Identification of a High-Affinity Pyruvate Receptor in Escherichia coli

Two-component systems are crucial for signal perception and modulation of bacterial behavior. Nevertheless, to date, very few ligands have been identified that directly interact with histidine kinases. The histidine kinase/response regulator system YehU/YehT of Escherichia coli is part of a nutrient-sensing network. Here we demonstrate that this system senses the onset of nutrient limitation in amino acid rich media and responds to extracellular pyruvate. Binding of radiolabeled pyruvate was found for full-length YehU in right-side-out membrane vesicles as well as for a truncated, membrane-integrated variant, confirming that YehU is a high-affinity receptor for extracellular pyruvate. Therefore we propose to rename YehU/YehT as BtsS/BtsR, after “Brenztraubensäure”, the name given to pyruvic acid when it was first synthesized. The function of BtsS/BtsR was also assessed in a clinically relevant uropathogenic E. coli strain. Quantitative transcriptional analysis revealed BtsS/BtsR importance during acute and chronic urinary-tract infections.

. Schematic depiction of the BtsS/BtsR (YehU/YehT) system in Escherichia coli. The scheme summarizes the regulatory network associated with signal transduction by the BtsS/BtsR two-component system, the influence of two-component system YpdA/YpdB and the global regulators CsrA and CRP. Membrane proteins are integrated in the cytoplasmic membrane (CM). Activating (↑) and inhibitory (⊥) effects are indicated. See text for details. metabolism in E. coli during growth in amino acid-rich media 11 , which was confirmed by monitoring the intracellular concentrations of serine and pyruvate (Fig. S2).
These data reveal that induction of BtsS-dependent yjiY expression coincides with the decline of serine in the medium and an extracellular accumulation of pyruvate (Fig. 3).

Extracellular pyruvate triggers yjiY expression under nutrient limitation.
In the next experiment we tested the influence of serine, pyruvate and related metabolites on yjiY expression in E. coli cells growing in low-nutrient environment. Since E. coli harbors a second two-component system, YpdA/YpdB, which responds to high concentrations of extracellular pyruvate (the threshold concentration that leads to induction was determined to be 600 µM) and positively regulates the BtsS/BtsR system 11 , we modified our reporter strain by deleting yhjX, which is sufficient to interrupt the feedback loop 1 . The resulting strain was then cultivated in 10-fold diluted (0.1x) LB medium for 1 h. At this time point, cells do not induce expression of yjiY, but experience soon carbon limitation (Fig. 4A, Fig. S1). However, expression of yjiY was rapidly triggered upon addition of pyruvate, and the induction level increased linearly with increasing pyruvate concentration (Fig. 4A). Addition of L-serine also induced yjiY expression, but only after a 20-min delay (Fig. 4B). Under these conditions the growth of E. coli did not differ significantly by addition of pyruvate or serine (Fig. S3). Only supplementation of 10 mM serine prolonged the exponential growth phase. Moreover, higher serine concentrations delayed yjiY expression for even longer, and decreased the level of induction attained (Fig. 4B). The threshold concentration of pyruvate required for detectable yjiY expression was 10 µM, and that for L-serine 50 µM. None of the other tested compounds (each of the other 19 amino acids, phosphoenolpyruvate, lactate, oxaloacetate, α-ketoglutarate, valeriate, propionate, acetate, malate) were able to induce yjiY in this context. These results suggest that extracellular pyruvate acts as a direct stimulus for BtsS/BtsR-mediated yjiY expression, whereas delayed yjiY induction in response to L-serine may depend on uptake of the amino acid, its conversion to pyruvate and excretion of the pyruvate into the culture medium.
