronounced. We did observe that virstatin
381
significantly decreased promoter activity, but to a lesser extent than previously described
382
(Figure 8 (21)). While Shakhnovich et al. saw, at greatest, 20% promoter activity in the presence
383
of virstatin at the aldA, acfA, and tcpI promoters, we observed a decrease, but not to that
384
degree. Interestingly, we did not see any effect of virstatin on ToxT activation at the single
385
toxbox, aldA promoter with the β-galactosidase assay (Figure 8). In the same vein, virstatin also
18
386
did not affect the binding affinity of ToxT for the aldA promoter (Figure 9E,F). In contrast, we
387
did see a negative effect of linoleic acid on both ToxT-dependent virulence gene promoter
388
activity (Figure 4) and ToxT binding affinity for PaldA, where the Kd increased from 11.25 nM to
389
48.67 nM (Figure 7E,F).
390
These data suggest that virstatin and linoleic acid, although both negative effectors of
391
ToxT activity, do not have the same mechanism for downregulating gene expression. Another
392
study performed bacterial two-hybrid assays to look at the effect of various UFAs on
393
dimerization and the data suggest another UFA, oleic acid, does affect ToxT dimerization (25).
394
However, linoleic acid was not tested and this study was done using only the NTD of ToxT
395
instead of intact, full-length ToxT, whose dimerization has never been experimentally observed,
396
at least in published reports. More testing using full-length ToxT in V. cholerae with this two-
397
hybrid system in the presence of linoleic acid would confirm whether or not linoleic acid is
398
involved in dimerization. As there was reduced binding of ToxT at the aldA promoter, it is likely
399
that linoleic acid changes the structural conformation of ToxT, leading to inhibited DNA binding.
400
Understanding the effect of host signals on bacterial pathogenesis is useful for complete
401
understanding of the V. cholerae virulence cascade, as many host signals are used by the
402
bacteria to sense the appropriate location in which virulence gene transcription should be
403
initiated. When passing through the small intestine, V. cholerae encounters many different
404
chemical signals, such as bile and bicarbonate, which either inhibit or activate ToxT-dependent
405
gene transcription. Bile and its UFA components are at high concentrations in the duodenal
406
lumen, in which ToxT is inactive (20, 25, 27, 37). Once the mucus layer is penetrated, UFA
19
407
concentration decreases, while the bicarbonate concentration increases. At this point, along
408
the epithelial surface, ToxT becomes active, virulence genes are expressed, and V. cholerae can
409
colonize. UFAs such as linoleic acid likely keep ToxT in a form unable to bind tightly to DNA in
410
the lumen, because production of TCP would lead to bacterial aggregation prior to penetration
411
of the mucus layer and colonization of the epithelial surface would become impossible.
412
According to this model, V. cholerae is able to use both negative and positive effectors of
413
virulence in order to identify and colonize its ideal niche. Our findings here show a direct effect
414
of linoleic acid on ToxT, giving useful insight into the mechanisms of ToxT gene regulation.
415
416 417
ACKNOWLEDGEMENTS The authors thank the members of the Withey laboratory for helpful discussions. This work was
418
supported by P.H.S. grants K22AI071011 and R56AI093622 (to J.H.W.), Bill and Melinda Gates
419
Foundation grant OPP1068124 and by Wayne State University funds.
420
20
421
422
423
a)
427 428 429 430 431 432
15000 10000 5000 0
WT 160μM LA
426
n.s.
WT
425
PtoxT::lacZ Expression (Miller Units)
424
b)
20000
433 434 435 436 437 438 439 440 441
FIGURE 1. Effect of linoleic acid on PtoxT::lacZ and ToxT expression. (A). Light gray bars, wildtype V. cholerae grown without linoleic acid; dark gray bars, wild-type V. cholerae grown with 64 µM linoleic acid. Statistical significance was determined by Student's t test. (B). Effect of linoleic acid and virstatin on ToxT protein levels. V. cholerae was grown under virulence inducing conditions (LB pH 6.5, 30°C, aeration) with DMSO, linoleic acid or virstatin (lanes 4, 5, 6, 7). Purified full length ToxT-6His migrates to ~32 kDa (lane 2) and a ΔtoxT strain shows no band (lane 3) were used as controls. Samples were normalized by OD600. The Western blot was probed with anti-ToxT antibody. This is representative of three separate experiments.
442
21
443 444
14
C CPM/OD600
150000
100000
50000
0
0
10
20
30
Time (minutes) 445 446 447 448 449 450
FIGURE 2. C14-radiolabeled linoleic acid is taken up by V. cholerae. Classical strain O395 cells were grown under virulence-inducing conditions for two hours and then the radiolabeled linoleic acid was added at time 0 and analyzed at times 5, 15, and 30 minutes post-addition, measured as CPM per optical density unit at 600nm. The equation of best fit is y = 24622 + 156786*(1 - e-.036x) and an R2-value of .989.
