Unsaturated Long Chain Free Fatty Acids Are Input Signals of the Salmonella enterica PhoP/PhoQ Regulatory System*

Background: The PhoP/PhoQ system governs crucial Salmonella typhimurium pathogenic traits. Results: A screening of natural compounds showed that long chain fatty acids present in the bacterial growth medium down-regulate the PhoP/PhoQ-dependent regulon. Conclusion: Long chain unsaturated fatty acids specifically inhibit PhoQ autokinase activity. Significance: The novel PhoQ input signal reveals a new scenario for the control of Salmonella virulence, providing a rationale for future antibacterial strategies. The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg2+ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the β-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.

The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg 2؉ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra-or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the ␤-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.
The Salmonella enterica serovar Typhimurium (S. Typhimurium) PhoP/PhoQ system consists of an orthodox two-component regulatory system, comprising PhoQ, a transmembrane sensor with bifunctional (histidine kinase/phosphatase) activity, and PhoP, a cytoplasmic response regulator (1). This twocomponent regulatory system serves as a master regulator of S. Typhimurium virulence. The regulon governed by the S. Typhimurium PhoP/PhoQ two-component regulatory system includes genes that are critical for Mg 2ϩ homeostasis (2) and those that provoke modifications of the LPS, which determine bacterial susceptibility to cationic antimicrobial peptides (2)(3)(4)(5). The PhoP/PhoQ system is also involved in the bacterial entry mechanism into the host cell, modulating the expression of the injectisome and of translocated effectors (6 -8). Once inside the cell, PhoP-modulated genes contribute to define the intracellular survival of Salmonella, being implicated in the intravacuolar proliferation capacity of the bacteria and in the traffic divergence of the Salmonella-containing vacuole from the canonical endocytic pathway (9 -11).
We have shown previously that the environmental availability of Mg 2ϩ signals on PhoQ and controls the switch between the kinase and the phosphatase activity of the sensor protein, modulating PhoP phosphorylation status and, thereby, the transcriptional activity of this response regulator (1,12). In addition to Mg 2ϩ limitation, which has been demonstrated to globally modulate the PhoP regulon (1,2,(12)(13)(14), sublethal concentrations of cationic antimicrobial peptides and mild acidic pH activate the expression of subsets of PhoP-regulated genes (14 -18). More recently, Lippa et al. (19) have shown that a decrease in the oxidizing activity of the periplasm can also stimulate PhoP/PhoQ through a DsbA/MgrB-dependent pathway. The multiplicity of signals that are able to act as an input of the PhoP/PhoQ system mirrors the intricate interaction of Salmonella with changing ambient conditions that this pathogen has to traverse through its life cycle.
Taking into consideration the relevant role of the PhoP/ PhoQ system in the adaptive capacity of S. Typhimurium to either host or non-host environmental challenges and the fact that the two-component regulatory system can only be found in prokaryotes, low eukaryotes, and plants, PhoP/PhoQ consti-tutes an ideal target to search for new compounds that would prevent and/or control Salmonella virulence in the mammalian host.
Plant extracts are sources of compounds with exceptional chemical diversity that constantly give rise to novel therapeutic agents (20,21). In this work, we carried out a screening of extracts from native plant species to identify naturally occurring molecules that would modulate PhoP/PhoQ activity. This search led us to determine that long chain unsaturated fatty acids (LCUFAs) 4 constitute specific input signals that downregulate the PhoP/PhoQ system activity. We demonstrate that this repression is due to the inhibition of PhoQ autophosphorylation capacity. Exogenously provided long chain fatty acids are transported across the bacterial membrane and converted to acyl-CoA derivatives that are substrates either for ␤-oxidation or for the synthesis of membrane phospholipids. Therefore, we examined whether the incorporation of the LCUFAs into these metabolic pathways was responsible for PhoQ autokinase repression. By the analysis of the effect of LCUFAs on mutant strains that are blocked in these metabolic routes, we demonstrate that neither esterification into membrane phospholipids nor degradation by the ␤-oxidative path is a prerequisite for LCUFAs to exert their repressive action on PhoQ activity. Our results indicate that free unsaturated fatty acids constitute specific signals detected by PhoQ, which provoke the inhibition of the sensor autokinase activity. In turn, this effect is transmitted downstream, resulting in the inactivation of the regulator PhoP with a concomitant down-regulation of PhoPactivated gene expression.
The fact that exogenously provided LCUFAs are able to specifically signal on PhoQ not only widens the array of potential environmental scenarios that Salmonella is capable of perceiving in vivo in order to orchestrate the expression of virulence phenotypes by means of the PhoP/PhoQ system, but also provides a rationale for the future design of new specifically targeted antibacterial strategies.
The critical micelle concentration (CMC) for oleic, strearic, palmitic, and linoleic sodium salts was calculated to be in the 1.8 -3.5 mM range (22,23). Although the exact CMC values under our experimental conditions have not been determined, from these orientative data, the concentrations used in the assays reported here are below the respective CMC (0.5 mg/ml corresponds to concentrations of Յ1.8 mM for the fatty acids used in this work).
Plant Material-Plants were collected in Pergamino, Buenos Aires Province, Argentina, during November 2005. Voucher specimens were deposited at the Universidad Nacional de Rosario Herbarium. Collected plants were Urtica urens L. Crude Extracts-A sample (200 g) of dried and powdered material (entire plant) was refluxed 3 times with 4 liters of methanol for 45 min. The extracts were combined after filtration, and the solvent was eliminated under reduced pressure by rotary evaporation.
Bacterial Strains, Cell Culture, and Growth Conditions-Bacterial strains used in this work are listed in supplemental Table S1. Bacteria were grown at 37°C in Luria-Bertani (LB) broth with shaking, with the addition of 0.7 mM isopropyl-␤-Dthiogalactopyranoside when appropriate, and/or the addition of fatty acids at the concentrations indicated in each assay. Ampicillin was used at a final concentration of 100 g ml Ϫ1 .