The sensor histidine kinase BtsS binds pyruvate with high affinity. BtsS cannot be autophosphorylated, possibly owing to a defective ATP-binding site within the G1 box 3 (data not shown). Therefore, we used the method of differential radial capillary action of ligand assays (DRaCALA) 12 to determine whether BtsS physically interacts with pyruvate as ligand. The technique is based on the ability of proteins that have been immobilized on a nitrocellulose membrane to bind a radiolabeled ligand, whereas unbound ligands undergo radial diffusion. DRaCALA allows rapid detection of both the total ligand and the ligand sequestered by proteins. The fraction of ligand bound to the protein, defined as F B , is calculated from the signal intensity of the area with protein (inner circle) and the total signal intensity of the area (outer circle) 12 . For the DRaCALA we used unsealed membrane vesicles prepared from E. coli cells overproducing BtsS (MV BtsS) and calculated an F B value of >0.15 (Fig. 5A). Control membrane vesicles (MV, lacking overproduced BtsS) were used and the low value of F B (0.05) reflected only minor, non-specific binding. Therefore this assay was judged to be suitable for membrane vesicles, and it clearly indicated binding of radiolabeled pyruvate to BtsS. We also tested 3 H-serine binding to BtsS in membrane vesicles using the DRaCALA technique. However, we only observed unspecific binding (data not shown). coli. E. coli MG1655/pBBR yjiY-lux was cultivated in LB medium, and growth (OD 600 ) and luminescence were monitored. At the times indicated, cells were harvested, and serine and pyruvate levels were quantified by hydrophilic interaction liquid chromatography. All experiments were performed in triplicate, and the error bars indicate the standard deviation of the means. The growth phases of E. coli are marked as following: lag phase (white), exponential growth (light grey) and stationary phase (dark grey). In addition, we tested MVs harboring a membrane-integrated truncated variant of BtsS (MV BtsS-TM) lacking its soluble domains, and found that this truncated sensor also binds pyruvate (F B > 0.15). Furthermore, we prepared sealed right-side-out vesicles (RSO BtsS), in which membrane proteins retain their native orientation 13 . BtsS in these vesicles also showed pyruvate binding, and no significant change in binding was observed when these vesicles were fragmented by sonication and had a random orientation (sonicated RSO BtsS) (F B values of each 0.18) (Fig. 5A). These data reveal that pyruvate binds to the external side of the membrane-spanning domain of BtsS.
The specificity of pyruvate binding to BtsS was then addressed by using a competition assay (Fig. 5B), in which several competitors were added in excess to the reaction mixture. Only unlabeled ("cold") pyruvate was able to prevent binding of radiolabeled pyruvate. L-serine, D-serine or glycine did not interfere with pyruvate binding to BtsS, suggesting that pyruvate binds specifically to BtsS.
Next we investigated the effects of varying the length and side-chain charge on ligand binding by BtsS. Using competition assays, we found that acetate, propionate and butyrate all failed to compete with pyruvate for binding, indicating the importance of the C-α keto group of pyruvate (Fig. 5C). The influence of polarity at the C-α position was then tested by addition of lactate and malate, which contain C-α hydroxyl groups. These molecules were able to decrease pyruvate binding by about 50% suggesting that a negative charge seems to be recognized. It should be noted that these compounds were tested in a 10 4 -fold excess over pyruvate. Phosphoenolpyruvate, which harbors a phosphoryl group at the C-α position, also competed with pyruvate, reducing binding by approximately 50%. In contrast, a positive charge at the C-α position in the form of the amino group in alanine had no effect, reducing pyruvate binding by 1% (Fig. 5C). Notably, none of these compounds were able to induce yjiY expression in vivo, which emphasizes the specificity of BtsS for pyruvate. The dissociation constant (K d ) for pyruvate binding to the histidine kinase was found to be 58.6 ± 8.8 µM (Fig. 5D).
To our knowledge this is the first application of DRaCALA to measure the interaction between a ligand and a membrane-embedded protein. Moreover, these assays unambiguously demonstrated that pyruvate binds specifically and with high affinity to BtsS.

