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].

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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 MgCl₂. 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.

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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).

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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 H₂O₂ (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.

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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 H₂O₂ 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).

TlpD directly senses hypochlorous acid through oxidation of a conserved cysteine

Though previous work had tested TlpD-dependent sensing of the ROS hydrogen peroxide and superoxide [26,27], and H. pylori is well known to inhabit and persist within inflamed tissue [1], no study had yet analyzed whether TlpD might respond to the inflammation oxidant HOCl. Addition of 500 μM HOCl to reconstituted TlpD-CheW-CheA complex dramatically decreases CheA autophosphorylation to the rates observed for CheW-CheA alone (Fig 3B). Functional assays using preoxidized TlpD show a similar decrease in CheA autophosphorylation as HOCl-treated complex, whereas HOCl treatment of CheW-CheA alone does not impair activity relative to untreated CheW-CheA, suggesting the source of inhibition of signaling complex activity is due to oxidation of TlpD (Fig 3C). A titration with HOCl revealed the concentration at which 50% of the kinase was inactivated, which we refer to as K_1/2 [41], to be approximately 110 μM (Fig 3D). A similar titration using the C340A mutant exhibited largely diminished activity even without the addition of HOCl, indicating the mutation may effectively mimic the “off” state of the receptor (Fig 3D). In these assays, no protein precipitation was observed, but because HOCl is known to oxidatively damage proteins and cause aggregation [42], HOCl-oxidized samples were tested for resurrection by the reductant TCEP, which was shown to effectively reactivate the complex (Fig 3D).

These data suggest the mechanism of HOCl sensing involves oxidation of TlpD C340 to a cysteine sulfenate, an oxidation product commonly formed in redox systems that utilize a redox-active cysteine and that is readily reversible by small molecule and protein reductants such as the thioredoxin/thioredoxin reductase system present in H. pylori [43,44]. Inspection of the E. coli CZB structure reveals a small channel that allows solvent to access the cysteine, which has not been previously reported (Fig 4A and 4B). The C340 S_γ is partially solvent exposed at the base of this cavity, reminiscent of an enzyme active site suitable for binding a small ligand (Fig 4B). In this site, two water molecules identified in the crystal structure could mimic the approximate positions for an oxygen and chloride of a hypochlorite molecule and are coordinated by the backbone NH of the following helical turn to be well positioned to react with the C340-S_ɣ (Fig 4B and Fig 4C). To determine if C340 can react with HOCl and become oxidized to a cysteine sulfenate, we treated wild-type TlpD and C340A mutant protein with HOCl and monitored formation of cysteine sulfenate by dimedone adduction and western blotting [45]. The results show potent formation of cysteine sulfenate for the wild-type protein but almost none for the C340A mutant (Fig 4D, S3A Fig). This indicates not only that HOCl can access and directly oxidize C340 in solution but also that C340 is tuned to be highly reactive with HOCl, as it is approximately 50-fold more readily oxidized than the four other TlpD Cys residues combined (Fig 4D, S3A Fig). Titration data exhibit a sigmoidal response and are fit well with a Hill coefficient of n = 2, suggesting positive cooperativity occurs across the receptor homodimer (Fig 4D and S3A Fig). As a secondary test of direct oxidation of C340 by HOCl, we performed mass spectrometry of reduced, H₂O₂-treated, and HOCl-treated TlpD to look for the formation of oxygen adducts. The spectra indicated little oxidation of C340 occurred for the reduced and peroxide-treated samples, but spectra of HOCl-treated TlpD showed C340 to be completely oxidized to sulfinate and sulfonate forms (Fig 4E and S3C–S3E Fig). Similar “overoxidation” in the absence of reductant in in vitro assays is seen to occur for other proteins with strongly redox-active Cys residues [46,47], but bacteria generally lack the capacity to reduce sulfinate and sulfonate forms [48], so we expect that cysteine sulfenate is the oxidation state of physiological relevance. Lastly, we tested two other representative CZBs, the CZB domain of the E. coli diguanylate cyclase DgcZ and the Salmonella enterica chemoreceptor McpA. We found them to be similarly reactive toward HOCl as TlpD, suggesting HOCl sensing is a conserved function of CZB domains (S4 Fig.).

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Fig 4. Structural and biochemical analyses of TlpD C340 oxidation.