Assays to Evaluate Potential Inhibitory Action of the Compounds under Study on Salmonella Growth-The potential inhibitory effect of plant extracts or fatty acids was evaluated by growing Salmonella Typhimurium wild-type strain in LB broth. The assay was carried out in sterile 96-well microtiter plates. Plant extracts in methanol or fatty acids (dissolved in ethanol for unsaturated or tergitol for saturated fatty acids) were diluted to give serial 2-fold dilutions that were added to LB, resulting in concentrations ranging from 0 to 6 mg ml Ϫ1 (for plant extracts) and from 0 to 2 mg ml Ϫ1 (for fatty acids). Neither the concentration range of plant extracts nor that of the fatty acids used in these experiments altered the pH of the medium. The final concentration of methanol, ethanol, or tergitol in the assays did not exceed 12, 4, or 4%, respectively, which was not detrimental for bacterial growth. Ampicillin was included as a positive control at 50 g ml Ϫ1 . The plates were incubated, for 18 h with agitation at 37°C, and optical density was measured at 630 nm (A 630 nm ), with the BioTek Synergy 2 multimode microplate reader. Inhibition of bacterial growth by each compound was evaluated by monitoring growth as detected by A 630 nm . No inhibition of bacterial growth was detected within the concentration range of either fatty acids or plant extracts assayed in this work.
Bacterial Growth Curves-Overnight cultures of S. Typhimurium wild-type, fadL, fadD, or phoPQ strains were diluted 1:100 in a sterile 96-well microplate (Greiner Bio-one) with fresh LB medium with or without the addition of commercial linoleic acid at a final concentration of 0.5 mg ml Ϫ1 , and the optical density at 600 nm was measured every 60 min with the BioTek Synergy 2 multimode microplate reader. Samples were processed by duplicates, and proper blank controls were used in each microplate.
Genetic and Molecular Biology Techniques-Plasmids used in this work are listed in supplemental Table S1. Plasmid DNA was introduced into bacterial strains by electroporation using a Bio-Rad apparatus as recommended by the manufacturer. Deletion of various genes and concomitant insertion of an antibiotic resistance cassette were carried out using Red-mediated recombination (24,25) in strain LB5010 (26). The mutations resulting from this procedure were transferred to the S. Typhimurium wild-type background by P22 transduction. Phage P22-mediated transductions were performed as described (27). To introduce the lacZ reporter gene, antibiotic resistance cassettes were removed using the temperature-sensitive plasmid pCP20 carrying the FLP recombinase (28). pCE36 was used to introduce the transcriptional lacZ fusion as described previously (25). The oligonucleotides used to perform these procedures were purchased from Sigma. Their sequences are listed in supplemental Table S2. Double mutant strains were generated by P22 transductions from previously obtained single mutant strains and selected by the adequate combination of antibiotic resistances, as listed in supplemental Table S1. Other conventional recombinant DNA techniques were performed according to standard protocols (29). Polymerase chain reaction-derived constructions and site-directed mutagenesis were all confirmed by PCR assays and DNA sequence analysis.
RNA Purification-Total RNA was extracted from mid-exponential phase cultures grown in LB medium at 37°C. Briefly, 8 ml of ice-cold 5% water-saturated phenol (pH 5.5) in ethanol was added to 50-ml cultures to stop RNA degradation. Cells were centrifuged at 5,500 ϫ g for 5 min at 4°C; resuspended in 5 ml of 0.5 mg ml Ϫ1 lysozyme-Tris-HCl (10 mM), EDTA (1 mM), pH 8.0, 1% SDS; mixed; and placed in a water bath at 64°C for 2 min. After incubation, 5.5 ml of 1 M sodium acetate, pH 5.2, was added. The sample was extracted twice with an equal volume of water-saturated phenol, pH 5.5, and incubated at 64°C for 6 min. The aqueous layer was extracted with an equal volume of chloroform and precipitated with ethanol. RNA was resuspended in water, treated with RQ1 RNase-free DNase (Promega), and subjected to a final step of RNA cleanup using an RNeasy kit (Qiagen) according to the manufacturer's protocol. DNA contamination in RNA preparations was assessed by performing a control PCR prior to RT-PCR analysis.
Semiquantitative RT-PCR-cDNA synthesis was performed using random hexamers, 2 g of total RNA, and 1 unit of Super-Script II RnaseH2 reverse transcriptase (Invitrogen). Five microliters of a 1:10 dilution of cDNA was used as the template for DNA amplification in RT-PCR (20 l), using primers PhoP-NTR and PhoP-NTF (supplemental Table S2). A primer set for the 16 S rRNA was used as a control to confirm that equal amounts of total RNA were used in each reaction mixture. The amplified fragment was 186 bp. The number of cycles was adjusted according to the level of expression of mRNA to ensure that the comparison was performed in the linear range of the amplification.
TLC Bioautography-The inhibitory activity of the assayed compounds on PhoP/PhoQ-dependent reporter gene expression was studied by TLC bioautography, a technique that combines chromatography separation with in situ activity determination. The amount of extract loaded on the TLC plate was 250 g of extract/band. After elution with a mixture of methanol/ dichloromethane (94:6), the solvents were removed under an air current, and the plate was subjected to autographic analysis. Chromatograms were placed in sterile Petri dishes with covers. TLC plates were overlaid with soft agar medium (LB with 0.6% agar) containing an inoculum of the reporter bacterial cell suspension from an overnight culture at a final concentration of 50 ϫ 10 5 cfu ml Ϫ1 , 0.33 mg ml Ϫ1 X-gal, and the addition of the adequate antibiotic when required. After solidification of the medium, the TLC plates were incubated overnight at 37°C for 18 h. Inhibition of the reporter gene expression was detected as light blue or white areas on a blue background given by the ␤-galactosidase activity-mediated cleavage of the X-gal substrate. Concomitantly, growth inhibition could also be detected by the presence of a transparent halo in contrast of the turbid appearance of the LB-agar layer that denoted bacterial growth.