451
22
b) 80
456
60
457 458 459 460 461 462
Percent
455
*
Membrane
a)
Cytoplasm
454
Ladder
453
Whole cell lysate
452
40 20 0
OmpU ToxT
25kDa – % Envelope
% Cytoplasm
1
2
3
4
463 464 465 466 467 468 469 470 471 472 473
FIGURE 3. C14-radiolabeled linoleic acid is able to enter the cell and enter the cytoplasm. (A). After a 2-hour subculture in virulence-inducing conditions, linoleic acid was added to the culture and allowed to sit for one hour. After one hour, V. cholerae was fractionated into an envelope and cytoplasmic portion by multiple freeze-thaw cycles. There is significantly more linoleic acid in the cytoplasmic portion as compared to the envelope portion. Error bars represent +/- standard error mean and statistical significance was determined by Student's t test, *, p
1
2
The mechanism for inhibition of Vibrio cholerae ToxT activity by the unsaturated fatty acid
3
components of bile
4
5
Sarah C. Plecha and Jeffrey H. Withey#
6
Department of Immunology and Microbiology
7
Wayne State University School of Medicine, Detroit, MI 48201
8
9
10
# To whom correspondence should be addressed: Jeffrey H. Withey, Department of
11
Immunology and Microbiology, Wayne State University, 540 E. Canfield, Detroit, MI 48201,
12
USA, Tel.: (313) 577-1316; Fax: (313) 577-1155; E-mail: [email protected]
13
14
15
16
Running Title: Mechanism for ToxT inhibition by bile
17
1
18
ABSTRACT
19
The gram-negative curved bacillus Vibrio cholerae causes the severe diarrheal illness
20
cholera. During host infection, a complex regulatory cascade results in production of ToxT, a
21
DNA-binding protein that activates the transcription of major virulence genes that encode
22
cholera toxin (CT) and toxin-coregulated pilus (TCP). Previous studies have shown that bile and
23
its unsaturated fatty acid (UFA) components reduce virulence gene expression and therefore
24
are likely important signals upon entering the host. However, the mechanism for the bile-
25
mediated reduction of TCP and CT expression has not been clearly defined. There are two likely
26
hypotheses to explain this reduction: 1) UFAs decrease DNA binding by ToxT or 2) UFAs
27
decrease dimerization of ToxT. The work presented here elucidates that bile/UFAs directly
28
affect DNA binding by ToxT. UFAs, specifically linoleic acid, can enter V. cholerae when added
29
exogenously and are present in the cytoplasm where they can then interact with ToxT.
30
Electrophoretic mobility shift assays (EMSAs) with ToxT and various virulence promoters in the
31
presence or absence of UFAs showed a direct reduction in ToxT binding to DNA, even in
32
promoters with only one ToxT binding site. Virstatin, a synthetic ToxT inhibitor, was previously
33
shown to reduce ToxT dimerization. Here we show that virstatin only affects DNA binding at
34
ToxT promoters with two binding sites, unlike linoleic acid which affects ToxT binding
35
promoters having either one or two ToxT binding site. This suggests a mechanism in which
36
UFAs, unlike virstatin, do not affect dimerization but affect monomeric ToxT binding to DNA.
37
38
IMPORTANCE Vibrio cholerae must produce the major virulence factors cholera toxin (CT) and toxin2
39
coregulated pilus (TCP) to cause cholera. CT and TCP production depends on ToxT, the major
40
virulence transcription activator. ToxT activity is negatively regulated by unsaturated fatty acids
41
(UFA) present in the lumen of the upper small intestine. This study investigates the mechanism
42
for inhibition of ToxT activity by UFAs and finds that UFAs directly reduce specific ToxT binding to
43
DNA at virulence promoters and subsequently reduce virulence gene expression. UFAs inhibit
44
ToxT monomers from binding DNA. This differs from the inhibitory mechanism of a synthetic
45
ToxT inhibitor, virstatin, which inhibits ToxT dimerization. Understanding the mechanisms for
46
inhibition of virulence could lead toward better cholera therapeutics. INTRODUCTION
47
48
V. cholerae, a gram-negative curved bacillus possessing a single polar flagellum, is the
49
causative agent of cholera. Cholera is a diarrheal disease contracted by consuming
50
contaminated food or water and is characterized by severe diarrhea that leads to dehydration,
51
and can ultimately cause death if left untreated. Each year, there are an estimated 1.4-4.3
52
million cholera cases and 20,000-142,000 deaths from the disease (1, 2). To cause disease, V.
53
cholerae must colonize the upper small intestine, where it expresses virulence genes including
54
those that encode the two most important virulence factors: toxin co-regulated pilus (TCP) and
55
cholerae toxin (CT). TCP is required for intestinal colonization, while CT is responsible for the
56
massive secretion of electrolytes and water into the lumen, causing diarrhea. Transcription of
57
these and other virulence genes are regulated by the major virulence transcription activator
58
ToxT.
59
Virulence gene expression is regulated by what is historically known as the ToxR regulon
3
60
(3, 4). toxT transcription is activated by two pairs of inner membrane proteins, ToxR/ToxS and
61
TcpP/TcpH, that bind upstream of toxT, as well as by a ToxT positive feedback loop (5-9). ToxT is
62
a 32-kDA member of the AraC/XylS transcriptional regulator family, having a conserved 100-
63
amino-acid DNA binding domain, consisting of two helix-turn-helix motifs, in its C-terminal
64
domain (CTD).
65
sequences called toxboxes that vary in configuration at individual genes (10-14). ToxT directly
66
controls transcription of genes encoding not only TCP and CT, but also other accessory virulence
67
genes including acfA, acfD, aldA, tagA, tarA, tarB, and tcpI (11-13, 15, 16). Except for the aldA
68
promoter, in which there is a single toxbox, there are two toxboxes present at ToxT-activated
69
promoters (11-14, 16). ToxT can bind to single toxboxes as a monomer but it is thought that full
70
activation only occurs upon ToxT dimerization on the DNA, at least at some genes (12, 13). The
71
ToxT N-terminal domain (NTD), does not share significant sequence homology with other
72
proteins, but has some structural similarity to the AraC NTD, which is the domain necessary for
73
AraC dimerization and arabinose binding (17). NTDs of ToxT have been shown to interact when
74
separated from the CTD, although the true role of dimerization in ToxT function is not fully
75
understood (16-22).