(A) The crystal structure of the E. coli CZB domain is shown, with the conserved residues of the Zn binding core and nearby W345 highlighted in gold (PDB code: 3t9o, numbering is for corresponding residues in HpTlpD based on sequence alignment). (B) The molecular surface in the same view as in panel A (sulfur is yellow; nitrogen is blue; oxygen is red; carbons are white; zinc is green) showing C340 is accessible to solvent. Three waters in the experimental structure are shown as small red spheres. (C) A close-up view of the CZB crystal structure is shown in which 2 water molecules bind in the pocket near C340, mimicking the predicted position of hypochlorous acid (O and Cl labeled and shown as transparent sticks). 2fo-fc electron density is shown as blue mesh, contoured at 1.5 σ. (D) Oxidation of 20 μM WT TlpD (open circles) or C340A mutant (crosses) to form Cys-SOH is shown at various HOCl treatments and monitored using an anticysteine-sulfenate antibody. Experiments were run in triplicate, and representative raw data are shown in the inset, with the image length reduced by 50%. Fits of the WT data to the Hill equation using either a coefficient of 1 (gray line) or 2 (black line) are shown. S3 Fig. Panel A shows these data plotted on a log scale. See S4 Fig. for similar experiments with the CZB domain of E. coli DgcZ and Salmonella McpA. (E) Data from MS/MS experiments show oxidation states of C340 following treatments of TlpD with 1 mM TCEP, H₂O₂, or HOCl. The 14-mer peptide containing C340 (noted in bold) was identified and found to be modified either by cysteine alkylation (gray, SH, indicating no oxidation), oxidation to a cysteine sulfinate (peach, SO₂⁻), or oxidation to a cysteine sulfonate (dark red, SO₃⁻). See also S3C–S3E Fig. and Method details. (F) Shown is an overlay of the WT E. coli CZB homodimer (dark blue, PDB code: 3t9o) and Cys→Ala mutant (light blue, PDB code: 4h54) crystal structures. Without the Cys-Zn bond, the region containing C340 and W345 (orange) locally unfolds and becomes disordered and solvent exposed. This structural rearrangement also promotes an approximately 3 Å shift in its dimer partner (wild-type dark gray, Cys→Ala light gray) in the helix adjoining the coiled-coil that may propagate to the CheW/CheA interface to deactivate the signaling complex. (G) Intrinsic protein fluorescence data (excited at 295 nm) for a titration of HOCl against 20 μM TlpD is shown. Treatments were for 30 minutes with 20 μM TlpD in PBS (pH 7) with addition of 0 μM (black), 100 μM (dark blue), 250 μM (light blue), 500 μM (teal), 750 μM (green), 1 mM (lime green), 1.1 mM (yellow), 1.5 mM (light orange), 2 mM (orange), 5 mM (red), or 10 mM (dark red) HOCl. (H) Data from panel F is quantified with the normalized RFU at 340 nm at each [HOCl] concentration fit to the Hill equation with a coefficient of 1 (gray) or 2 (black). Representative equivalent experiments performed with C340A are shown as crosses. (I) Time courses comparing fluorescence quenching at 340 nm (excited at 295 nm) of 20 μM TlpD by peroxide (2 thick dashed lines) and HOCl (5 thin solid lines) at concentrations of 0 μM, 100 μM, 250 μM, 500 μM, 1 mM, 2 mM, and 5 mM, colored as in panel C. (J) Fluorescence experiments are shown of oxidized TlpD treated with TCEP (excited at 295 nm); 20 μM TlpD samples were preoxidized (solid lines) with HOCl at concentrations of 500 μM (blue) and 1 mM (lime) for 30 minutes prior to reduction by equimolar concentrations of TCEP for 30 minutes (dashed lines), resulting in recovery of fluorescence intensity (upward-pointing arrows). CheA, chemotaxis protein A; CheW, chemotaxis protein W; Cys-SOH, cysteine sulfenic acid; CZB, chemoreceptor zinc-binding; DgcZ, diguanylate cyclase Z; HpTlpD, Helicobacter pylori transducer-like protein D; PDB, Protein Data Bank; RFU, relative fluorescence units; TCEP, Tris(2-carboxyethyl)phosphine; TlpD, transducer-like protein D; WT, wild type.