Bioguided Fractionation-26 g of crude extract was dissolved in 1.0 liters of MeOH/H 2 O (9:1) and extracted with hexane (2 ϫ 500 ml). The MeOH was evaporated from the aqueous phase under reduced pressure, and H 2 O was added to a final volume of 200 ml. The resulting solution was extracted with dichloromethane (3 ϫ 200 ml). The organic phases (hexane and dichloromethane) were dried with anhydrous Na 2 SO 4 , and the solvents were evaporated at reduced pressure to give the corresponding hexane (HEX) and dichloromethane (DCM) fractions. The DCM fraction was chromatographed on silica gel (Analtech) with a dichloromethane/methanol gradient (99:1 to 90:10). The active fraction was chromatographed onto a TLC silica gel 60 F 254 preparative plate (Merck) with dichloromethane/methanol (95:5). The active fraction was analyzed by NMR and GC-MS.
To rule out a potential repressive action on the PhoP/PhoQ system activity due to the presence of Mg 2ϩ , the content of this divalent cation was measured in L. amplexicaule extracts by flame photometry. The Mg 2ϩ content was determined to be 23.0, 4.4, and 0.8 M for a 4.0 mg/ml solution of the L. amplexicaule methanolic crude extract and DCM and HEX fractions, respectively.
Mass Spectrometry and NMR Analysis-1 H NMR spectra were recorded on a Bruker Avance II at 300 MHz in CDCl 3 , in the presence of TMS (0.00 ppm) as the internal standard. GC-MS was performed using a PerkinElmer Life Sciences Autosystem XL gas chromatograph coupled to a TurboMass mass spectrometer. The column was as follows: SE-30, 25 m ϫ 0.22 mm inner diameter (Scientific Glass Engineering, Ringwood, Victoria, Australia). Prior to analysis, the fatty acids present in the samples were derivatized to methyl esters according to the procedure described by Lepage and Roy (30). Briefly, a solution of the samples in HCl-containing methanolic medium was heated at 80°C for 150 min. The solution was diluted with 6% Na 2 CO 3 , and the derivatives were then extracted with hexane.
␤-Galactosidase Activity Assays-For the ␤-galactosidase activity assays, bacteria were grown overnight with shaking at 37°C in LB with 5 mM MgCl 2 , L. amplexicaule extract fraction, and/or each commercial fatty acid at the final concentration indicated in each experiment. In the case of strains harboring a plasmid vector, isopropyl-␤-D-thiogalactopyranoside was added at a final concentration of 0.7 mM. Ampicillin was used at 100 g ml Ϫ1 final concentration in the bacterial growth medium when appropriate. ␤-Galactosidase activity was determined as described (31).
Preparation of Membranes Enriched in PhoQ or EnvZ-Membranes to test the autokinase activity of the sensors PhoQ or EnvZ were prepared as described previously (1). Briefly, overnight cultures of S. Typhimurium strains PB4663, PB10359, and PB9079 were used to inoculate LB containing 100 g ml Ϫ1 ampicillin and the corresponding fatty acid from a commercial source at the final concentration indicated in each experiment. These were then grown at 37°C to logarithmic phase (A 630 nm ϭ 0.6), and protein expression was induced by the addition of 0.7 mM isopropyl-␤-D-thiogalactopyranoside for an additional 3 h with shaking. Cells were collected, washed twice with 10 mM Tris-HCl (pH 8.0), and resuspended in a solution containing 20 mM Tris-HCl (pH 8.0), 20% sucrose, 5 mM EDTA, and 150 g ml Ϫ1 lysozyme. After a 40-min incubation at 4°C, 20 mM MgCl 2 was added, and cells were centrifuged for 20 min at 9,000 ϫ g. The pellet was resuspended in ice-cold 10 mM Tris-HCl (pH 8.0) and subjected to sonication. Nonruptured spheroplasts were removed by a brief centrifugation at 5,500 ϫ g, and the membrane fraction was recovered after a 40-min centrifugation at 21,000 ϫ g. The membranes were washed subsequently with 10 mM Tris-HCl (pH 8.0), 2 M KCl, 10 mM Tris-HCl, 5 mM EDTA, and 10 mM Tris-HCl by 40-min centrifugation rounds at 21,000 ϫ g. Each time, the supernatant was discarded. Finally, the membranes were resuspended in 25 mM Tris-HCl (pH 8.0), 50 mM KCl. All procedures were carried out at 4°C. Protein concentration was determined by the bicinchoninic acid assay (Sigma) using bovine serum albumin as a standard.
Autokinase Activity Assays and Immunodetection Analysis-For the autokinase assay, membranes (50 g of total protein) harboring PhoQ or EnvZ were incubated for 10 min at 37°C in a 30-l reaction mixture containing 25 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM MgCl 2 , 50 M nonlabeled ATP, and 0.16 Ci l Ϫ1 [␥-32 P]ATP (PerkinElmer Life Sciences). Reactions were started by the addition of the reaction mixture and stopped by the addition of 6 l of 5ϫ SDS-PAGE sample buffer (2.5% ␤-mercaptoethanol, 9% glycerol, 10% SDS, 600 mM Tris-HCl (pH 6.8), 0.006% bromphenol blue). The amount of radiolabeled PhoQ or EnvZ increased proportionally to the amount of membrane protein used in the range from 0.16 to 2.6 g l Ϫ1 ; this indicated that PhoQ was not in excess at 1.67 g l Ϫ1 , the membrane concentration used in the autophosphorylation assay. Samples were heated for 3 min at 65°C. All reactions were analyzed by SDS-PAGE (12% polyacrylamide), transferred to nitrocellulose, and then subjected to Western blot or autoradiography analysis. Western blot membranes were incubated with rabbit anti-PhoQ Cyt or anti-EnvZ cyt poly-clonal antibodies, obtained as described (1), and developed by incubation with protein A conjugated with phosphatase, coupled to a chromogenic reaction using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates. Autoradiographies and Western blot membranes were densitometrically scanned using ImageJ software (32) to perform quantitative determinations.