Located upstream of all ToxT-activated genes are 13-bp degenerate DNA
76
There are a number of host and environmental factors that affect ToxT activity, including
77
temperature, pH, bile and bicarbonate (23-27). Bile is produced by the liver and then
78
subsequently stored in the gallbladder. Upon eating, bile is released into the duodenum where
79
it acts to solubilize lipids. Bile itself is a complex, heterogeneous mixture that includes bile salts,
80
cholesterol, bilirubin, and saturated and unsaturated fatty acids (UFAs). V. cholerae encounters
81
bile early during infection and it is proposed to be a natural effector of ToxT as it is found in the 4
82
same places that V. cholerae colonizes (24, 25, 27). V. cholerae has reduced virulence gene
83
expression in the presence of bile and/or UFAs, with increased motility gene expression, biofilm
84
formation, the induction of efflux pumps, and increased amounts of outer membrane proteins
85
OmpU, OmpT and TolC (24, 25, 28-31). Bile and its UFA components have been previously
86
shown to decrease CT and TCP production, but the mechanism of this effect on virulence gene
87
expression levels is not fully understood (24, 25). Bile also has another role in that it is able to
88
act as a bactericide for the benefit of the host, but enteric bacteria, such as V. cholerae, are not
89
only adapted to live in the presence of bile, but potentially can recognize bile as a signal to
90
ensure survival in the host (24, 27). Another ToxT inhibitor is a synthetic molecule called
91
virstatin (21, 22). The mechanism of action for virstatin has been proposed to be through
92
inhibition of ToxT dimerization (21). Whether UFAs and virstatin inhibit ToxT activity via the
93
same mechanism is unknown.
94
In this study we show that UFAs, specifically the unsaturated fatty acid linoleic acid, are
95
able to enter the bacterial cytoplasm where ToxT is also present. We report that linoleic acid
96
causes decreased transcription of ToxT-controlled virulence genes assessed. The mechanism by
97
which this occurs is decreased binding affinity at ToxT-activated promoters in the presence of
98
linoleic acid, regardless of the number or configuration of the toxboxes. Our results indicating
99
that UFAs decrease ToxT binding to DNA even at promoters having one ToxT binding site
100
suggest a mechanism in which UFAs affect the ability of monomeric ToxT to bind to DNA, in
101
contrast to the results we observed using virstatin. Thus these two ToxT inhibitors exhibit
102
somewhat different modes of action. These results give us a clearer understanding of the
103
regulatory networks controlling virulence gene expression during human infection. 5
MATERIALS AND METHODS
104 105
V. cholerae strains and growth conditions. All V. cholerae strains used in the study are
106
derived from classical biotype O395. Strains were maintained in Luria Broth (LB) containing
107
20% glycerol and stored at -70°C. All promoter::lacZ fusions used for β-galactosidase assays
108
were made in plasmid pTL61T as described in previous studies (26, 32). Overnight cultures were
109
grown for ~16 hours at 37°C in LB and then diluted 1:40 into LB pH 6.5 at 30°C for virulence
110
inducing conditions in the presence or absence of freshly prepared .05% bile (sodium choleate),
111
160µM linoleic acid, or 100µM virstatin. V. cholerae strains were grown with antibiotic
112
concentration of 100µg/mL streptomycin and strains with the plasmid pTL61T were grown with
113
100µg/mL ampicillin.
114
14
C linoleic acid uptake. V. cholerae classical biotype strain O395 was grown overnight in
115
LB at 37°C, subcultured 1:40 in LB pH 6.5, and grown for two hours in the absence of linoleic
116
acid. At two hours, 0.1μCi
117
added for each milliliter of the subculture. Upon addition of the radiolabeled linoleic acid, 1 mL
118
of the culture was immediately centrifuged, and the supernatant saved to compare to cell
119
pellet cpms. The cell pellet was washed 3 times with 1 mL of PBS and centrifuged each time.
120
Cell pellets were resuspended in 100μL PBS and added to 5mL Scintillation cocktail (Fisher
121
Scientific). The same procedure was followed for other aliquots of the subculture at times 5, 15
122
and 30 minutes. After uptake, cpm was measured for each time point using an LS6000IC liquid
123
scintillation counting system (Beckman).
124 125
14
C-radiolabeled linoleic acid (58.2mCi/mmol) (Perkin-Elmer) was
14
C linoleic acid fractionation assay. After overnight growth, V. cholerae O395 classical
biotype was subcultured 1:40 in the absence of linoleic acid. After 2 hours, 0.1μCi
14
C-
6
126
radiolabeled linoleic acid (58.2mCi/mmol) (Perkin-Elmer) was added to one milliliter of the
127
subculture and incubated at room temperature. After one hour, the bacteria were harvested by
128
centrifugation and washed three times in PBS. Bacteria were then resuspended in 20mM Tris-
129
HCl, pH 8.5 and 500 mM NaCl) and freeze-thawed in an ethanol and dry ice bath, then thawed
130
at 37°C. This process was repeated for a total of three freeze-thaw cycles. The bacteria were
131
then fractionated by centrifugation for 10 min at 15,000 x g to separate the membrane and
132
cytoplasm. The cytoplasm was taken as the supernatant and the pellet was resuspended in
133
PBS. Each fraction was then measured for cpm of each fraction using a LS6000IC liquid
134
scintillation counting system (Beckman).
