The gastric pathogen Helicobacter pylori requires a noncanonical cytosolic chemoreceptor transducer-like protein D (TlpD) for efficient colonization of the mammalian stomach. Here, we reconstituted a complete chemotransduction signaling complex in vitro with TlpD and the chemotaxis (Che) proteins CheW and CheA, enabling quantitative assays for potential chemotaxis ligands. We found that TlpD is selectively sensitive at micromolar concentrations to bleach (hypochlorous acid, HOCl), a potent antimicrobial produced by neutrophil myeloperoxidase during inflammation. HOCl acts as a chemoattractant by reversibly oxidizing a conserved cysteine within a 3His/1Cys Zn-binding motif in TlpD that inactivates the chemotransduction signaling complex. We found that H. pylori is resistant to killing by millimolar concentrations of HOCl and responds to HOCl in the micromolar range by increasing its smooth-swimming behavior, leading to chemoattraction to HOCl sources. We show related protein domains from Salmonella enterica and Escherichia coli possess similar reactivity toward HOCl. We propose that this family of proteins enables host-associated bacteria to sense sites of tissue inflammation, a strategy that H. pylori uses to aid in colonizing and persisting in inflamed gastric tissue.

Funding: Research reported in this publication was supported by the National Institutes of Health ( https://www.nih.gov ), NIDDK under award numbers R01DK101314 5 (S.J.R., M.R.A and KG.), and F32DK115195 (A.P.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we have taken advantage of the solubility of TlpD to reconstitute the full (TlpD, CheW, CheA) chemotaxis signaling complex in vitro to directly assay equilibrium and kinetic parameters and to test the effects of putative ligands on receptor signaling. We show TlpD is active and promotes CheA autophosphorylation without the addition of any ligand. Our biochemical characterization of TlpD provides no evidence to support direct sensing of zinc, pH, hydrogen peroxide, or superoxide. Instead, we show that in vitro the strong oxidant hypochlorous acid (HOCl, bleach), the major oxidative product generated by neutrophilic inflammation, potently and reversibly inactivates the signaling complex through a universally conserved cysteine in the TlpD CZB to elicit a chemoattractant signaling response, and that reactivity toward HOCl is a conserved feature of CZB domains from other bacteria. We demonstrate a mechanism by which the cysteine forms a redox “Cys-Zn switch” that is tuned to be reactive toward HOCl and much less reactive toward H 2 O 2 and superoxide. Lastly, we perform in vivo assays that show H. pylori tolerates millimolar concentrations of HOCl and uses TlpD for chemoattraction responses to HOCl at physiologically relevant concentrations. We propose this mechanism has evolved to facilitate H. pylori chemoattraction to sites of inflammation and persistence in neutrophil-rich gastric glands.

A clear consensus on the molecular function of TlpD has remained elusive. Single receptor knockout strains of H. pylori show defects in colonization of the stomach antrum, a preferred niche for the bacterium and the region of highest inflammation, and of these, tlpD strains are by far the most impaired [ 10 ]. The first molecular study of TlpD revealed the protein to possess a novel chemoreceptor zinc-binding (CZB) domain that is prevalent among chemoreceptors and diguanylate cyclases of numerous bacteria [ 25 ]. The domain uses a rare 3His/1Cys motif to coordinate the bound zinc with high affinity, and the zinc cannot be removed even by the strong zinc chelator N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) [ 25 ]. Crystal structures of a wild-type and Cys→Ala mutant CZB domain were solved for diguanylate cyclase Z (DgcZ) (previously called YdeH) from Escherichia coli. Those authors suggested CZBs function as zinc sensors and showed for DgcZ that the zinc could be chelated out only by millimolar concentrations of ethylenediaminetetraacetic acid (EDTA) [ 18 ]. Additionally, studies using chemotaxis assays to monitor H. pylori swimming patterns have implicated TlpD as a redox sensor, suggesting it responds to extracellular sources of ROS. However, there are discrepancies in the literature regarding this point. Using the rate of swimming reversals as a metric for chemotaxis, recent work reported a TlpD-dependent increase in reversals from paraquat-generated superoxide [ 26 ]. A different study observed a decrease in reversals with superoxide [ 27 ]. Additionally, millimolar concentrations of hydrogen peroxide (H 2 O 2 ) were reported to increase reversal rates [ 26 ], whereas in vivo concentrations of H 2 O 2 are thought to rarely exceed the low micromolar range [ 28 , 29 ]. TlpD sensing of ROS was also suggested to function as an “energy sensor” by responding to cytosolic oxidants produced through metabolism [ 27 , 30 ]. However, a molecular mechanism has yet to be proposed for how TlpD may function as a sensor.