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Comparison of the wild type and Cys→Ala CZB crystal structures shows that in the mutant a region containing 22 residues, including the position of the conserved cysteine, locally unfolds and becomes disordered (Fig 4F). We speculate that oxidation of C340 would promote a similar conformational change, leading to signal transduction by shifting the upstream coiled-coil region (Fig 4F). Fortuitously, TlpD contains a single conserved Trp residue (W345) located near C340 that should participate in this conformation change (Fig 4A and Fig 4F). Therefore, fluorescence of W345 was used as a native probe to detect the receptor’s conformation change in response to C340 oxidation. Strong Trp fluorescence is observed in untreated TlpD samples but is effectively quenched by HOCl concentrations in the μM to mM range, supporting that oxidation induces a conformation change like that observed in the crystal structures (Fig 4G). Similar to the apparent cooperativity observed for oxidation of C340 (Fig 4D), the conformation change exhibits a sigmoidal response (Fig 4H). The C340A mutant is about 5-fold less sensitive to HOCl treatment than wild type, showing this conformation change is dependent on the presence of C340 (Fig 4H). To ensure that loss of fluorescence was not due to protein denaturing, a similar titration was monitored by CD, which shows TlpD remains folded up to at least 10 mM HOCl and partially folded even at 100 mM HOCl (S3B Fig). Trp fluorescence quenching time courses revealed HOCl reacts to completion within the sample mixing time (<60 seconds), whereas quenching by hydrogen peroxide requires 6- to 7-fold higher concentrations and is very slow, with the reaction requiring approximately 20 minutes to reach completion (Fig 4I). Quenched fluorescence of oxidized TlpD was recovered by reduction with TCEP (Fig 4J). Together, these data support a model in which HOCl oxidation of TlpD C340 stimulates a conformational change in the receptor, although further analysis will be required to understand how these structural changes may regulate the distant CheA interface.

Hypochlorous acid decreases H. pylori swimming reversals

HOCl can reach concentrations as high as 5 mM at sites of inflammation [49], and so prior to investigating H. pylori sensing of HOCl in vivo, we first determined the range of HOCl concentrations H. pylori can survive and remain motile. We used a video tracking assay to measure swimming velocities in response to acute treatments of HOCl in the absence of chemotaxis for an H. pylori cheA mutant (strain G27), which is motile but cannot respond to chemotaxis signals, and a motile E. coli lacking all chemoreceptors (strain UU1250; Fig 5A). We found that H. pylori is extremely resilient against HOCl treatment, with full loss of motility observed only for treatments exceeding approximately 7 mM, whereas E. coli lost motility in treatments exceeding 2 mM HOCl (Fig 5A).

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Fig 5. H. pylori swimming behavior in response to HOCl treatment.

(A) Shown is the mean swimming velocity following treatment with various concentrations of HOCl using the chemotaxis-null E. coli strain UU1250 (triangles, gray line) and H. pylori cheA (circles, black lines). The concentration of HOCl at which no motile bacteria were detected is noted with “n.d.” Error bars shown are the sample standard deviation. Experiments were performed in triplicate, with n > 30 bacteria tracks (see Method details). (B) Shown are representative western blots of WT and tlpD H. pylori cell lysate from cells treated with either PBS buffer (pH 7) or 1 mM HOCl in PBS buffer (pH 7). Cells from liquid cultures were treated for 30 seconds in the presence of dimedone, then quenched by methionine to inactivate residual HOCl. Blots of cell lysate were probed with α-Tlp primary antibody (pink), stripped and re-probed with α-cysteine-sulfenate primary antibody (green), and visualized with chemiluminescence. Bands appearing in the α-Tlp blot corresponding to the molecular weights of TlpA, TlpB, and TlpD monomer and disulfide-linked dimer are noted with red triangles. Five prominent bands in the α-cysteine-sulfenate blot of WT H. pylori are numbered, with bands 1 and 3 noted with circles that appear at the same molecular weight as the TlpD monomer and dimer bands. Bands 1 and 3 are absent in the tlpD mutant. Images shown are false-colored and overlaid and merged with ImageJ. Data shown in this panel are from lanes 4 and 5 of the full blots; see S5 Fig and S6 Fig for full blots with 4 to 5 independent experiments for each treatment and Method details. (C) Representative swimming tracks of WT H. pylori are shown following the addition of PBS buffer (pH 7), 1 mM urea in PBS buffer (pH 7), or 1 mM HOCl in PBS buffer (pH 7). Tracks are colored according to reversal rate on a spectrum of green (low) to red (high). In these figures, 1 pixel corresponds to 0.65 microns. Note that these images are intended to visualize the swimming behavior of the bacterial swarm but that individual reversals cannot be seen at this scale. (D–F) Reversal rates for WT and mutant H. pylori are shown for various treatments. Experiments were conducted in triplicate, and each dot corresponds to a single swimming bacteria track captured during a 30 second video, with the mean reversal rate for each treatment indicated with a dark black horizontal line. The baseline reversal rate for the WT in PBS buffer is shown as a dotted black line. For clarity, only data points in the 0 to 8 reversals/second range are displayed. A small percentage of outliers exist outside of this range and are included in the calculation of the mean shown. Because data are not normally distributed, p-values were calculated using the nonparametric Kolmogorov-Smirnov two-sample test [50] comparing each data set to WT in buffer (D), tlpD in buffer (E), or tlpABC in buffer (F). p < 0.001 are indicated with *** and p > 0.05 are noted as n.s. All experiments were performed using H. pylori strain G27 or mutants created in that background. See also S7 Fig for the definition of a swimming reversal applied in these analyses and control HOCl treatments with a cheA mutant. See S2 Table for numerical mean and distribution values. n.s., not significant; TlpA, transducer-like protein A; TlpB, transducer-like protein B; TlpD, transducer-like protein D; WT, wild type.