135
Western blot analysis. Immunodetection of ToxT was performed using the same
136
procedure as previously described (26). Briefly, bacteria were harvested after inducing
137
conditions grown with DMSO only, 160µM linoleic acid, or 100µM virstatin.
138
normalized at an optical density of 600nM and resuspended in 2x protein buffer. Samples were
139
then boiled, separated by 14% SDS-PAGE, blotted, and blocked for 30 minutes in TBS-Tween
140
buffer. After blocking, the blots were incubated overnight in a 1:2,500 dilution of rabbit
141
polyclonal anti-ToxT serum, washed three times in TBS buffer, and secondary goat anti-rabbit
142
IgG conjugated to alkaline phosphatase (IgG-AP) was used at a dilution of 1:5000 (Southern
143
Biotech). Blots were washed again and then developed using immune-BCIP (MP Biomedicals).
Cells were
144
To ensure complete fractionation, immunodetection of cytoplasmic ToxT and the outer
145
membrane protein OmpU was performed. After bacteria was separated into cytoplasmic and
146
membrane fractions, with whole cell lysate used as a control, a 14% SDS-PAGE gel was run,
147
blotted, and blocked as described above. The membrane was then incubated overnight in anti7
148
ToxT serum, to detect the cytoplasmic fraction, and 1:500 dilution of goat polyclonal anti-
149
OmpU serum to detect the membrane fraction. Blots were washed three times in TBS-Tween
150
buffer, and secondary rabbit anti-goat IgG-AP (to detect OmpU) followed by secondary goat
151
anti-rabbit IgG-AP (to detect ToxT) were used. Blots were washed again and then developed
152
using immune-BCIP.
153
β-galactosidase assays. Strains to be analyzed were grown overnight in LB at 37°C and
154
then subsequently subcultured 1:40 into LB pH 6.5 in the presence or absence of 160µM
155
linoleic acid dissolved in DMSO. Cultures were grown under virulence inducing conditions
156
(aeration at 30°C) for 3 hours and then analyzed.
157
performed following the same established protocol (33).
The β-galactosidase assay was then
158
qPCR. As in previous assays, after overnight growth, V. cholerae classical biotype O395
159
was subcultured 1:40, either with or without 160µM linoleic acid. RNA from three biological
160
samples in each condition was extracted using the RNeasy Bacteria Protect Mini Kit (Qiagen)
161
and manufacturer’s protocols were followed. DNA contamination was removed using an on-
162
column RNase-free DNase kit (Qiagen) and confirmed free of DNA by the absence of bands
163
using logarithmic PCR. To measure the relative mRNA levels of tcpA and aldA the following
164
primers were used: forward tcpA (5’-ACGCAAATGCTGCTACACAG-3’) reverse tcpA (5’-
165
CCCCTACGCTTGTAACCAAA-3’), and forward aldA (5’-TTGGTGGGCATCCTAACAAT-3’) reverse
166
aldA (5’-ACACCGGCACCTAAACCATA-3’). qRT-PCR was performed using one-step SYBR green
167
Mastermix (Invitrogen) and the following program: cDNA synthesis for 10min. at 55° C,
168
denaturing step for 5min. at 95° C, followed by 35 cycles of 95° C for 10 s, then 55° C for 30 s.
169
The level of each mRNA was normalized to the level of rpoB using primers forward rpoB (5’8
170
ACCTGAAGGTCCAAACATCG-3’) and reverse rpoB (5’-CAAAACCGCCTTCTTCTGTC-3’). Relative
171
levels of transcript with the addition of linoleic were calculated using 2-ΔΔCT and analyzed
172
comparing the ΔΔCT values as previously described (34).
173
Protein Purification. Maltose binding protein-ToxT fusion (MBP-ToxT) and MBP-CRP
174
purification was performed as previously described using E. coli strain BL21(DE3) with the
175
plasmid pMAL-c2E carrying either MBP-ToxT or MBP-CRP (32, 35). Briefly, after MBP-ToxT or
176
MBP-CRP induction by IPTG, cells were lysed by French press, run over an amylose column, and
177
fractions containing MBP-ToxT or MBP-CRP were saved and dialyzed into 50mM Na2HPO4 (pH
178
8.0), and 100mM NaCl and then dialyzed into the same solution containing 20% glycerol to save
179
as freezer stock at -80° C.
180
Electrophoretic mobility shift assays (EMSA). EMSAs were performed as previously
181
described (35). Purified MBP-ToxT, or MBP-CRP with cAMP (New England Biolabs), was
182
incubated with DNA probes made by PCR from the promoter sequence of interest using one
183
primer radiolabeled with γ-32P (Perkin-Elmer) by T4 polynucleotide kinase (New England
184
Biolabs). Binding reactions contained various amounts of MBP-ToxT with constant 10 µg/mL
185
salmon sperm DNA as nonspecific competitor, 10mM Tris-acetate (pH 7.4), 1mM Potassium
186
EDTA (pH 7.0), 100mM KCl, 1mM dithiothreitol (DTT), 0.3mg/mL bovine serum albumin (BSA)
187
and 10% glycerol in a volume of 30µL. To each reaction, a constant concentration of the labeled
188
DNA probe was added. In reactions containing linoleic acid, the final concentration was 32µM
189
for each reaction, and for reactions containing virstatin, the final concentration was 50µM,
190
except at the aldA promoter where a higher concentration of 100µM was used. All other
191
reactions contained 3.33% (1μL in 30μL) DMSO as a solvent control. Binding reactions were 9
192
incubated for 30 minutes at 37°C and then loaded into a 6% polyacrylamide gel at 4°C. Gels
193
were dried for 1 hour and then analyzed by autoradiography.