Interestingly, previous work has shown that the cytosolic chemoreceptor TlpD is most highly expressed in H. pylori, constituting about half of the total chemoreceptor pool [ 20 ] ( Fig 1A ). Such cytosolic or “soluble” chemoreceptors are widespread and common, accounting for approximately 15% of all bacterial and 45% of archaeal chemoreceptors, but their functions are mostly unknown, and to date only a few cytosolic chemoreceptors have been mapped to their ligands [ 23 ]. Like membrane-bound homologues, cytosolic chemoreceptors form nanoarrays [ 24 ] and therefore represent an intriguing, fully soluble model system to better understand chemoreceptor function. No study has yet described the assembly of cytosolic signaling units in terms of the dissociation constants or reaction kinetics for a cytosolic receptor with CheW and CheA.

H. pylori chemotaxis is mediated by four chemoreceptors, transducer-like proteins (Tlps) A-D ( Fig 1A ). Environmental chemicals that elicit a chemoresponse by H. pylori include acid as a chemorepellent [ 9 , 20 ] and urea as a chemoattractant [ 21 ], which both serve to direct H. pylori from the stomach lumen to the gastric epithelium. Tlp deletion mutants have implicated certain chemoreceptors in cellular sensing processes, but only two chemoeffectors have been confirmed in biochemical experiments to act through direct binding to a receptor: TlpB sensing of urea [ 20 , 21 ] and TlpC sensing of lactate [ 22 ], though the latter receptor is not conserved and apparently dispensable. Some progress has been made for understanding direct ligand sensing by TlpA, for which recent crystal structures suggest binding of a small hydrophobic ligand [ 20 ]. Sensing of pH remains more complicated to understand, because genetic knockouts indicate signals are integrated from TlpA, B, and D [ 9 , 20 ], and no direct sensing mechanism is known for TlpD.

(A) H. pylori inner-membrane–bound (yellow arc) chemoreceptors TlpA-C and cytosolic TlpD control the autophosphorylation of CheA to CheA-Pi, which then transfers the phosphate to CheY, and CheY-Pi can interact directly with the flagella rotor (pink) to cause a temporary reversal in flagellar rotation. Flagella reversals cause direction changes in swimming trajectory (dotted lines). Black arrows show signal transduction for a chemorepellent response. (B) Two sets of chemoreceptor trimers of homodimers (light and dark blue, denoted as Tlp) associate with the scaffold protein CheW (gray) and a CheA dimer (light and dark orange) to form the core signaling unit and modulate CheA autophosphorylation (illustrative model based on PDB code: 3ja6)[ 16 ]. (C) A relatedness tree of TlpD protein sequences from Helicobacter (blue), Deferribacteraceae (purple), and other species. H. pylori TlpD (arrow) is nearly identical to species found in dolphins and cheetas (red dot). (D) A theoretical model of a HpTlpD monomer constructed by i-Tasser with an N-terminal region of unknown structure (light green), the canonical coiled-coil domain of chemoreceptors that interfaces with CheW and CheA (light blue), and a CZB domain (dark blue). Sequence conservation among 459 TlpD homologues is mapped onto the model with 100% conservation highlighted in red and >95% conservation in orange. Cys residues present in the SS1 strain of HpTlpD are noted with green circles. See Zähringer and colleagues [ 18 ] for a theoretical model of the TlpD dimer. (E) Conservation of each Cys residue in TlpD is shown with 2 Weblogo plots [ 19 ]; the left shows conservation among all sequences of HpTlpD (394 sequences, >92% sequence coverage, >98% sequence identity), and the right plot is conservation among nonpylori TlpD sequences (68 sequences, >91% coverage, >40% sequence identity). Only C340 is universally conserved among all TlpD homologues (noted with * in panels E and D). CheA, chemotaxis protein A; CheA-Pi, phosphorylated CheA; CheW, chemotaxis protein W; CheY, chemotaxis protein Y; CheY-Pi, phosphorylated CheY; CZB, chemoreceptor zinc-binding; HpTlpD, Helicobacter pylori TlpD; PDB, Protein Data Bank; TlpA-C, transducer-like proteins A-C; TlpD, transducer-like protein D.