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Western blot experiments were performed to investigate if exogenous HOCl can enter H. pylori cells and oxidize cysteine thiols. Liquid cultures of wild-type H. pylori and a tlpD mutant (strain G27) were treated with either PBS buffer or 1 mM HOCl in buffer in the presence of dimedone, which is cell permeable, quenched with methionine, and then cell lysates were examined for the presence of cysteine sulfenate (Fig 5B and S5 and S6 Figs). Western blots of samples were probed with α-Tlp antibody [26], then stripped and reprobed with α-cysteine-sulfenate. The HOCl-treated wild-type cells exhibit approximately five prominent bands corresponding to protein targets that are oxidized by HOCl to form cysteine sulfenate, suggesting that HOCl is sufficiently cell permeable to oxidize cytosolic thiols and that these targets are rather specific (Fig 5B). Interestingly, two of the five cysteine-sulfenate bands in the wild-type blot appear at approximately the same molecular weight as TlpD monomer and disulfide-linked TlpD dimer [27], and these bands are absent in the tlpD mutant blot (Fig 5B and S5 and S6 Figs). These data suggest that TlpD is one of only a few protein targets of HOCl cysteine oxidation; however, detecting the oxidation of a single cysteine residue in vivo is technically challenging, and additional analyses are needed to establish the identities of these cysteine-sulfenate protein bands.

We next showed that H. pylori swimming reversal rates are altered by HOCl exposure. Chemorepellants such as acid stimulate a build-up in CheA-Pi that results in more frequent flagella reversals and an increased rate of direction changes that, here, we refer to as “reversals”; conversely, chemoattractants reduce cellular CheA-Pi to decrease swimming reversal rate and promote smooth swimming [26]. We used automated particle tracking [51] to track almost all motile bacteria from each experiment and characterize their trajectories according to a standard definition for swimming reversal, thereby eliminating observer bias and increasing statistical power over manual tracking methods (see Method details and S7A Fig). Wild-type strain G27 H. pylori treated with PBS buffer showed a diverse range of reversal rates, with an average reversal rate of 1.77 s⁻¹ (n = 669; Fig 5C left, S1 Movie). Addition of 1 mM of the chemoattractant urea [21] shifted the swimming behavior to be mostly smooth swimming, reducing the average reversal rate to 1.21 s⁻¹ (n = 1,263; Fig 5C center, S2 Movie). Addition of 1 mM HOCl similarly promoted smooth swimming and decreased the reversal rate to also be 1.21 s⁻¹, consistent with chemoattraction behavior (n = 765; Fig 5C right, S3 Movie). In comparison, by our metrics, a chemotaxis-null cheA mutant exhibited a much lower reversal rate of 0.32 s⁻¹ and was unresponsive to HOCl (S7B Fig, S4 and S5 Movies).

Experiments conducted for three concentrations of HOCl in the range of 250 μM to 1 mM resulted in a dose-dependent decrease in reversal rate for wild type (Fig 5D). Similar assays were performed with a tlpD mutant, which exhibited a very low baseline reversal rate, as observed previously by Collins and colleagues and Schweinitzer and colleagues [26,30], and reversals were not further decreased by HOCl treatment (Fig 5E, S6 and S7 Movies). Despite its low reversal rate, previous work has shown tlpD mutants are not chemotaxis-null and retain chemoattraction to urea through TlpB [9]; however, quantification of reversal rates is inherently poor at identifying further decreases when the baseline is already near zero. To further investigate if these decreases in reversal rates from HOCl treatments are TlpD-dependent, assays were performed with a tlpABC mutant, which possesses TlpD as its sole chemoreceptor, and this mutant showed dose-dependent decreases in reversal rates similar to wild type (Fig 5F, S8 and S9 Movies). Calculations of average reversal rates and sample distributions are listed in S2 Table.