194
Binding curve analysis. Autoradiographs were analyzed using ImageJ software (NIH) as
195
previously described with nonspecific binding omitted from further analysis (32). Briefly, to
196
determine the Kd for samples containing either linoleic acid or virstatin and compared to
197
protein bound to DNA without inhibitor, the percent of protein bound with labeled DNA was
198
determined
199
%Bound=Bmax*[Protein]h/(Kdh + [Protein]h) with Bmax constraint set to 100 using Graphpad Prism
200
5 software. The Kd values for each condition were compared to each other using the extra sum
201
of squares F test to determine if the two values were statistically different.
202
for
each
lane.
This
was
then
fit
to
the
equation
RESULTS
203
ToxT protein is produced in the presence of linoleic acid. Previous work has
204
demonstrated that UFAs inhibit virulence gene expression by acting through ToxT (25). Oleic
205
acid has been previously shown to negatively regulate TCP and CT expression levels (17). In
206
preliminary experiments, we determined that linoleic acid had the strongest negative effect on
207
ToxT activity of several different UFAs tested (data not shown), and previous studies have
208
shown that linoleic acid makes up the largest proportion of unsaturated fatty acid content in
209
human bile (36). To date, direct interaction of linoleic acid and ToxT has not been observed,
210
although a palmitoleic acid molecule was visualized within the ToxT NTD in the solved crystal
211
structure (17). To begin our study, we investigated whether ToxT protein production was
212
affected by the addition of linoleic acid in the media. It had been previously determined that
213
there are comparable levels of toxT expression in cultures grown with and without linoleic acid 10
214
(25). To confirm this, we analyzed the β-galactosidase production from a toxT::lacZ reporter
215
plasmid in V. cholerae grown in the presence or absence of linoleic acid (Figure 1A). When V.
216
cholerae was grown under virulence-inducing conditions (LB pH=6.5, aeration) in the presence
217
of linoleic acid, the amount of β-galactosidase activity indicated that there was no statistically
218
significant difference (P=.799) in toxT expression whether the culture had linoleic acid or lacked
219
it. This is consistent with previous data confirming that linoleic acid does not
220
affect toxT transcription.
221
To further examine the effect of linoleic acid on ToxT, and confirm that there were no
222
effects of linoleic acid on translation, we carried out Western blot analysis to assess the ToxT
223
protein levels (Figure 1B). V. cholerae was grown under virulence-inducing conditions in the
224
presence or absence of linoleic acid and cell extracts were harvested. ToxT-specific polyclonal
225
antibodies were used to detect protein levels. No differences in ToxT levels were observed
226
regardless of the presence or absence of linoleic acid, as a ToxT-specific band was visible in
227
Western blots at the same intensity under both conditions (Figure 1B, lanes 4 and 5). This band
228
was not detected in the ΔtoxT control strain (Figure 1B, lane 3). Thus, ToxT was stably
229
produced regardless of the presence of linoleic acid while bacteria were grown under virulence-
230
inducing conditions.
231
Linoleic acid enters the V. cholerae cytoplasm. As ToxT protein levels were roughly
232
equivalent with or without linoleic acid, we next assessed whether a direct interaction between
233
linoleic acid and ToxT would be possible. Such an interaction would require that linoleic acid be
234
imported into the bacterial cytosol. To determine whether this occurs, we performed an uptake
235
assay using radiolabeled linoleic acid (Fig. 2). V. cholerae was subcultured under virulence11
236
inducing conditions for two hours and then 14C-labeled linoleic acid was added to the culture.
237
Aliquots of the culture were taken at times 0, 5, 15, and 30 minutes after addition of
238
radiolabeled linoleic acid and radioactivity was quantified by a scintillation counter. The amount
239
of radioactivity found in the cell pellet was compared to the supernatant. Results from this
240
assay show that linoleic acid enters the bacteria with a best fit equation for one-phase
241
association of y = 24622 + 156786*(1 - e-.036x) and an R2-value of .989.
242
These data, however, do not indicate whether linoleic acid is present inside the
243
bacterium or simply associated with the cell surface or outer membrane. To determine whether
244
linoleic acid enters the cytosol,
245
grown under virulence-inducing conditions and then the bacteria were fractionated into
246
membrane and cytoplasmic portions. This allowed us to see where in the bacterium linoleic
247
acid was localized: the membrane, the cytoplasm, or both. Cultures were incubated for one
248
hour with the radiolabeled linoleic acid. These cultures were then pelleted and washed and the
249
bacteria were separated into a cytoplasm and envelope fraction. The 14C-linoleic acid in each
250
fraction was then quantified using a scintillation counter. Results of this experiment show that
251
most of the linoleic acid is able to enter the cell (Figure 3A). A control Western blot was
252
performed to ensure complete fractionation of the bacteria and showed no ToxT in the
253
membrane fraction and no OmpU in the cytoplasmic fraction (Figure 3B). Directly comparing
254
the amounts of linoleic acid in the two fractions showed that about 65% of the total 14C-linoleic
255
acid was present in the cytoplasmic fraction, while 35% was present in the membrane fraction.
256
These data suggest that a significant fraction of extracellular linoleic acid can enter the cytosol,
257
where it could then interact with ToxT.