BtsS/BtsR importance during urinary tract infection. To understand the potential implications of
BtsS/BtsR for pathogenesis, we therefore turned our attention to uropathogenic E. coli (UPEC) that colonize a nutritionally demanding environment -the mammalian bladder. However, responses to nutritional stress are of the utmost importance for the survival of both commensal and pathogenic bacteria within a given host.
UPEC strains are the primary causative agent of urinary-tract infections (UTIs) worldwide, accounting for over 85% of all reported episodes 14 . In the bladder, bacteria adhere to the apical surface of the epithelium and are internalized, before replicating to form biofilm-like pods within host bladder cells 15,16 . Subsequent to this transient intracellular cascade, adverse outcomes such as chronic colonization can ensue as a result of an aberrant host immune response 17 . Previous studies have demonstrated that successful UTI requires aerobic respiration and amino acid utilization [18][19][20] . To investigate the significance of BtsS/BtsR in bladder infection, we first created a btsS/btsR mutant in the UPEC cystitis isolate UTI89. Using the same reporter fusion, we demonstrated that this strain, unlike the wild-type parental UTI strain, failed to induce transient expression of yjiY (Fig. 6A). Both pyruvate and L-serine are present in human and murine urine, and levels are elevated in diabetic populations [21][22][23] . Given that BtsS/BtsR responds directly to extracellular pyruvate levels and indirectly to L-serine, we asked whether BtsS/BtsR is active in the bladder lumen during acute and chronic UTI.
Mice were infected with the cystitis isolate UTI89 and RNA samples were extracted from bladder homogenates at 6 h post infection (coinciding with the acute stage of UTI), and at 2 weeks post infection from mice that went on to develop chronic cystitis. At this stage, the majority of bacteria are found on the bladder epithelial surface in the form of an extracellular biofilm 17 . Subsequent TaqMan-based qPCR analysis compared the expression of yjiY in the corresponding cDNA samples to the expression of yjiY in the starting bacterial inoculum. Our results demonstrated robust expression of yjiY in both the acute and chronic stages of infection from at least three separate mice per time-point (Fig. 6B). Taken together, these data suggest that, in the murine UTI model BtsS/ BtsR responds to serine/pyruvate fluctuations and could play a role in promoting the infection process.

Discussion
Although numerous studies continue to demonstrate the importance of histidine kinase-mediated signal transduction in bacterial physiology, the natural ligands have been identified for very few histidine kinases [24][25][26] . This study presents compelling evidence demonstrating that the histidine kinase BtsS is a high-affinity receptor for extracellular pyruvate. Induction of the BtsS/BtsR target gene yjiY, which codes for a "carbon starvation" CstA-like transporter, correlates with the depletion of serine in complex medium, explaining the observation that BtsS/BtsR senses low serine. At the same time, while serine is being depleted from LB medium, E. coli excretes a substantial amount of pyruvate from overflow metabolism into the medium. Shortly afterwards, the concentration of extracellular pyruvate returns to its basal level (Fig. 3 and ref. 11). In previous studies, we demonstrated activation of a different system, YpdA/YpdB in response to high levels of extracellular pyruvate 11 and showed that induction of YpdA/YpdB enhances BtsS/BtsR activation 1 . The current work provides a comprehensive characterization of how BtsS/BtsR directly responds to extracellular pyruvate levels and fine-tunes the metabolic fitness of E. coli under low-nutrient conditions. Sequence comparison between YpdA and YehU did not identify putative amino acids involved in pyruvate binding. In non-pathogenic E. coli, each of these signaling systems induces the expression of exactly one gene, which codes for a transporter. One (YhjX) belongs to the major facilitator superfamily, and is assumed to be a low-affinity carboxylate antiporter 27 . The other one (YjiY) belongs to the CstA-like transporters, which are characterized by an unusually large number of transmembrane helices (17 in the case of YjiY). Neither transporter has been characterized thus far. It is hypothesized that both are involved in nutrient uptake, but with different affinities. The interconnectivity between the high-affinity pyruvate signaling system BtsS/BtsR with the putative low-affinity pyruvate signaling system YpdA/YpdB by a positive and a negative feedback loop 1 would provide E. coli with a network that could tailor pyruvate uptake in each individual cell according to its availability. Switching between low-and high-affinity phosphate transporters based on the needs of the individual cell was recently demonstrated for S. cerevisiae 28 , and seems to be a widely distributed strategy for the maintenance of nutrient homeostasis as stocks of essential nutrients decline 29 .