H. pylori is attracted to hypochlorous acid

Although the measurement of swimming reversal rates in the presence of a chemoeffector provides useful insight into how bacterial swimming is altered, it is a proxy for the cellular levels of CheA-Pi and does not directly assay chemoattraction in terms of localization of bacteria toward a point source. HOCl degrades at room temperature and is reactive with a variety of organic molecules, so commonly used point-source chemotaxis experiments such as capillaries and soft agar plates were not feasible. Instead, we utilized an assay in which a highly calibrated micropipette can continuously deliver fresh treatment solution to establish a microgradient [21,52]. We tested wild-type H. pylori strains PMSS1 and G27 for HOCl chemoattraction and indeed found a rapid increase in the bacteria localization to a point source of 5 to 10 mM HOCl, with the bacteria visible in frame after 30 seconds of treatment increasing by approximately 100%; the response was reproducible in experiments performed from different cultures on different days, although the magnitude of the responses showed some variation (Fig 6A, S8 and S9 Figs, and S10 and S11 Movies). The rate and magnitude of this attraction was similar to control experiments performed with urea, and HOCl chemoattraction was eliminated for a cheA mutant (S8D Fig and S15 Movie). To test the reversibility of the HOCl attraction, the HOCl point source was removed after establishing an attraction response and the bacteria were observed to disperse (S9 Fig and S12 Movie). HOCl chemoattraction was eliminated for a tlpD G27 mutant, indicating that TlpD is required for HOCl sensing (Fig 6C and 6D, S10 Fig, and S13 Movie). A weaker chemoattraction was retained for a tlpABC G27 mutant with an increase in bacteria of approximately 70% over buffer after 40 seconds, suggesting TlpD is required and sufficient for HOCl chemoattraction (Fig 6C and 6E, S10 Fig, and S14 Movie).

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Fig 6. H. pylori TlpD-dependent chemoattraction to sources of HOCl.

(A) Data from point-source chemotaxis experiments [21] are shown for H. pylori strains PMSS1 (red) and G27 (blue) using a highly calibrated micropipette for chemoeffector delivery. Three-second motility traces are shown prior to treatment (0–3 seconds) and following 30 seconds of treatment (27–30 seconds) with buffer (500 mM sodium phosphate [pH 6.7]) or 10 mM HOCl (diluted into buffer [pH 6.7]). The position of the micropipette is highlighted in yellow in the treatment panels. (B) Data from panel A is quantified over time with the average percentage of bacteria in the field of view prior to treatment normalized to be 100%. Treatment with buffer (pluses) or 10 mM HOCl (circles linked by lines) begins at time zero, noted with the yellow micropipette. Each data point shown is the average over 2 seconds of video (60 frames). (C) Experiments performed as in panel A with HOCl for the tlpD G27 (gray, 3-second time traces) and tlpABC G27 (pink, 5-second time traces) mutants. (D) Quantified data for tlpD ± 10 mM HOCl. (E) Quantified data for tlpABC experiments ± 10 mM HOCl. For panels D through E, each data point shown is the normalized bacteria count per frame. See supplementary materials for videos of these experiments and statistical analysis. (F) Shown is a synthesis of the data from this work as the basis for a new model for how TlpD functions to sense HOCl as a chemoattractant. By default, TlpD is active and promotes swimming reversals (blue circle) by stimulating CheA autophosphorylation (gray arrows). TlpD is deactivated by HOCl sensing, leading to smooth swimming and chemoattraction (green arrows). The inflammation product HOCl oxidizes a specially tuned C340 to form a cysteine sulfenate (panel 1). Oxidation of C340 causes the Cys to detach from the Zn-binding core, and a large conformational change occurs in the receptor (panel 2). This structural rearrangement deactivates the receptor so that it no longer stimulates CheA autophosphorylation in the chemotaxis signaling complex (panel 3). Decreasing the cellular pool of CheA-Pi leads to increased smooth swimming and chemoattraction to the HOCl source. CheA, chemotaxis protein A; CheA-Pi, phosphorylated chemotaxis protein A; CheW, chemotaxis protein W; CZB, chemoreceptor zinc-binding; TlpD, transducer-like protein; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000395.g006

Cys340 participates in a Cys-Zn switch with exquisite sensitivity and specificity for HOCl