H. pylori utilizes chemotaxis to seek sites optimal for growth and colonization within the hostile environment of the stomach [ 9 – 11 ]( Fig 1A ). Bacterial chemotaxis involves a well-studied phospho-relay system prevalent across bacteria and archaea, and functions through a conserved mechanism [ 12 ]. Chemoreceptor proteins typically possess a periplasmic ligand-sensing domain to recognize small molecules and transduce signals across the inner membrane to a cytosolic coiled-coil region to regulate ATP-dependent autophosphorylation of the histidine kinase CheA [ 13 ] ( Fig 1A ). Typically, as the cellular pool of phosphorylated CheA (CheA-Pi) is increased, the flagella rotors frequently reverse and alter the swimming bacterium’s trajectory, leading to chemorepulsion; decreases in CheA-Pi result in smooth swimming and chemoattraction ( Fig 1A ). Chemoreceptors in H. pylori and other bacteria oligomerize to form trimers-of-receptor dimers ( Fig 1B ) to build repeating hexagonal arrays that serve to amplify ligand-induced signals up to 50-fold, whereby a single activated receptor can initiate the signal transduction cascade [ 13 – 15 ]. The minimal core unit for signaling is thought to contain two trimers-of-receptor dimers, a CheA dimer, and two CheW scaffold proteins [ 14 , 16 , 17 ] ( Fig 1B ).

Helicobacter pylori is a bacterial pathogen and persistent colonizer of the human stomach and can cause gastritis, ulcers, and stomach cancer [ 1 ]. The health burden caused by H. pylori is particularly large because it infects about half the world’s population, with nearly 100% infection rates in some developing regions, and drug resistance to first-line antibiotics is increasing [ 1 , 2 ]. Despite triggering a robust inflammation response and bursts of reactive oxygen species (ROS) from immune cells, H. pylori avoids clearance and persists for many decades [ 3 ]. The chronic inflammation induced by H. pylori infection is thought to be a major factor in causing disease [ 4 ]. Not only is H. pylori not eradicated by inflammation, the pathogen, in fact, has been shown to navigate to sites of injury and may capitalize on tissue damage by using host iron extracted from blood hemoglobin and transferrin [ 5 – 8 ].

Results

TlpD homologues possess a universally conserved Zn-binding cysteine To identify conserved regions important to TlpD function, we performed a protein sequence BLAST search to retrieve putative homologues [31]. This search revealed that TlpD is almost exclusively found in the family Helicobacteraceae, with most homologues being from mammal-associated strains and a few TlpDs from reptile-associated strains (Fig 1C). As expected, TlpD sequences from H. pylori were highly similar, with >98% sequence identity across 394 isoforms in the nonredundant sequence database. To better understand sequence conservation on a structural level, the i-Tasser program [32] was used to generate a homology model of the receptor monomer, because no experimental structure for TlpD is available (Fig 1D). This model predicts three general domains: an N-terminal region of low-sequence conservation and unknown function, the canonical chemoreceptor coiled-coil that interacts with CheW and CheA, and a C-terminal CZB domain. Mapping positions of high conservation onto the model reveals that the CheW/CheA interface (common to all chemoreceptors) is highly conserved, as are positions in the CZB domain near the Zn-binding residues (Fig 1D). H. pylori TlpD is unusual for a chemoreceptor, because it contains an abundance of Cys residues (4–5 depending on the strain): C35, C103, C117, C308, and C340 (Fig 1D). Four of the Cys positions are conserved among H. pylori TlpDs, but only C340 within the CZB domain is conserved across all homologues (Fig 1E).