Although nutrient sensing is crucial for host-microbe and microbe-microbe interactions, the majority of studies in non-pathogenic E. coli strains have focused on metabolic engineering, aiming to understand how processes such as elevated intracellular pyruvate levels affect metabolite distribution 30,31 or how central mutations trigger/alter metabolic fluxes [32][33][34] . In recent years, technological advances have permitted detailed analyses of in vivo metabolism 35 , revealing its complexity and its influence on virulence and pathogenesis 36 . These studies have shown pyruvate to be one of the major factors connecting cellular metabolism to cell division 37 . Moreover, pyruvate levels are thought to reflect the quantitative relationship between carbon and nitrogen availability in the cell, and affect amino acid biosynthesis 38 .
Investigations of how metabolic decisions determine pathogen fitness within host niches are increasingly uncovering opportunities for the development of robust and pathogen-specific drugs. Different E. coli pathotypes cause various clinical syndromes, depending on their genetic makeup and expression patterns. They obviously differ extensively from each other and from commensal E. coli. For example, uropathogenic and enterohemorragic E. coli differ significantly in genome content, and employ different strategies to infect their niches -the bladder and the gut, respectively. Very recent work has pointed to the need for aerobic respiration and amino acid utilization for optimal colonization of the urinary tract by UPEC [18][19][20] . Pyruvate is a crucial molecule required for fueling the aerobic arm of the tricarboxylic acid cycle, which is used by UPEC during infection 18,19,39 . Thus, it is reasonable to postulate that UPEC has the capacity to sense and respond to pyruvate fluctuations within the urinary tract. Here we demonstrate that BtsS/BtsR mediates yjiY expression during infection by a clinically relevant UPEC isolate, which strongly suggests that BtsS/BtsR constitutes part of the metabolic circuitry engaged during UTI. Thus understanding how UPEC can sense the metabolic signature of the bladder lumen, the kidney and the urothelial cell cytosol (in which they form intracellular bacterial communities) will be vital for a complete understanding of pathogen strategies and the design of more effective therapeutics. However, the study in Salmonella enterica Serovar Typhi and Typhimurium showed no detectable effect on the ability of yehUT mutant strain to invade cultured epithelial cells or induce colitis in a murine model 40 .
In summary, this study has uncovered a signal transduction network that responds with exquisite sensitivity to extracellular pyruvate levels and fine-tunes carbon utilization in E. coli strains. Furthermore we provide direct evidence for receptor-ligand interactions between BtsS and pyruvate, and demonstrate that BtsS/BtsR mediates yjiY expression during UTI. Future studies will focus on understanding the contribution of this system to pathogenic E. coli intra-host fitness and dissecting molecular determinants that drive the fine-tuning of bacterial fitness in response to external metabolic cues.

Methods
Strains, plasmids, and oligonucleotides. In this study we used the E. coli strains MG1655 41 , E. coli MG1655 ΔyhjX 1 , E. coli MG1655 ΔyjiY 1 , BL21(DE3) 42 , E. coli KX1468 43 and the cystitis isolate UTI89 44 . Plasmids used in this study include the transcriptional promoter-luciferase fusion construct for P yjiY activity (pBBR yjiYlux) 3 . For protein production we used the arabinose-inducible expression vectors pBAD24 45 , pBAD24-yehU 3 and pBAD24-yehU-TM (YehU (BtsS) amino acids 1-205). DNA fragments for plasmid construction were amplified from genomic DNA by PCR. Scarless deletion of btsS/btsR genes in UTI89 was performed using the method published by Murphy and Campellone 46 , and verified via PCR with oligonucleotides flanking the deleted loci. All oligonucleotide sequences are available on request.