14
C-labeled linoleic acid was added to V. cholerae cultures
12
258
Linoleic acid decreases ToxT activation of virulence gene transcription. tcpA and ctxAB
259
expression have been previously shown to be inhibited by the addition of oleic acid to the
260
media, but the effects of linoleic acid were not assessed in those studies (17). We assessed
261
whether the inhibitory effect of linoleic acid that we observed extended to a variety of ToxT-
262
activated promoters with various ToxT binding site (toxbox) configurations, including tcpA,
263
ctxAB, acfA, aldA, acfD, tagA, and tcpI. All of these promoters contain two toxboxes in either
264
direct (tcpA,ctxAB) (13) or inverted repeat configurations (acfA, acfD, tagA, tcpI) (12-14) except
265
for aldA, which contains only one toxbox (16). Plasmid-borne promoter-lacZ fusions were
266
added to the wild-type V. cholerae classical strain as well as an otherwise isogenic ΔtoxT strain
267
to measure transcriptional activity at these promoters with and without 160µM linoleic acid
268
(Figure 4). At each of these promoters, linoleic acid had a statistically significant negative effect
269
that prevented ToxT from fully activating transcription at the promoters in wild-type O395. No
270
effect of linoleic was observed in the ΔtoxT strain, confirming that linoleic acid acts through
271
ToxT (Figure 4).
272
As the aldA promoter activity even in the absence of linoleic acid was much lower than
273
at the other promoters, we performed quantitative real-time PCR (qPCR) to compare
274
expression levels of this promoter to expression levels of the tcpA promoter upon the addition
275
of linoleic acid. Bacteria were cultured using the same methods as the β-galactosidase assay
276
and then the RNA was extracted. qPCR was then performed using primers for tcpA, and aldA,
277
while using rpoB as the housekeeping gene control. The qPCR results showed that both tcpA
278
and aldA were downregulated upon the addition of linoleic acid to the media (Figure 5).
13
279
Linoleic acid decreases ToxT-DNA binding affinity. EMSA was used to determine if
280
linoleic acid is able to directly affect ToxT binding to DNA at the ToxT-activated promoters. This
281
method allows the binding pattern of ToxT to be characterized and gives a rough estimation of
282
the equilibrium dissociation constant, Kd, which represents the concentration of ToxT required
283
for binding 50% of the DNA at equilibrium. As the concentration of active ToxT varies from
284
preparation to preparation, the Kd values can only be compared per experimental condition,
285
and are only an estimate. The Kd values with and without linoleic acid give a direct comparison
286
of the effect of the fatty acid on the given promoter, as a higher Kd value indicates a lower
287
binding affinity of ToxT for the DNA and a lower Kd value indicates higher binding affinity.
288
To begin, we used the primary promoter in the tcp operon, PtcpA, as our 32P-labeled DNA
289
probe. Using purified MBP-ToxT, we added increasing amounts of protein to binding reactions
290
containing a constant concentration of labeled DNA probe. Additionally, either DMSO alone
291
(Figure 6A, lanes 1-7) or 32 μM linoleic acid dissolved in DMSO were added to the reactions. A
292
representative EMSA is shown in Fig. 6A. Densitometry analysis was performed to calculate the
293
percentage of bound DNA at each concentration of MBP-ToxT and a binding curve was drawn
294
for each set of reactions to determine the percentage of bound DNA at each concentration of
295
MBP-ToxT (Figure 6B). In the case of MBP-ToxT binding to PtcpA DNA, the Kd without linoleic acid
296
was 9.99 nM and the Kd with linoleic acid was 33.11 nM. These values are significantly different,
297
and indicate a lower ToxT binding affinity for PtcpA in the presence of linoleic acid. These data
298
strongly suggest there is likely a direct interaction between linoleic acid and ToxT.
299
To confirm that linoleic acid does not generally inhibit protein-DNA interactions, we
300
used cyclic AMP receptor protein (CRP) as a control, together with the region upstream of tcpP 14
301
to which CRP binds, as described above. We performed an EMSA comparing CRP binding with
302
and without the addition of linoleic acid (Figure 6C) and performed densitometric analysis to
303
determine the binding curves and Kd values. The binding curve and Kd values were statistically
304
the same for CRP binding to PtcpP with or without linoleic acid (Figure 6D).
305
We next determined the binding curves and Kd values at other ToxT-activated
306
promoters to determine whether different toxbox configurations may impact the effect of
307
linoleic acid on ToxT binding to DNA (Figure 7). Equilibrium binding experiments on PtcpA, which
308
has two direct repeat toxboxes, showed a decrease in DNA binding upon addition of linoleic
309
acid. Other promoters have direct and inverted repeat toxbox configurations, and PaldA has only
310
one toxbox (Figure 7.) If linoleic acid could decrease the binding affinity of ToxT for PaldA, it
311
could indicate the mechanism for ToxT inhibition did not involve effects on dimerization, which
312
had been previously proposed as the mechanism for decreasing virulence gene expression (18).
313
PctxAB contains two directly repeated toxboxes like PtcpA, albeit in the opposite orientation and
314
having different spacing (35). The rest of the ToxT-activated promoters previously mentioned,
315
PacfA, PacfD, PtagA, and PtcpI each contain toxboxes oriented in inverted repeat configuration as
316
well as variable spacing between the toxboxes (12-14).