Analysis of Lipids by TLC-Overnight cultures of S. Typhimurium wild-type, fadL, and fadD strains were diluted 1:100 in fresh LB medium containing commercial linoleic acid at a final concentration of 0.5 mg ml Ϫ1 and 0.075 Ci ml Ϫ1 [1-14 C]linoleic acid. Cells were grown at 37°C for 5 h with shaking. Then cells were harvested by centrifugation at 3,000 ϫ g for 10 min, washed three times with phosphate-buffered saline (PBS, pH 7.4), and resuspended in 200 l of the same buffer. Lipids were extracted over a weekend by the method of Bligh and Dyer (33) and spotted onto a TLC silica gel 60 F 254 plate (Merck). Lipids were separated using a solvent system consisting of diethyl ether/petroleum ether/acetic acid (70:30:2, v/v/v). The TLC plates were exposed overnight to a Storage Phospho Screener screen, and product formation was detected and analyzed using an Amersham Biosciences Storm Imaging System equipped with Storm Scanner Control. Total lipids were developed with 10% CuSO 4 in 8% H 3 PO 4 as a charring reagent. Densitometry was calculated for the major spots observed by TLC.
Statistical Analysis-Statistical analysis of all quantitative data shown was performed using one-way analysis of variance and the Holm-Sidak test with an overall significance level of 0.05.

Plant Extracts with PhoP/PhoQ System Inhibitory Action-
We performed a systematic screen from four plant extracts (please see "Plant Material" under Experimental Procedures) in a search for compounds that could modulate the activity of the PhoP/PhoQ system in Salmonella. The methanolic total extract of each plant species was first screened by a dot agar overlay bioautography assay. Briefly, the crude methanolic extract was spotted onto a TLC plate and analyzed for activity without development of the plate. Inhibitory activity was detected by overlaying the plate with an aqueous solution of soft agar containing X-gal and a homogeneous suspension of the S. Typhimurium strain that harbors a reporter transcriptional lacZ fusion to virK, a previously characterized PhoP-activated gene (12,34). Those extracts that produced a light blue or white halo (intact X-gal substrate) over the blue background of the hydrolyzed/oxidized X-gal were further analyzed (see "Experimental Procedures" for details). Among tested extracts, the L. amplexicaule-derived extract showed the largest inhibition zone. 5 With this L. amplexicaule extract, we also performed ␤-galactosidase activity assays in liquid medium to quantitatively assess the inhibitory action on the expression of the PhoP-dependent reporters. We tested the activity of seven different PhoP-activated genes with transcriptional fusions to lacZ when bacteria were grown in LB (PhoP/PhoQ-activating condition) or in LB supplemented with 5 mM MgCl 2 (PhoP/ PhoQ-repressing condition) or a 1 or 4 mg ml Ϫ1 concentration of the L. amplexicaule methanolic extract. We also tested the PhoP/PhoQ-unrelated transcriptional reporters tppB::lacZ and golT::lacZ as controls; tppB is a two-component OmpR/EnvZcontrolled gene, whereas golT is the transcriptional regulator of an operon involved in Salmonella resistance to transition metals (35)(36)(37). As shown in Fig. 1A, the expression of all PhoPactivated genes assayed was down-regulated in those cultures where the semipurified fraction of L. amplexicaule was added to LB, compared with LB with no additions, whereas golT or tppB gene expression was unaffected, showing that the observed inhibitory effect was specific for the genes that belong to the PhoP/PhoQ regulon.
Next, increasing polarity solvents (hexane and dichloromethane) were used to fraction L. amplexicaule dry extract. Both the HEX and DCM fractions showed inhibitory activity by either bioautographic or liquid ␤-galactosidase activity assays (Fig. 2, B and C, respectively). The DMC fraction was subjected to sequential bioactivity-guided purification by chromatographic steps (silica gel column followed by preparative TLC; see "Experimental Procedures") to obtain a semipurified bioactive fraction. We excluded bactericidal activity and verified that subinhibitory concentrations of the plant extracts were used (see "Experimental Procedures"). The composition of this bioactive fraction recovered from the preparative TLC was simul-taneously determined by GC-MS and by 1 H NMR analysis (supplemental Figs. S1 and S2). Both analyses revealed that the fraction was mainly composed of fatty acids of 16 -30-carbon length chain, 16 and 18-carbon species being the most abundant constituents.
To examine whether long chain fatty acids were responsible for the observed effect, the action of single unsaturated fatty acids with chain lengths of 16 and 18 carbons (C16 and C18) from commercial source was tested. C16 or C18 unsaturated fatty acids added to the bacterial LB growth medium downregulated the expression of virK ( Fig. 2A), whereas they did not exert a repressive effect on the expression of tppB (Fig. 2B). The four LCUFAs assayed down-regulated virK expression when a concentration higher than 0.25 mg ml Ϫ1 was added to LB. To explore whether a fatty acid moiety was enough to repress PhoP-activated genes, equivalent concentrations of palmitic (C16:0) or stearic (C18:0) saturated fatty acids added to LB were tested. As shown in Fig. 2, C and D, the saturated fatty acids did not alter either virK or tppB expression levels compared with LB alone, indicating that LCUFAs exert a specific inhibitory effect on the PhoP/PhoQ system activity. To further substantiate this observation, we also assayed the action of linoleic acid as a representative LCUFA on ppiA expression, used as reporter of the activity of the CpxA/CpxR two-component system (38).  (PhoP-repressing condition). B, 500, 250, or 125 g of DCM or HEX fractions of L. amplexicaule was loaded onto a TLC plate, and the separation was carried out with dichloromethane/methanol (95:5). LB-agar with a suspension of the reporter virK::lacZ strain was layered over the chromatogram as described under "Experimental Procedures." The arrow points at the area where clear halos (intact X-gal) over the colored hydrolyzed X-gal background were detected in each lane. C, ␤-galactosidase activity from virK::lacZ was carried out as in A using LB added with a 4 mg ml Ϫ1 concentration of the L. amplexicaule DCM or HEX fractions, as indicated. ␤-Galactosidase activity was measured as described under "Experimental Procedures." Results are the average of three independent assays performed in duplicate, and error bars correspond to S.D. Fig. S3, the expression of ppiA was modulated by pH and by ethanol (which was also used as the C18:2-solubilizing agent), previously described signals detected by the Cpx system (38,39), but it was not affected by the action of the fatty acid in the bacterial growth medium. Additionally, as shown in Fig. 3A, PhoP-activated genes other than virK were down-regulated by linoleic acid (C18:2), confirming the specific action of the LCUFAs on the PhoP-regulon. (The well known repressing action of extracellular millimolar concentrations of Mg 2ϩ on PhoP-activated genes was tested as a control.). We also assayed the combined action of Mg 2ϩ and C18:2. The results shown in Fig. 3B indicate that, when simultaneously present in the Salmonella growth medium, Mg 2ϩ and C18:2 are able to exert a repressive action that is higher than that observed when each inhibitor is separately tested, whereas no synergic effect is observed. This result led us to conjecture that LCUFAs might have a different target site of action on the PhoP/PhoQ system from that of divalent cations.