Molecular biological techniques.
Plasmid and genomic DNAs were isolated using a HiYield plasmid minikit (Suedlaborbedarf) and a DNeasy blood and tissue kit (Qiagen), respectively. DNA fragments were purified from agarose gels using a HiYield PCR cleanup and gel extraction kit (Suedlaborbedarf). Q5 DNA polymerase (New England BioLabs) was used according to the supplier's instructions. Restriction enzymes and other DNA-modifying enzymes were also purchased from New England BioLabs and used according to the manufacturer's directions.
Growth conditions. All strains were grown overnight in LB medium. After inoculation, bacteria were routinely grown in LB, LB supplemented with the indicated amino acids or diluted LB medium [containing 1% (w/v) NaCl] under agitation (200 rpm) at 37 °C. For solid medium, 1.5% (w/v) agar was added. Where appropriate, media were supplemented with antibiotics (ampicillin sodium salt, 100 µg/ml; kanamycin sulfate, 50 µg/ml; gentamicin sulfate, 50 µg/ml). For infection studies in mice, UTI89 was inoculated into 10 ml of LB medium and grown without shaking at 37 °C for 24 h. This culture was used to seed a fresh flask with 10 ml of a 1:1000 dilution, which was incubated for another 24 h to maximize expression of type 1 pili, as previously described 47 . The growth phases of E. coli marked in graphs were according to the definition in ref. 48. In vivo expression studies. In vivo expression of yjiY was quantified by means of luciferase-based reporter-gene assays, using bacteria that had been transformed with plasmid pBBR yjiY-lux. Cells from an overnight culture were transferred to fresh medium to give a starting optical density at 600 nm (OD 600 ) of 0.05, and Scientific RepoRts | 7: 1388 | DOI:10.1038/s41598-017-01410-2 incubated under aerobic growth conditions at 37 °C while OD 600 and luminescence were continuously monitored. The optical density was determined in a microplate reader (Tecan Sunrise) at 600 nm. Luminescence levels were determined in a luminescence reader (Centro LB960; Berthold Technology) for 0.1 s and are reported as relative light units (RLU; counts s −1 ).

Identification of quorum sensing-like molecule. The cells of E. coli MG1655
ΔyjiY/pBBR yjiY-lux were cultivated in M9-minimal medium supplemented with 20 mM gluconic acid and OD 600 and luminescence were constantly monitored. Cells were harvested shortly before, at and after the induction of yjiY. The supernatant was sterile filtrated (Stericup ® , Filter Unit Millipore Express ® PLUS(PES), 0.22 μm and separated by high pressure liquid chromatography (HPLC) using column C18-Hypersil Gold, with the gradient 1-100% (v/v) acetonitrile in 40 min. Fractions were collected, concentrated and used in reporter strain E. coli KX1468 43 /pBBr yjiY-lux grown in M9-minimal medium supplemented with 20 mM succinate. The fraction with the highest induction potential was then analyzed via UPLC-UHR-ToF-MS using a Waters XBridge Amide column.