317
As linoleic acid was shown above to decrease promoter activity for all of these ToxT-
318
activated promoters, we determined whether linoleic acid also affected the DNA binding
319
affinity using EMSA with each promoter region. For PctxAB, containing direct repeats, the Kd
320
without linoleic acid is 4.436 nM, and with linoleic acid is 10.05 nM, indicating decreased
321
binding affinity of ToxT (Figure 7A and 7B). As a representative of the inverted repeat toxbox
322
promoters, we chose to look at PtagA. Here, densitometry and binding curve analysis again 15
323
revealed decreased binding affinity of ToxT for the promoter upon the addition of linoleic acid,
324
with a Kd of 4.078 nM without linoleic acid, and 27.78 nM with linoleic acid (Figure 7C and 7D).
325
Lastly, we examined ToxT binding to the single toxbox promoter, PaldA. Figure 7E shows the
326
autoradiograph of ToxT binding to PaldA with and without linoleic acid. The Kd once again was
327
increased, going from 11.25 nM without linoleic acid to 48.67 nM with linoleic acid (Figure 7F).
328
These data, along with the previously described decrease in promoter activity with the addition
329
of linoleic acid as indicated by the β-galactosidase assays, suggest that linoleic acid decreases
330
binding at each promoter regardless of toxbox configuration and spacing.
331
Another negative ToxT effector, virstatin, acts differently than linoleic acid. The results
332
above suggest a direct negative effect of linoleic acid on ToxT binding to DNA. We next
333
examined whether this was also true for another negative ToxT regulator, virstatin. Previous
334
studies suggested that virstatin negatively affects the ability of ToxT to dimerize (21). To begin,
335
we looked at ToxT levels using Western blot analysis to compare V. cholerae grown with or
336
without virstatin (Figure 1B, lanes 6 and 7). The ToxT levels were unchanged, confirming that
337
ToxT production is not affected by virstatin. We then performed β-galactosidase assays on the
338
previously described ToxT-regulated genes to assess the effects of virstatin (Figure 8). These
339
data show decreased ToxT activity at all promoters except for aldA, where virstatin had no
340
significant effect. A previous study suggested that the aldA promoter was affected by virstatin
341
(21). We then performed EMSAs to determine whether virstatin affected the DNA binding
342
ability of ToxT at tcpA, ctxAB, and aldA, as representatives of the different toxbox
343
configurations (Figure 9). Virstatin decreased binding at tcpA and ctxAB promoters, as indicated
344
by analysis of the binding curve and Kd, (Figure 9A-D) but did not decrease ToxT binding at the 16
345
aldA promoter even at a higher concentration of virstatin (Figure 9E). In fact, the Kd significantly
346
decreased from 21.83nM to 11.79nM with the addition of virstatin, indicating a higher binding
347
affinity of ToxT for the aldA promoter when virstatin is present (Figure 9F).
348
349
350
DISCUSSION
351
The activation of virulence gene expression relies on the major virulence transcription
352
activator, ToxT. Previous studies have looked at the involvement of bile, UFAs, and virstatin on
353
V. cholerae virulence gene expression and ToxT dimerization (17, 21, 22, 24, 25, 27). Most of
354
this work with negative effectors has been done only on tcpA and ctxAB; here, in addition to
355
these genes, we also examined the effects of negative effectors on ToxT activity at aldA, acfA,
356
acfD, tagA, and tcpI. All ToxT-controlled promoters showed a decrease in expression in the
357
presence of linoleic acid. Prior to entry into the mucus layer of epithelial cells and colonization,
358
V. cholerae must pass through the lumen of the small intestine where high concentrations of
359
bile and UFAs are present. Linoleic acid needs to be taken up by V. cholerae and enter the
360
cytoplasm in order to directly interact with ToxT. Here, we show that linoleic acid is able to
361
enter the cell and that the majority of it enters the cytoplasm. One caveat is that there may be
362
periplasmic components present in the cytoplasmic portion in our fractionation studies.
363
However, the observation that a majority of linoleic acid was in this fraction, combined with our
364
other observations that linoleic acid directly impacts DNA binding by ToxT in vitro and ToxT
17
365
activity in vivo, strongly suggest that at least some linoleic acid is present in the bacterial
366
cytosol where it directly interacts with ToxT.
367
Previous work has described the effect of negative ToxT effectors on virulence gene
368
expression, but their direct effect on ToxT DNA binding affinity had not been previously
369
assessed. It has been proposed that the mechanism of action for virstatin and UFAs is inhibition
370
of ToxT dimerization (18, 21). The binding sites for ToxT, toxboxes, and their configurations
371
have been extensively studied (12,13,16,35), but whether toxbox configuration factors into the
372
inhibition of ToxT activity by linoleic acid had not been examined. Here we show that the
373
decrease in binding affinity in response to linoleic acid can be seen at each ToxT-activated
374
promoter that we examined, regardless of toxbox configuration. This suggests that linoleic acid
375
has a general effect on DNA binding by ToxT monomers. Previous work strongly suggested that
376
ToxT binds to DNA as a monomer (12, 13, 16) and that ToxT does not behave as a typical
377
obligatorily dimeric activator, such as AraC.