The molecular basis for TlpD’s specific responsiveness to HOCl is the preferential reactivity of Cys340 with HOCl versus the chemically similar H₂O₂. Such specificity can be understood based on differences between the reactivity of these species with protein residues. H₂O₂ in the micromolar range almost exclusively reacts with cysteine, and to a lesser extent with methionine, whereas the targets of HOCl are broader and exhibit extremely fast reaction rates; a detailed kinetic analysis measured the reaction rate of HOCl with Cys to be 3.0 × 10⁷ M⁻¹ s⁻¹ and 3.8 × 10⁷ M⁻¹ s⁻¹ for Met [53]. That same study showed cysteines bound to zinc are by far the most reactive protein residue with HOCl, achieving a near diffusion-limited rate of 9.3 × 10⁸ M⁻¹ s⁻¹, almost 3 orders of magnitude greater than cysteine alone [53], and in fact also exhibit decreased reactivity with hydrogen peroxide and oxygen [54]. Previous work has identified numerous other proteins containing zinc-bound cysteines that are targets for HOCl oxidation including Hsp33 [53] and alcohol dehydrogenase, which is 3,000-fold more reactive with HOCl than peroxide [55]. The example we found to be most similar to TlpD is the activation of human neutrophil collagenase that occurs by HOCl oxidation of a single zinc-bound Cys, a mechanism coined as a “Cys-Zn switch” [56,57]. Together, our data indicate the molecular function of TlpD is modulated by reaction of HOCl with C340 to switch between 2 conformations: (1) a “reduced” and Cys-zinc-bound state that is fully folded and activates CheA, and (2) an “oxidized” cysteine-sulfenate state with a partially unfolded CZB domain that does not activate CheA and that can be reduced and resurrected by cellular reductants (Fig 6F).

HOCl is a prevalent host-produced oxidant encountered by H. pylori

HOCl, which is produced by the granulocyte-specific enzyme myeloperoxidase from H₂O₂ and chloride anion, reaches levels of up to 5 mM at sites of inflammation [49,58,59]. In contrast, maximal gastric concentrations of extracellular H₂O₂ are thought to be 1 to 15 μM and perhaps rarely reach approximately 100 μM in the presence of oxidants from dietary sources such as coffee [28,29]. The concentration of superoxide is approximately 2 μM, and it decomposes rapidly, with a half-life on the order of milliseconds [60]. Previous analyses of H. pylori oxidant sensing [26,27] used concentrations of H₂O₂ and superoxide at a much higher concentration than what occurs in vivo. Additionally, although triggered human neutrophils produce several ROS during inflammation, including H₂O₂ and superoxide, it seems these are all funneled into the production of HOCl as the major product, because quantitative analyses show essentially all H₂O₂ generated is converted to HOCl [49,61].

HOCl is a potent disinfectant that can kill bacteria at micromolar concentrations but is also sufficiently cell permeable to oxidize intracellular thiols at sublethal doses [59,62]. In addition to examples of cytosolic proteins that are targets for HOCl oxidiation discussed above, such as Hsp33 and alcohol dehydrogenase, the bacterial transcription factor NemR within the cytosol uses a reactive Cys residue to selectively sense HOCl to trigger expression of antioxidant enzymes [63]. We showed that H. pylori is exceptionally resistant to HOCl, likely due to its strong antioxidant defenses including three peroxiredoxins, and high expression of its catalase, KatA [64]. KatA in particular has been shown to be key for H. pylori to survive ROS bursts from triggered immune cells, and evidence indicates this resilience leads to the chronic release of ROS that damages gastric tissue and results in disease [3]. Our finding that H. pylori is attracted to sources of HOCl by TlpD suggests that H. pylori does not just tolerate but can actively seek out sources of HOCl, which would be produced by neutrophils at sites of inflammation. Neutrophils are a hallmark of H. pylori infection and are actively recruited in response to H. pylori induction of interleukin-8 [65]. Neutrophils concentrate near the stem precursor and cell zones of the gastric gland epithelium [66], which undergoes proliferation in response to H. pylori gastric gland colonization [67]. H. pylori colonization of the gastric glands is an important strategy for the bacteria’s persistent infection of the stomach [68] and requires chemotaxis [52,69], and therefore chemoattraction to HOCl may constitute a strategy H. pylori uses to persist in neutrophil-infiltrated gastric glands.