Production of BtsS-6His in membrane vesicles. E. coli BL21(DE3) cells were transformed with the vector pBAD24-yehU 3 , which codes for BtsS-6His, and grown in LB medium at 37 °C to an OD 600 of 0.5. Recombinant gene expression was induced by addition of 0.2% (w/v) arabinose. After 3 h of further incubation, cells were harvested by centrifugation, disrupted and fractionated. At each step, the pellet was resuspended in buffer consisting of MES/NaOH (pH 6), 10% (v/v) glycerol and 10 mM MgCl 2 . To produce BtsS-6His in right-side-out vesicles, cells were cultivated as described before. After harvesting the cells by centrifugation, lysozyme (50 µg/ml) and EDTA (10 mM) were added for 45 min at room temperature to remove the cell wall. After low-speed centrifugation, the spheroplasts were resuspended in buffer consisting of 100 mM MES/NaOH (pH 6), 30% (w/v) sucrose, 20 mM MgSO 4 , DNase and RNase, and then diluted 100-fold into pre-warmed buffer (100 mM MES/NaOH (pH 6)) to trigger osmotic lysis. After centrifugation, the pellet was resuspended in 100 mM MES/NaOH (pH 6), 10% (v/v) glycerol and 10 mM MgCl 2 . Formation of spheroplasts and right-side-out vesicles was monitored under the microscope. His-tagged BtsS was detected by Western blot analysis with an anti-His antibody (Qiagen) and an alkaline phosphatase-conjugated anti-mouse antibody as the secondary antibody. Extraction and determination of extra-and intracellular metabolites. The reporter strain E. coli MG1655/pBBR yjiY-lux was cultivated in LB medium, and OD 600 and luminescence were constantly monitored. At selected time points, cells were harvested and the supernatants were saved. Cells were washed with 10% (v/v) glycerol, and subsequently cell pellets and supernatants were analyzed via hydrophilic interaction liquid chromatography (HILIC). Acetonitrile (ACN), methanol (MeOH), ammonium acetate and ammonium hydroxide (all of LC-MS grade) were obtained from Sigma-Aldrich (Sigma-Aldrich GmbH). Water was purified using a Merck Millipore Integral water purification system to a resistance of 18 MΩ and TOC < 5 ppb. Sodium pyruvate and serine were also obtained from Sigma-Aldrich and dissolved in water at a concentration of 100 mM and further diluted with ACN. Cell pellets were extracted with 200 µl ice-cold water/MeOH (50/50, v/v). Samples were vortexed vigorously and lysed in an ice-cold sonic bath for 15 min. After centrifugation at 20,000 × g and 4 °C for 10 min, supernatants were transferred to autosampler vials.

Protein-ligand interactions.
Pyruvate and serine were quantified using a modified version of the method published by Yuan et al. 49 . Separation was achieved using a Waters XBridge Amide column (3.5 µm, 100 mm × 4.6 mm ID) and an ACN/ water gradient using a Waters Acquity UPLC system coupled to a Bruker maXis UHR-ToF-MS (Bruker Daltonics). Eluent A consisted of 5% (v/v) ACN, 95% (v/v) water, 20 mM ammonium hydroxide, 20 mM ammonium acetate, pH 9.0 and eluent B was pure ACN.
Metabolites were eluted with the following gradient: After 3 min of 85% B, a linear decrease to 2% B over 9 min, isocratic hold for 3 min and return to initial conditions for 1 min and a 7-min re-equilibration time. Sample (5 µl) was injected via partial loop injection.

Murine infections.
A cohort (15) of 7-to 8-week old female C3H/HeN mice was infected with strain UTI89 via transurethral catheterization as described in Hung et al. 50 . Five mice were sacrificed at 6 h after infection, marking the acute stage of UTI in this murine model 51 . Bladders were excised for CFU enumeration and RNA extraction. The remaining 10 mice were monitored for chronic infection using longitudinal urinalysis, as previously described 52 . Mice that consistently shed more than 100,000 CFUs/ml of urine were separated from the rest of the cohort, and flagged as chronically infected. These mice were sacrificed at 4 weeks post infection and bladders were obtained for CFU enumeration and RNA extraction.
To ensure the proper and humane treatment of animals, all animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the Vanderbilt University Medical Center's Institutional Animal Care and Use Committee (IACUC), who approved all protocols. Statistical analyses were performed using two-tailed Mann-Whitney t-test.