378
We also looked at virstatin and its method of action on ToxT. A previous study examined
379
virulence gene promoter activity in the presence of virstatin; however, unlike this earlier study,
380
the effect of virstatin in our hands was not as pronounced. We did observe that virstatin
381
significantly decreased promoter activity, but to a lesser extent than previously described
382
(Figure 8 (21)). While Shakhnovich et al. saw, at greatest, 20% promoter activity in the presence
383
of virstatin at the aldA, acfA, and tcpI promoters, we observed a decrease, but not to that
384
degree. Interestingly, we did not see any effect of virstatin on ToxT activation at the single
385
toxbox, aldA promoter with the β-galactosidase assay (Figure 8). In the same vein, virstatin also
18
386
did not affect the binding affinity of ToxT for the aldA promoter (Figure 9E,F). In contrast, we
387
did see a negative effect of linoleic acid on both ToxT-dependent virulence gene promoter
388
activity (Figure 4) and ToxT binding affinity for PaldA, where the Kd increased from 11.25 nM to
389
48.67 nM (Figure 7E,F).
390
These data suggest that virstatin and linoleic acid, although both negative effectors of
391
ToxT activity, do not have the same mechanism for downregulating gene expression. Another
392
study performed bacterial two-hybrid assays to look at the effect of various UFAs on
393
dimerization and the data suggest another UFA, oleic acid, does affect ToxT dimerization (25).
394
However, linoleic acid was not tested and this study was done using only the NTD of ToxT
395
instead of intact, full-length ToxT, whose dimerization has never been experimentally observed,
396
at least in published reports. More testing using full-length ToxT in V. cholerae with this two-
397
hybrid system in the presence of linoleic acid would confirm whether or not linoleic acid is
398
involved in dimerization. As there was reduced binding of ToxT at the aldA promoter, it is likely
399
that linoleic acid changes the structural conformation of ToxT, leading to inhibited DNA binding.
400
Understanding the effect of host signals on bacterial pathogenesis is useful for complete
401
understanding of the V. cholerae virulence cascade, as many host signals are used by the
402
bacteria to sense the appropriate location in which virulence gene transcription should be
403
initiated. When passing through the small intestine, V. cholerae encounters many different
404
chemical signals, such as bile and bicarbonate, which either inhibit or activate ToxT-dependent
405
gene transcription. Bile and its UFA components are at high concentrations in the duodenal
406
lumen, in which ToxT is inactive (20, 25, 27, 37). Once the mucus layer is penetrated, UFA
19
407
concentration decreases, while the bicarbonate concentration increases. At this point, along
408
the epithelial surface, ToxT becomes active, virulence genes are expressed, and V. cholerae can
409
colonize. UFAs such as linoleic acid likely keep ToxT in a form unable to bind tightly to DNA in
410
the lumen, because production of TCP would lead to bacterial aggregation prior to penetration
411
of the mucus layer and colonization of the epithelial surface would become impossible.
412
According to this model, V. cholerae is able to use both negative and positive effectors of
413
virulence in order to identify and colonize its ideal niche. Our findings here show a direct effect
414
of linoleic acid on ToxT, giving useful insight into the mechanisms of ToxT gene regulation.
415
416 417
ACKNOWLEDGEMENTS The authors thank the members of the Withey laboratory for helpful discussions. This work was
418
supported by P.H.S. grants K22AI071011 and R56AI093622 (to J.H.W.), Bill and Melinda Gates
419
Foundation grant OPP1068124 and by Wayne State University funds.
420
20
421
422
423
a)
427 428 429 430 431 432
15000 10000 5000 0
WT 160μM LA
426
n.s.
WT
425
PtoxT::lacZ Expression (Miller Units)
424
b)
20000
433 434 435 436 437 438 439 440 441
FIGURE 1. Effect of linoleic acid on PtoxT::lacZ and ToxT expression. (A). Light gray bars, wildtype V. cholerae grown without linoleic acid; dark gray bars, wild-type V. cholerae grown with 64 µM linoleic acid. Statistical significance was determined by Student's t test. (B). Effect of linoleic acid and virstatin on ToxT protein levels. V. cholerae was grown under virulence inducing conditions (LB pH 6.5, 30°C, aeration) with DMSO, linoleic acid or virstatin (lanes 4, 5, 6, 7). Purified full length ToxT-6His migrates to ~32 kDa (lane 2) and a ΔtoxT strain shows no band (lane 3) were used as controls. Samples were normalized by OD600. The Western blot was probed with anti-ToxT antibody. This is representative of three separate experiments.
442
21
443 444
14
C CPM/OD600
150000
100000
50000
0
0
10
20
30
Time (minutes) 445 446 447 448 449 450
FIGURE 2. C14-radiolabeled linoleic acid is taken up by V. cholerae. Classical strain O395 cells were grown under virulence-inducing conditions for two hours and then the radiolabeled linoleic acid was added at time 0 and analyzed at times 5, 15, and 30 minutes post-addition, measured as CPM per optical density unit at 600nm. The equation of best fit is y = 24622 + 156786*(1 - e-.036x) and an R2-value of .989.
451
22
b) 80
456
60
457 458 459 460 461 462
Percent
455
*
Membrane
a)
Cytoplasm
454
Ladder
453
Whole cell lysate
452
40 20 0
OmpU ToxT
25kDa – % Envelope
% Cytoplasm
1
2
3
4
463 464 465 466 467 468 469 470 471 472 473
FIGURE 3. C14-radiolabeled linoleic acid is able to enter the cell and enter the cytoplasm. (A). After a 2-hour subculture in virulence-inducing conditions, linoleic acid was added to the culture and allowed to sit for one hour. After one hour, V. cholerae was fractionated into an envelope and cytoplasmic portion by multiple freeze-thaw cycles. There is significantly more linoleic acid in the cytoplasmic portion as compared to the envelope portion. Error bars represent +/- standard error mean and statistical significance was determined by Student's t test, *, p