As shown in supplemental
We also measured the transcriptional activity of the PhoP reporter gene from bacteria grown in LB plus C18:2 or from these preconditioned bacteria after the fatty acid has been removed by replacing the growth medium by fresh LB. As shown in supplemental Fig. S4A, the repression of virK expression could be relieved, provided the LCUFA was removed from the growth medium, indicating that the inhibitory action of LCUFAs on the PhoP/PhoQ system activity is reversible. The reversibility of the repression exerted by Mg 2ϩ and the null effect on tppB expression levels obtained in all tested conditions are also shown as controls (supplemental Fig. S4, A and B).
The Transcriptional Activity of phoPQ Is Down-regulated by the Action of C18:2-Taking into account that phoPQ expression is subjected to autoregulation as part of the PhoP/PhoQ regulon (40), the concerted down-regulation in the expression of PhoP-activated genes allowed us to predict that the transcriptional activity of phoP would be turned off when bacteria are grown in the presence of LCUFAs. As shown in Fig. 3C, when bacteria were grown in LB with added C18:2, phoP transcription levels assessed by semiquantitative RT-PCR decreased an estimated 40% compared with those obtained in LB with no additions. The repressing action of Mg 2ϩ on phoP expression (75% repression) and the transcriptional levels of 16 S rRNA, which were unaffected in the three conditions assayed, were also analyzed as controls (Fig. 3C).
LCUFAs Specifically Inhibit PhoQ Autokinase Activity-To define the PhoP/PhoQ transduction mechanism step that was affected by LCUFAs, we first examined the action of these compounds on PhoQ autokinase activity. With this aim, we isolated PhoQ-enriched membrane vesicles derived from the S. Typhimurim phoQ Ϫ strain that expresses PhoQ from the pUHE-21-2::phoQ plasmid, as we described previously (1) (also see "Experimental Procedures"). This assay system proved to preserve a PhoQ functional protein in its native bacterial membrane environment, avoiding conformational or functional artifacts that would result from the solubilization of the protein with detergents and reconstitution in artificial proteoliposomes. The Salmonella-derived vesicles were prepared from bacteria grown in LB with or without the addition of the indicated concentration of LCUFA, as described under "Experimental Procedures." To determine PhoQ autophosphorylation activity, vesicles were incubated with a [␥-32 P]ATP-containing reaction medium, and samples were analyzed by SDS-PAGE followed by autoradiography, as described previously (1). As shown in Fig. 4A, PhoQ autophosphorylation was inhibited in a dose-dependent man- ner when bacteria were grown in LB containing increasing concentrations of C18:2. Inhibition of the PhoQ autokinase activity was detected when a concentration as low as 50 g ml Ϫ1 C18:2 was used. Moreover, and consistent with the above shown ␤-galactosidase activity assays, the inhibitory action on PhoQ autokinase activity was observed for LCUFAs but not when saturated fatty acids of equivalent carbon chain lengths were tested (Fig. 4B). When EnvZ (an orthodox Salmonella histidine kinase sensor of the two-component family that has analogous structural and functional properties to PhoQ (41, 42)) autokinase activity was examined, following the same protocol employed for PhoQ, no detectable effect of saturated or unsaturated fatty acids on the autokinase activity of this sensor protein was observed (Fig.  4C), indicating that LCUFAs act selectively on the PhoQ sensor autophosphorylation activity. These last results also allow us to discard a potential nonspecific inhibitory action of the LCUFAs on the activity of the conserved catalytic domain of histidine kinases that would prevent autophosphorylation in the presence of ATP. Additionally, we verified that the addition of C18:2 in the autokinase reaction medium did not exert any effect on PhoQ autophosphorylation capacity (supplemental Fig. S5). The action of equivalent final concentrations of the fatty acid solvents employed added to the bacterial growth medium, tergitol or ethanol, was also tested to verify that they exerted no action on the autokinase activity of the sensor proteins (Fig. 4, B and C, lanes T and E, respectively). To exclude a potential action of fatty acids on PhoQ or EnvZ expression, we simultaneously monitored the protein level attained by each sensor by Western blot, and the ratio between the levels of labeled versus immunodetected bands was calculated by densitometry; the plot with the calculated ratios is shown below each autophosphorylation assay image in all autokinase assays.
We have demonstrated previously that millimolar concentrations of Mg 2ϩ repress the PhoP/PhoQ system activity by inducing the phosphatase activity of PhoQ (1). The results shown in Fig. 4D corroborate our previous results demonstrating that the addition of increasing concentrations of Mg 2ϩ to the Salmonella growth medium, which are inhibitory to the expression of PhoP-dependent genes, does not affect the autophosphorylation levels of PhoQ and also indicate that the addition of the cation in combination with C18:2 to the growth medium does not interfere with the repression exerted by C18:2 on PhoQ autophosphorylation levels. This last result allows us to conclude that the inhibitory effect exerted by LCUFAs when present in the Salmonella growth medium targets PhoQ autokinase capacity.