This behavior is consistent with other proinflammatory strategies of H. pylori, such as its degradation of host-secreted extracellular antioxidant glutathione [70], and the fact that its colonization levels are reduced in mice treated with the ROS scavenger ursodeoxycholic acid [71] or sulforaphane, which stimulates host antioxidant protein production through Nuclear factor erythroid 2-related factor 2 [72]. In this regard, H. pylori belongs to a growing list of bacterial pathogens and pathobionts that can promote and benefit from host inflammation, thereby setting up positive feedback loops that can drive chronic inflammation [73,74]. For example, Salmonella-induced inflammation increases the concentrations of colonic tetrathionate and nitrate, which it recognizes as chemoattractants, and it can use these inflammation products as electron acceptors in anaerobic respiration to outcompete fermentation-restricted members of the native microbiota [75–78]. Additionally, other molecular oxidants not tested in this study could be sensed as chemoeffectors by TlpD through a similar mechanism, such as aldehydes and quinones, which are also known to be reactive toward zinc-bound cysteines [79,80], and might provide an explanation for the reported role of TlpD in energy taxis [30,81].

Interpreting conflicting data of H. pylori responses to ROS

In contrast to our finding of H. pylori’s TlpD-dependent chemoattraction to HOCl, previous studies have proposed that TlpD senses ROS as chemorepellents [27,81–83]. Differences both in experimental methodologies and interpretations of results likely contribute to these divergent conclusions. First, we note that measuring H. pylori chemotaxis can be challenging and often relies on indirect measurements of swimming behavior, such as reversal rates in a uniform chemical solution. Our data indicates that reversal rates are highly variable within a single population of H. pylori (Fig 5C–5F), which could contribute to inconsistent results using this assay (e.g., opposite reported responses of H. pylori to paraquat [25, 26]). We found that greater than 100 tracks were required for the mean reversal rate to be well defined (S7C Fig). To better understand H. pylori responses to ROS, we have focused on ROS species at concentrations reported in the gastric environment. We confirmed our findings of TlpD-dependent, HOCl-induced smooth swimming with a point-source chemotaxis assay that demonstrated localization of H. pylori toward HOCl (Fig 6, S8–S10 Figs, and S11–S14 Movies).

Secondly, we note the challenges of making inferences about a gene’s function based on the pleiotropic phenotypes of mutant bacteria lacking that gene. tlpD-deficient strains have reported colonization defects in mice and gerbils [9,10,81,82], defective chemotactic responses to pH and ROS [9,26,27], and growth defects in the presence of ROS [81]. The mechanistic basis for the colonization defects could thus be due to any combination of the mutant strain’s altered chemotaxis to multiple chemicals in the host environment and its increased sensitivity to ROS. For example, the finding that tlpD-mutant strains colonize wild-type mice and gerbils poorly could be alternatively interpreted as evidence of the mutant’s failure to avoid toxic ROS, its failure to establish persistent infection of HOCl-rich gastric glands, or its inherent sensitivity to ROS.

H. pylori integrates multiple chemical cues to persist in the gastric environment it shapes

H. pylori is a notoriously persistent colonizer, often retained for an individual’s lifetime, and previous work highlights the bacteria’s ability to adaptively modify the stomach environment [84–87]. Interestingly, some of the chemoeffectors sensed by H. pylori’s limited chemoreceptor repertoire reflect the environment of the H. pylori-infected, as opposed to a naïve stomach, such as low concentrations of urea [21] and presence of autoinducer-2 [88]. Now, we add to this picture chemoattraction to HOCl produced by H. pylori-induced neutrophil infiltration, a hallmark of the H. pylori-infected stomach. TlpD seems to be especially important for colonizing the stomach antrum, which has been described in some work as the area of highest inflammation [9,10]. Decreasing host epithelial hydrogen peroxide and immune cell superoxide, which are both precursors for HOCl production, shifts the wild-type H. pylori infection away from the antrum toward the corpus [83], which could reflect the loss of HOCl as a chemoattractant for locating inflamed regions of the stomach. Clearly, additional studies are needed to elucidate the importance of HOCl sensing for H. pylori biology and infection. More generally, CZB domain containing proteins are encoded by the genomes of numerous host-associated bacterial species [25], suggesting the broad utility of HOCl sensing for bacteria living together with animals that produce HOCl as an antimicrobial response.

Contact for reagent and resource sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Arden Perkins (ardenp@uoregon.edu).