Reconstituted chemoreceptor signaling complex retains function in vitro Recombinant H. pylori TlpD, the scaffold protein CheW, and the histidine kinase CheA were expressed and purified for use in functional assays to directly test the effects of mutations and potential ligands using radio-ATP labeling to monitor autophosphorylation of CheA (see Method details). In this assay, activation of CheA increases CheA autophosphorylation which promotes swimming reversals and chemorepulsion; decreasing CheA activity increases smooth swimming and chemoattraction (Fig 1A). Autophosphorylation kinetics of HpCheA alone revealed a K M for ATP of 136 μM, similar to the approximately 300 μM K M reported for E. coli CheA; however, the basal level of k cat was exceptionally slow at 8.25 × 10−3 min−1 (compared with 1.56 min−1 for E. coli)[33](Fig 2A). We next determined the concentration of TlpD required to achieve maximal dimerization, because the receptor dimer is the core building block for the chemotaxis signaling complex [17]. The dimer K D of our TlpD recombinant construct was found by analytical ultracentrifugation to be 188 nM, consistent with fluorescence anisotropy experiments that showed that at approximately 16 μM and above TlpD is mostly dimerized (Fig 2B and S1 Fig). Based on these results, assays with TlpD were generally run at concentrations of 20 μM or higher so that the properties of the physiologically relevant dimer were observed rather than the inactive monomer. Addition of stoichiometric concentrations of TlpD, CheW, and CheA resulted in synergistic activation of the chemotaxis complex; CheW was able to partially activate CheA alone, and activation was further increased with the addition of TlpD (Fig 2C). These results confirmed that the recombinant system was functional and activated CheA in a manner similar to other described chemotaxis systems [34]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. In vitro reconstitution of the TlpD, CheW, and CheA chemotaxis signaling complex. (A) Kinetics of HpCheA with varying concentrations of ATP are shown. Experiments were conducted in triplicate with 4 μM CheA and varying amounts of ATP in 50 mM Tris (pH 7.5), 100 mM NaCl, and 10 mM MgCl 2 . The average k obs for three replicate time courses are shown at various concentrations of ATP (black dots), error bars are the sample standard deviation, and these measurements are fit to the Michaelis-Menten curve (black line). (B) Representative analytical ultracentrifugation data (black axes) are shown for TlpD at 1 μM (black dotted black line) in PBS buffer (pH 7) with 1 mM TCEP. At this concentration, peaks corresponding to the TlpD monomer and dimer occur near 3.5 [S] and 5.2 [S], respectively. The average dimer K D for the recombinant TlpD construct calculated across various protein concentrations was found to be 188 nM (S1 Table). Shown in red on secondary axes are fluorescence anisotropy data for a titration of TlpD under identical conditions with experiments run in triplicate. See also S1 Fig. for a comparison of the TlpD dimer K D with the N-terminal His tag present or cleaved off and simulated data showing the expected monomer and dimer populations expected based on measured K D values. (C) Shown on top are representative raw data from radio-ATP labeling experiments of 15-minute reactions with CheA alone (“A”) and additions of CheW (“AW”) and TlpD (“AWD”). Below are reactions of 1 mM ATP and 4 μM CheA (black circles), +8 μM CheW (blue triangles), and +8 μM CheW; 24 μM TlpD (red squares) run in triplicate and fit to a pseudo–first-order reaction curve (solid lines). (D) CheW was titrated against 4 μM CheA and resulting k obs measurements were fit to a binding isotherm to estimate a kinetically-defined K D of 14.6 μM for the CheA↔CheW interaction. (E) TlpD was titrated against 4 μM CheA and 40 μM CheW and fit to a binding isotherm as in panel C to approximate the thermodynamics of the CheA, CheW↔TlpD interaction to have a K D of 15.2 μM. (F) A titration of TlpD against 4 μM CheA shows no activation (gray squares). For CheA in the presence of saturating [CheW], a 2.7-fold activation occurs (blue line), and with saturating [CheW] and [TlpD], this is increased to a 14.6-fold activation (red line) over CheA alone (black line). See S1 Table for a summary of reaction parameters and statistics. CheA, chemotaxis protein A; CheA-Pi, phosphorylated CheA; CheW, chemotaxis protein W; HpCheA, Helicobacter pylori CheA; S, Sedverg; TCEP, tris(2-carboxyethyl)phosphine; TlpD, transducer-like protein D. https://doi.org/10.1371/journal.pbio.3000395.g002 We next leveraged this system to estimate kinetically defined dissociation constants for the components and to determine the required order of complex assembly using radio-ATP labeling. Titration of CheW against CheA revealed an estimated K D of 14.6 μM for formation of the CheW-CheA complex, similar to that of E. coli at 17 μM [35], and maximal activation of 2.7-fold (Fig 2D). Titration of TlpD against CheW-CheA showed an estimated K D of 15.2 μM and maximal 14.6-fold activation of the complex (Fig 2E). Previous work has shown for E. coli that CheW is required for the receptor to activate CheA [36], but a recent study suggested TlpD could activate CheA independently [37]. However, that study used nonstoichiometric ratios for the chemotaxis components and only 2 μM TlpD; based on our measurements of the TlpD dimer K D , approximately 12% to 20% of the receptor could be in its inactive monomer form at this concentration (S1 Fig). Direct activation of CheA by TlpD was tested with a titration of TlpD against CheA without CheW, but in our hands no activation occurred even at 200 μM receptor (Fig 2F). Instead, the data support a sequential activation of the complex whereby CheA and CheW first associate to achieve 18% activation, and then TlpD can bind and promote full 100% activation of the chemotaxis complex (Fig 2F). To our knowledge, this is the first characterization of dissociation constants and autophosphorylation rates of a cytosolic chemotaxis complex and indicates in this case that the chemoreceptor TlpD is “on” by default and able to activate CheA even without the addition of other ligands. Kinetic parameters and dissociation constants are summarized in S1 Table.