The Inhibitory Action of LCUFAs on the PhoP/PhoQ System Does Not Require a Functional ␤-Oxidative Metabolic Pathway-In either Escherichia coli or Salmonella enterica, exogenously provided long chain fatty acids are incorporated by the fatty acid ␤-oxidative metabolic route to finally render acetyl-CoA (43,44). Lesley and Waldburger (45) have shown previously that acetyl-CoA exerts an inhibitory effect on the autokinase activity of PhoQ. To explore whether the increase in the intracellular concentration of acetyl-CoA or of an intermediate metabolite upon S. Typhimurium growth in LCUFAs was responsible for the observed PhoQ autokinase down-regulatory effect, we generated Salmonella mutant strains harboring deletions in fadL, fadD, or fadBA. These genes code for the proteins responsible for critical steps along the fatty acid ␤-oxidative pathway: transport across the envelope, activation to an acyl-CoA derivative, and iterative dehydrogenation cycles of the fatty acid, respectively (46). fadL, fadD, and fadBA are all part of the FadR regulon. FadR is a transcriptional regulator that plays a dual role in fatty acid metabolism (47). Upon interaction with a fatty acyl-CoA, FadR derepresses the genes involved in ␤-oxidation, including fadL, fadD, and fadBA, whereas it activates the expression of fatty acid biosynthesis key enzymes (46). Therefore, to corroborate the metabolic response of the S. Typhimurium strain under analysis in the presence of LCUFAs, we generated lacZ-fused transcriptional reporters of the aforementioned fad genes. By ␤-galactosidase activity assays, we demonstrated that, as predicted, fadBA expression was induced when C18:2 was added to the bacterial growth medium (Fig. 5). fadL-lacZ or fadD-lacZ reporter constructs retain their respective promoter regions intact, whereas coding regions were removed and replaced by lacZ. These gene deletions block the entrance or the CoA activation of the fatty acid, abrogating its conversion to the corresponding acyl-CoA derivative. Consistently, the expression of fadD and fadL remained unchanged in the presence of fatty acids compared with LB with no additions (Fig. 5). We also examined the transcriptional activity of the fad genes in a wild-type versus a phoP background to exclude a potential regulatory effect by the PhoP/PhoQ system on their expression. As shown in Fig. 5, the expression of the fad genes was unaffected in the phoP mutant strain when compared with the otherwise isogenic wild-type strain grown  in LB either with or without the addition of C18:2 or Mg 2ϩ . Moreover, we verified that the addition of C18:2 to the LB medium did not significantly alter the growth pattern of phoPQ or fad mutant strains, compared with the wild-type strain (supplemental Fig. S6, A and B).
In the fadL or fadBA genetic backgrounds, C18:2 added to the bacterial growth medium rendered inhibition of virK expression levels equivalent to those observed in the wild-type strain (Fig. 6). virK transcriptional inhibition was observed when either wild-type or fad strains were grown in LB ϩ Mg 2ϩ , whereas the transcriptional activity of the tppB::lacZ reporter was unaltered in the three fad backgrounds in all conditions tested (Fig. 6). However, in the fadD strain, an estimated 7-fold reduction in the basal levels of virK expression was observed (Fig. 6, LB with no addition), precluding an accurate evaluation of the effect of the unsaturated fatty acid. Identical results were obtained when other PhoP-activated genes were assayed in the same fad backgrounds (not shown). Despite this last observation, which will be further examined, our results indicate that the inhibition of PhoP-activated genes by C18:2 does not require the integrity of the unsaturated fatty acid ␤-oxidative pathway.
Taken together, these results show that LCUFAs present in the bacterial growth medium exert a repressive action on the PhoP/PhoQ system activity that is independent of the metabolic degradative fate of these compounds in the cell and rule out the potential involvement of an accumulation of acetyl-CoA or of an intermediate metabolite of the ␤-oxidative pathway as responsible for the observed inhibition of PhoP-activated gene expression by LCUFAs.
As previously mentioned, FadD is required to activate the translocated exogenous unsaturated fatty acids into CoA-conjugated thioesthers. These acyl-CoA compounds can be either degraded by the ␤-oxidative path or used as fatty acid donors for esterification into membrane phospholipids by the PlsB/ PslC-dependent pathway (48). In consequence, the observed reduction in virK expression in a fadD mutant strain can conceivably be attributed to lower levels of PhoQ protein expres-sion, to a reduced PhoQ autokinase activity due to a change in the phospholipid composition of the membrane where the sensor is anchored, or to the accumulation of LCUFAs incorporated from the growth medium that cannot be vectorially transported to the cytoplasm and therefore inhibit phoQ expression by the phoPQ autoregulatory loop.
To explore this issue, we performed the autokinase activity assay using PhoQ-containing vesicles obtained from a fadD mutant strain (fadD/pUHE21-2::phoQ) grown in LB with or without the addition of C18:2. In this assay, because PhoQ expression was driven from the pUHE21-2lacI q promoter, PhoQ expression is independent of the PhoP-modulated autoregulatory mechanism. As shown in Fig. 7, a reduction of PhoQ autokinase activity levels can be detected in the fadD mutant when compared with the wild-type strain in LB with no additions. As tested by simultaneous PhoQ immunodetection, this decrease correlates with a reduction in PhoQ steady-state levels in the fadD strain relative to the wild-type strain (Fig. 7, bottom). This result might be due to a deficient assembly or to increased degradation of PhoQ in this genetic background and contributes to explain the low basal transcriptional expression of virK observed for the fadD mutant strain in the above shown ␤-galactosidase activity assays. Nevertheless, we observed undetectable PhoQ autophosphorylation levels in the samples obtained either from the wild type or from the fadD strain when C18:2 was present in the bacterial growth medium, relative to those obtained from bacteria grown in LB. Together, our results clearly show that exogenously provided free LCUFAs exert a repressive action on PhoQ autokinase activity, irrespective of their metabolic fate in the bacterial cell.