Experimental model and subject details

Method details

Mass spectrometry

20 μM of pure recombinant TlpD in PBS buffer (pH 7) was prepared for analysis by treatment with 1 mM TCEP, H₂O₂, or HOCl for 1 hour. Subsequently, the samples were alkylated by the addition of 5 mM iodoacetamide and allowed to react for 30 minutes at room temperature sheltered from light. The sample was then buffer-exchanged with fresh buffer three times by centrifugation using a 10 kDa cutoff and flash frozen in liquid nitrogen prior to analysis. Mass spectrometric analysis of TlpD was performed as a service by Dr. Larry David at Oregon Health and Science University’s (OHSU) Proteomics Shared Resource Facility. As per their request, we include the following statement: “Mass spectrometric analysis was performed by the OHSU Proteomics Shared Resource with partial support from NIH core grants P30EY010572, P30CA069533, and S10OD012246.” Protein samples were subjected to digestion with LysC, and 3 μg of digest was analyzed on a Q-Exactive HF instrument using a 75 μm × 250 mm nanospray C18 column. MS/MS data were searched with Sequest against an E. coli database supplemented with the TlpD protein sequence. False discovery was controlled using a reversed sequence database and only q-scores below 0.05 were accepted. C340 was located within the peptide NCRLGKWYYEGAGK (567.94415 Da), and carbamidomethylated (+57.02146 Da), sulfinate (+31.98983 Da), and sulfonate (+47.98474 Da) forms were detected. Ion extractions were performed for the +3 charge states for the 3 forms of this peptide using a 2 ppm tolerance, and peaks were integrated to obtain absolute values for quantification and comparison of modifications across experiments (Fig 4E and S3C–S3E Fig).

Fluorescence, CD spectroscopy, and analytical ultracentrifugation

For Zn-chelation assays, we used the fluorescent probe Zinpyr-1 (Abcam, Eugene, OR) with excitation at 492 nm and maximum emission near 527 nm; 5 mM stock solutions of Zinpyr-1 were prepared in DMSO and diluted to 50 μM final concentration for experiments with zinc controls and TlpD in a buffer of 100 mM Tris (pH 7) and 300 mM NaCl. Samples were loaded into a 2 × 2 mm quartz cuvette (Starna Cells Inc. Atascadero, CA) and fluorescence was monitored at 20°C using a FluoroMax-3 Spectrofluorometer (HORIBA Scientific, Austin, TX). For time courses, fluorescence was measured every 60 seconds over the course of 3 hours. Identical samples containing only buffer were used as blanks, and final spectra shown are blank-subtracted spectra, but these were minimally different (S2 Fig).

For intrinsic protein fluorescence assays with TlpD and the C340A mutant, we used an excitation wavelength of 295 nm, to optimize signal from the single Trp345, and monitored emission in the 300 to 400 nm range (Fig 4G). Samples were prepared in a buffer of 25 mM NaCl and 20 mM Tris (pH 7), and experiments were conducted at 20°C. Samples were pretreated for 1 hour prior to measuring fluorescence for endpoint fluorescence spectra of TlpD HOCl treatments (Fig 4G–4I), or subsequently treated with TCEP for 30 minutes (Fig 4J). Fluorescence anisotropy experiments for recombinant TlpD in PBS buffer (pH 7) and 1 mM TCEP were collected with excitation at 295 nm and monitoring emission at 340 nm (Fig 2B). Intrinsic fluorescence was too weak at low protein concentrations to be reliably detected, and so a K_D value could not be calculated from these data but were still useful to indicate that the protein is maximally oligomerized by approximately 16 μM, consistent with monomer/dimer ratios predicted from AUC experiments (Fig 2B and S1 Fig)

Protein CD was performed for TlpD with a Jasco J-810 spectropolarimeter using a 1 mm path length quartz cuvette. All spectra for fluorescence and CD experiments were corrected for background by subtracting signal from buffer-only samples. To obtain a melting and cooling curve, 50 μM TlpD in a buffer of 150 mM NaCl and 17.5 mM sodium citrate (pH 7) was heated from 25°C to 95°C in 0.5°C increments per 5 minutes and then cooled back to 25°C (S2D Fig). For HOCl titration and unfolding experiments, 5 μM TlpD was treated with various concentrations of HOCl for 10 minutes in a buffer of PBS (pH 7), then desalted with a Micro Bio-Spin Column (Bio-Rad) into a buffer of 150 mM NaCl and 17.5 mM sodium citrate (pH 7), and then analyzed by CD at 20°C (S3B Fig).

Analytical ultracentrifugation experiments were performed for recombinant TlpD and TlpD(-His) using a Beckman Proteome Lab XL-I centrifuge and a 60 Ti 4-cell rotor. Interference experiments were performed using 1, 2, and 3 × 10 μM TlpD and 3 × 28 μM TlpD(-His) in PBS (pH 7) and 1 mM TCEP at 20°C, with a total volume of 400 μl at 55,000 rpm. Data were processed with SedFit [94] using a continuous distribution model with rmsd of the final data sets <0.016 (Fig 2B and S1 Table).

Quantification and statistical analysis