TlpD is not directly sensitive to exogenous Zn++, pH, hydrogen peroxide, or superoxide Two lines of evidence led us to hypothesize that the zinc atom bound to the CZB domain might be a cofactor in ligand sensing. First, due to its resistance to zinc chelation, TlpD was previously estimated to bind zinc with subfemtomolar affinity [38], which seems inconsistent with a sensor that is activated by exogenous zinc, considering that ligand affinity in other bacterial chemoreceptors is typically in the micromolar range [12,20,39]. Inductively coupled plasma (ICP) mass spectrometry analysis of recombinantly-grown TlpD previously showed TlpD to be zinc-loaded following purification without addition of zinc [25]. We verified that addition of 6 to 24 μM zinc (0.25× to 1× concentration relative to TlpD) to the functional assay did not substantially impact autophosphorylation rates, and higher concentrations, far beyond those considered to be physiologically relevant, caused the proteins to precipitate (S2A Fig). To further test the feasibility of a reversible zinc-sensing function, we attempted to remove zinc from TlpD by chelation with the fluorescent probe Zinpyr-1, which binds zinc with nanomolar affinity, but no chelation was observed, in support of earlier work by Draper and colleagues [38] (S2B Fig and S2C Fig). We also heat-denatured TlpD, and only a submolar equivalent of zinc was released, even after 3 hours of incubation with Zinpyr-1 (S2C Fig). Circular dichroism (CD) of the protein solution indicates that the lack of chelation is not due to the protein refolding (S2D Fig), suggesting that even when the polypeptide is denatured, zinc is sufficiently buried to prevent removal. Given the absence of data demonstrating the reversibility of zinc binding or impact on complex signaling activity, TlpD may not directly detect exogenous Zn++ as a chemotaxis ligand. The crystal structure of the E. coli CZB, in which the cysteine residue equivalent to the conserved TlpD C340 was mutated to an alanine (Protein Data Bank [PDB] code: 4h54), provides a second line of evidence in favor of Zn as a cofactor in ligand sensing. In this structure, the zinc remained bound by the 3-His core in the absence of the Cys S γ [18], hinting that in the wild-type protein the Cys side chain might be released from the zinc in response to some molecular cue. The importance of C340 in promoting CheA autophosphorylation was demonstrated using our functional assay; compared with wild-type TlpD, the C340A mutant caused a 6.8-fold loss in CheA activation, suggesting that the Cys S γ is required for full activation by TlpD (Fig 3A). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Chemotaxis signaling responses to ROS. (A) Reaction time courses with reconstituted signaling complex are shown using either wild-type TlpD (CheW and CheA present, red squares) or C340A (CheW and CheA present, violet diamonds), with single time courses for CheA alone (black circles) and CheA and CheW (blue triangles) for reference. Data were run in triplicate and are fit with pseudo–first-order curves (solid lines). (B) Shown are functional assay time courses following 1-H pretreatments at pH 7 with either PBS buffer (red), 500 μM H 2 O 2 (pink), TCEP (dark blue), or hypochlorous acid (HOCl, orange), using TlpD, CheW, and CheA (n = 3). Chemoeffectors were diluted in PBS buffer (pH 7). (C) Functional assays are shown with 250 μM HOCl either added directly to the reconstituted complex (CheW and CheA present, light orange boxes), using preoxidized TlpD (CheW and CheA present, dark orange boxes), or HOCl treatment of CheA and CheW alone with no TlpD added (light green triangles), relative to the untreated reconstituted signaling complex (TlpD, CheW, and CheA, red boxes; n = 3). Samples were treated with HOCl or buffer as indicated for 30 minutes and subsequently quenched with 1 mM methionine prior to addition of other reaction components. (D) A series of functional assays with reconstituted signaling complex titrating with HOCl is shown with k obs values from reaction time courses plotted against [HOCl] on a log scale (TlpD, CheW, and CheA, orange boxes). Fitting these data to a binding isotherm with a Hill coefficient of 2 yields a K 1/2 for inhibition of TlpD-activation of CheA activity by HOCl of 110 μM (orange line). An identical titration was performed using the C340A mutant (violet diamonds and line). Recovery of inactivated complex with wild-type TlpD by reduction with TCEP is shown in dark blue and plotted on a secondary axis. Complex was pretreated for 1-H with 125 μM HOCl and then treated with varying [TCEP] for 30 minutes. See also S2 Fig. for additional analyses of responsiveness to zinc, pH, and paraquat. AW, complex formed by CheA and CheW; AWD, complex formed by CheA, CheW, and TlpD; CheA, chemotaxis protein A; CheA-Pi, phosphorylated protein CheA; CheW, chemotaxis protein W; K 1/2 , the concentration at which 50% of the kinase was inactivated; ROS, reactive oxygen species; TCEP, tris(2-carboxyethyl)phosphine; TlpD, transducer-like protein D. https://doi.org/10.1371/journal.pbio.3000395.g003 We also explored a potential role of the zinc-binding core of TlpD in direct pH sensing, because protonation of C340 could cause it to dissociate from the bound Zn. In vivo data show TlpD participates in both an acid chemorepulsion and chemoattraction to basic pH [9], and if this mechanism involved direct sensing by TlpD, we would expect this to be reflected in our in vitro functional assay as decreasing CheA activation as a function of increasing pH. However, functional assays across different pH did not replicate in vivo responses (see Method details and S2E Fig). Activity was similar across pH 6.6 to 7.8, the normal range that TlpD would be expected to experience within the well-buffered cytosol [40]. Together, these data led us to conclude that, although TlpD is important for acid sensing in vivo, the receptor apparently does not directly sense pH. Finally, we tested if C340 might directly sense the oxidants tested previously by Collins and colleagues and Behrens and colleagues [26,27]. Functional assays were performed with 500 μM H 2 O 2 or 500 μM of the reductant TCEP but no change in activity was observed (Fig 3B). Addition of paraquat at concentrations used in previous H. pylori chemotaxis assays [27] also did not alter activation of the complex, suggesting TlpD does not directly sense superoxide (S2F Fig).