DISCUSSION
The PhoP/PhoQ regulatory system is central to the pathogenic properties of Salmonella Typhimurium. The fact that PhoP/PhoQ belongs to the so-called two-component family signal transduction systems, which are absent in mammalian organisms, makes it an ideal target for the development of anti- microbial strategies. In this work, while searching for compounds of natural sources that could modulate the activity of Salmonella PhoP/PhoQ signal transduction system, we found that LCUFAs could reversibly down-regulate PhoP/PhoQ activity. The action of these fatty acids was detected when bacteria were grown in the presence of a subinhibitory concentration of either L. amplexicaule-derived extracts that contain LCUFAs in their composition or C16 or C18 chain length unsaturated fatty acids from a commercial source.
We demonstrated that free LCUFAs specifically repress the PhoP/PhoQ system because they down-regulated the expression of both the phoPQ operon and PhoP-activated genes at the transcriptional level, whereas these compounds exerted no effect on genes controlled by other signal transduction mechanisms. Moreover, we showed that equivalent concentrations of saturated fatty acids of the same carbon chain length as the assayed LCUFAs did not alter the expression of PhoP-dependent genes.
The transcriptional inhibition of phoP-dependent genes could be attributable to a down-regulation of the activated status of the transcriptional regulator PhoP, which is dictated by the PhoQ sensor activity. When the autophosphorylation ability of PhoQ-harboring vesicles obtained from bacteria grown in LB containing concentrations of linoleic acid (C18:2) that were non inhibitory for bacterial growth was measured, we found that the sensor protein autokinase activity was severely repressed, in a dose-dependent manner. This repression was replicated when other LCUFAs were assayed, whereas no effect was detected when saturated fatty acids of equal chain length were tested. No fatty acid-dependent effect was observed when the functionally and structurally related sensor EnvZ autokinase activity was assayed, ruling out a potential broad action of these compounds on the catalytic domain of Class I histidine kinases (as classified in Ref. 49). The absence of effect also observed when the activity of transcriptional reporters dependent on either the EnvZ/OmpR or CpxA/CpxR system was determined contributed to reinforce this notion. Additionally, these results also show that the in vivo or in vitro inhibition of the PhoP/PhoQ system by LCUFAs is unlikely to be the result of nonspecific detergent effects on these sensor proteins. Besides, saturated fatty acids with similar CMC values showed no inhibitory action, and the repressive action of C18:2 could be detected when concentrations as low as 50 g ml Ϫ1 were assayed (Fig. 4A), a concentration far below the estimated CMC for this fatty acid in aqueous solution.
We have previously demonstrated that Mg 2ϩ extracellularly supplied to the Salmonella growth medium specifically induces PhoQ phosphatase activity while exerting no effect on PhoQ autokinase activity (1). When the transcriptional expression levels of a representative PhoP-activated gene from bacteria grown in the simultaneous presence of C18:2 and Mg 2ϩ were measured, we found that they exerted stronger repression compared with the values obtained when the action of each single factor was analyzed, suggesting that their inhibitory actions were independent. In support of this, we herein showed that this divalent cation did not interfere with the repressive action of C18:2 on PhoQ autophosphorylation levels. Although we cannot rule out the possibility that fatty acids somehow simultaneously induce PhoQ phosphatase capacity, our results clearly indicate that Mg 2ϩ and C18:2 exert a differential signaling action on the PhoQ sensor protein and that both signals can act in concert to down-regulate the expression of the PhoP-dependent regulon.
The average 50% LCUFA-mediated repression obtained for PhoP-dependent gene transcription levels did not entirely correlate with the strongest inhibition (Ն95%) obtained from the in vitro PhoQ autokinase activity assays. Whereas the in vitro autokinase assay only detects PhoQ autophosphorylation capacity, the level of expression from a given PhoP-regulated gene reflects the global transduction process. Phosphodonors other than PhoQ, such as acetyl-phosphate or other histidine kinases as a result of cross-talk, could be responsible for PhoP phosphorylation levels (50). In contrast, irrespective of the source of phospho-PhoP, when PhoQ phosphatase activity is triggered by high extracellular Mg 2ϩ , 75-85% inhibition can be detected in the expression of PhoP-activated genes, supporting the notion that LCUFAs and Mg 2ϩ differentially target PhoQ catalytic activities.
Exogenously supplied fatty acids can be incorporated and metabolized by Salmonella through the fatty acid ␤-oxidative pathway. We hypothesized that the repressing effect of LCUFAs on the PhoP/PhoQ system could be the result of an increased intracellular concentration of a ␤-oxidation intermediate metabolite, of acetyl-CoA (the ␤-oxidation end product), or of the increased incorporation of unsaturated fatty acids in membrane phospholipids. Therefore, we tested the action of FIGURE 7. The inhibition of the autokinase activity of PhoQ by LCUFAs is independent of fadD. 0.5 mg ml Ϫ1 linoleic acid (18:2) was added to the Salmonella wild-type or fadD strains growth medium. Membranes obtained by cell fractionation, harboring PhoQ, were incubated for 10 min at 37°C in a reaction medium containing [␥-32 P]ATP, as described under "Experimental Procedures." The autophosphorylation reactions were analyzed by SDS-PAGE (12% polyacrylamide) and transferred to nitrocellulose, followed by autoradiography (top) or immunodetection with anti-PhoQ Cyt polyclonal antibodies (middle). The phosphorylation levels and the protein expression levels of PhoQ in each condition assayed were determined by densitometry and the ratio (labeled against immunodetected) was plotted (bottom); the value obtained with the addition of ethanol was taken as 100%. 18:2, linoleic acid; E, ethanol. Data shown represent results from three independent experiments. C18:2 when the function of key components of the Salmonella fatty acid ␤-oxidation pathway (FadL (transmembrane transporter that mediates the lateral passage of fatty acids to the bacterial periplasm (51)), FadD (which couples translocation to import of the LCFA into the cytoplasm by its activation to a CoA thioester), and FadBA (acyl-CoA dehydrogenase complex)) has been abrogated by mutagenesis of their respective encoding genes, fadL, fadD, and fadBA.
First, the possibility of a potential cross-regulatory action of PhoP over the expression of the fad genes assayed was discarded. We here demonstrate that either the presence of the FadL long chain fatty acid membrane translocator or the FadBA downstream dehydrogenase complex is dispensable for LCUFAs to exert their repressive action on the PhoP/PhoQ system. Even when in a fadL strain translocation across the membrane would conceivably be bypassed by passive diffusion or by transbilayer flip-flop of the LCUFAs at slow rates (52), FadD activity is still necessary to convert the transported LCUFA to its activated CoA-derivative, which can be used as a membrane phospholipid acyl donor. In the fadD genetic background, and simultaneously circumventing a possible effect on the autoregulatory control of PhoQ expression, we showed that PhoQ autokinase activity was repressed by the action of C18:2. Therefore, we can rule out that the PhoQ inhibitory action of exogenously supplied LCUFAs is due to a FadD-mediated change in the phospholipid composition of the bacterial envelope membranes that would perturb the catalytic activity of membrane-anchored PhoQ protein. These findings also indicate that the basal down-regulation in the PhoP-regulated gene expression observed in the fadD mutant strain (Fig. 6) is attributable to a reduction in PhoQ autokinase activity caused by accumulation of LCUFAs together with a decrease in PhoQ protein levels due to phoPQ autoregulation combined with diminished PhoQ or stability in the fadD background.
As mentioned before, a fadD mutant strain is unable to channel fatty acids into the ␤-oxidative or the phospholipid synthesis pathways (with the sole exception of the 2-acylglycerophosphoethanolamine acyltransferase/acyl carrier protein synthetase pathway, which can preferentially incorporate saturated fatty acids at very low rates (53,54)). In consequence, when LCFUAS are provided in the bacterial growth medium, it is reasonable to speculate that the interaction of PhoQ with accumulated LCUFAs would be responsible for the induction of a conformational change that switches off the autokinase activity of the sensor protein. Accordingly, it has been shown recently that either E. coli or Sinorhizobium melilotti fadD mutant strains accumulate unsaturated free fatty acids, and it was proposed that fadD is also required for utilization of endogenous free fatty acids that could have been released as a result of membrane lipids remodeling (55). To back this conjecture in our system, we compared the esterified versus the free fatty acid levels of Salmonella wildtype, fadD, and fadL strains when they were grown in the presence of labeled [ 14 C]C18:2 by TLC analysis. As shown in supplemental Fig. S7, free fatty acids represent 33% of the total label incorporated into the bacterial cell in the fadL and 93% in the fadD mutants in comparison with 3% in the wild-type strain. This result supports the notion that free LCUFA accumulation either by a blockage in the vectorial transport to the bacterial cytoplasm or by an exogenous oversupply of the metabolic capacity of the cell would be the active species that act as signals repressing the autokinase activity of PhoQ. Because no obvious recognition motif for free unsaturated fatty acids can be found by in silico predictive analysis of PhoQ amino acid sequence or from its structural features, further work is under way in our laboratory to determine the biochemical basis of the LCUFA-PhoQ interaction.
In this context, it is worth highlighting that in Vibrio cholerae, the causative agent of cholera, an acute intestinal infection, a virulence mechanism involving detection of unsaturated free fatty acids has been described. Chatterjee et al. (56) have shown that unsaturated fatty acids isolated from bile at a concentration of 0.03% are able to inhibit the expression of virulence factors, whereas they enhance V. cholerae motility. Later, the work of Lowden et al. (57) demonstrated that direct binding of unsaturated free fatty acids (including oleic and linoleic acids that repress the transcription of the tcp toxin-co-regulated pilus and of the ctx cholera toxin-encoding genes, at a concentration of 0.02%) to ToxT provokes a conformational alteration of this virulence master regulator by decreasing its ability to bind DNA. More recently, Antunes et al. (58) have found that small hydrophobic molecules present in freshly extracted bovine bile were able to repress PhoP activity, although they could not assess the chemical identity of the compound. In addition, Goldberg et al. (59) have shown that the presence of a polar residue in the second transmembrane helix of PhoQ, which would stabilize a structural water-filled cavity in the sensor protein, is essential for the ability of PhoQ to switch from kinase-to phosphatase-dominant conformations. They showed that the replacement of hydrophilic to hydrophobic residues in transmembrane domains abrogated PhoQ kinase without affecting its phosphatase capacity. It is tempting to speculate that the hydrophobic environment generated by accumulation of LCUFAs could provoke an analogous impact on PhoQ conformation.
Finally, free LCUFAs are present in environmental niches that Salmonella has to traverse along the infection process in the mammalian host. LCUFAs can be found in the digestive tract, forming part of bile components, in the ruminal content and in the mammalian intestine due to degradation of fatty acid-containing nutrients by lipolytic enzymes. Few reports provide accurate information about free LCUFA content in mammalian fluids, and most of them are expressed as percentage relative to total fatty acids (60 -62). However, from these data, it is clear that free LCUFAs levels in these animal contents vary depending on the metabolic, dietary, and health status of the organism. Salmonella can be found colonizing the hepatobiliary tract, and the relevance of the association between the presence of Salmonella in bile or gallstones and the bacterial chronic carriage has been established previously (63)(64)(65)(66). Chaterjee et al. (56) reported that linoleic acid represents 14%, whereas oleic acid represents 23%, of total fatty acids present in ox bile. In bile of cholelithiasis patients, Tsuchiya et al. (67) reported concentrations of individual free LCUFAs in the 0.23-2.0 mM range, which is in agreement with PhoP/PhoQ activity inhibitory values shown in this work.
Together with our results, these previous data allow us to speculate that the presence of LCUFAs, such as linoleic acid, might function as a signal that, in combination with divalent cations, aids Salmonella in distinguishing between extracellular and intracellular environments. In response to these signals, the PhoP/PhoQ system will remain turned off. Conversely, once inside the intravacuolar ambient of the host cell, the absence of repressing cues together with triggering conditions, such as acidic pH and the presence of cationic peptides, will turn on the system. This will allow Salmonella to induce the expression of virulence factors that counteract the defense mechanisms of the infected cell, favoring bacterial survival and dissemination.