Why is urea important to helicobacter

There has been great interest in H. Most experts agree that the urea breath test will be the test of choice to establish H. Symptomatic patients who had been determined to have H. In addition, symptomatic patients were studied who had undergone upper endoscopy but were negative for H. Antral biopsies were taken for histological techniques and CLO tests. The discordant CLO test, histology, or breath test patients were submitted to a second blinded histological examination. Sera were assayed for H.

Incorporation of urease into a well-characterized and highly immunogenic purified H. In the first step, patient samples and controls were diluted with sample diluent and incubated with beads coated with highly purified H.

Specific antibodies were bound to the beads. After removal of the unbound material by a washing step, the antigen-antibody complex on each bead was detected with peroxidase POD -conjugated goat anti-human IgG antibody.

After removal of unbound conjugate, the beads were incubated with a substrate solution containing tetramethylbenzidine TMB and hydrogen peroxide. A blue color developed, the intensity of which was proportional to the amount of H.

The enzymatic reaction was stopped by the addition of acid, and absorbance values were determined at nm. For quantitative results a standard curve was obtained by dilution series of the positive control and plotting each absorbance value versus the corresponding standard value.

The concentrations of IgG antibody to H. Antral mucosal biopsy specimen was inserted into a homemade urease test tube 4. Urease reagent was made up to ml with bacto-yeast extract 0. It was sterilized by filtration, dispensed into 0. If the urease enzyme of H. The urease test tube was examined over the next 24 h. Aliquots of 0. Subjects were fasted for at least six hours prior to the test.

They had to remove false teeth if present , and cleanse their mouth with antiseptic solution thymol, salol, menthol, saccharin, fuchsin, water and ethanol provided by the Pharmacy Division of the Hospital; a baseline breath sample was collected and identified as Time 0.

Then, they swallowed 5 u Ci of 14 C- urea dissolved in 20 ml of water. However, when was given directly into the esophagus in Hp- subjects, isotope excretion did not increase more than 50 cpm above the baseline value. Therefore, patients rinsed their mouth again two min after they had swallowed 14 C-urea, spitting the water out and making sure that none was ingested.

Breath samples were collected at 5, 10, 15, 20 and 30 min. Patients were instructed to blow through tubing attached to a safety trap into a scintillation vial containing 2. They had to blow until the solution became colorless indicating the collection of 1 mmol of CO 2. Once the breath samples had been collected, scintillation fluid 10 ml- 5. In addition to equilibrium trans-pore free energy profiles, this approach also provides transport kinetics, as well as the atomic resolution mechanistic details of urea conduction without bias.

Simulations of mutants provide further mechanistic insights into the function of the channel. The scarcity of urea in the pore illustrates the low urea affinity of the channel.

The crystal structure did not show density for periplasmic loop 1 PL1 , possibly because of the fact that the 6-histidine tag had to be inserted into this loop because N- and C-terminally tagged protein was not active 5. To construct a complete Hp UreI hexamer, the loop was copied and transposed onto the other protomers. A complete list of simulation systems is given in Supplementary Table S1.

Knowledge of the oligomeric state of Hp UreI in the membrane is important for understanding how the tetrahedral dodecameric 1. It may also play a role in cooperative pH gating. To investigate the nature of the unusual hexameric ring arrangement, we carried out unrestrained simulations of a single protomer embedded in a lipid bilayer.

This resulted in rapid deviation from the native state, ultimately leading to pore collapse within several hundreds of nanoseconds, irrespective of urea concentration Supplementary Fig. These results were obtained independently with two different force fields OPLS and CHARMM , suggesting that the hexameric arrangement is essential for maintaining proper helix conformations for each loosely packed protomer and represents the native state in the bilayer.

Our simulations support experimental observations that Hp UreI has a remarkably low binding affinity for urea 8. This low occupancy is chiefly due to the absence of a high affinity binding site that could form simultaneous hydrogen bonds with the single carbonyl and two amino groups of planar urea. Although approximately half of the side chains lining the channel are able to form hydrogen bonds, urea entry is unfavourable. This is due to the strong polarity of urea. With a liquid state dipole moment of 6—8 Debye D 11 the experimental value for liquid water is 2.

Our analysis gives a slightly higher bulk value of water molecules in the first hydration shell. A similar compensation mechanism has been observed for aquaporin, bacterial glycerol facilitator, and urea transporter UT channels 7 , However, confinement of the polar urea molecule carries an additional energetic penalty because of the reduction in translational and rotational entropy, which is associated with the strong preferential alignment observed during transport through the pore Fig.

Analysis of the averaged spatial alignment of the urea dipole moment, which is zero in bulk water due to rapid tumbling, revealed a net alignment of up to 5. This compares with the theoretical maximum of 8 D for a perfectly immobilized urea molecule.

In the channel, urea molecules preferentially orient with their carbonyl group pointing towards the central lipid plug Fig. Rapid tumbling of urea and water results in net-zero dipole moments in bulk solution.

However, inside the pore urea molecules are strongly aligned, with the amino groups pointing away from the central lipid plug. The figure shows that protein side chain contacts cannot fully compensate the loss of water molecules from the hydration shell of urea.

The profile shows two main barriers to transport separated by a local free energy minimum. The right panel shows the superposition of the urea carbon atom over the course of the simulation blue , demonstrating visually that urea fully samples all parts of the channel.

To prevent the pore collapse mentioned above, the backbone atoms of the six transmembrane helices were restrained over the entire duration of the simulations. Mutation studies have shown that histidine residues on the periplasmic loops are essential for pH gating 16 , suggesting that opening and closing of the channel might be controlled by reversible folding of one or both loops into the periplasmic vestibule of the channel. However, in the absence of an experimental loop structure, the conclusions drawn from the simulations remain speculative.

A C C residue Trp, in blue is shown for reference. This simulation captured 10 spontaneous conduction events see Supplementary Fig. Grey et al. In contrast, ion channels, which typically have much higher conduction rates, also have much longer dwell times. A recent simulation study found that for voltage-gated sodium channels the dwell time is approximately twice as long as the time between conduction events Comparison of the motion of channel-lining residues and the atomic detail conduction mechanism showed that the spontaneous transport of urea closely resembled that determined in the hexamer simulation see Supplementary Discussion and Supplementary Figs S6 and S7.

Each protomer in the Hp UreI crystal structure revealed two closely spaced constrictions near the center of the bilayer that obstruct the solute pathway 5. The first constriction C P in the channel, located at the end of the periplasmic vestibule, is formed by the side chains of the conserved residues Leu6, Val9, Phe84, Trp, and Trp This constriction is very hydrophobic and presumably acts as the proton filter.

In the crystal structure, both constrictions were found to be too narrow to allow the passage of urea or even water through the channel. The simulations reveal in atomic detail how these constrictions open and allow the passage of urea and water Fig. Each aromatic has only a small number of accessible states defined by the angle of its aromatic ring normal with respect to the membrane normal Supplementary Fig. Representative equilibrium states of the ring orientations are shown in the circular panels.

Tyr88 has one state, Phe84 and Trp have two states, whereas Trp has four. The opening motion is towards the periplasm, in the direction of the approaching urea.

Control simulations of restrained protomers before opening of C P showed no conduction, suggesting this rearrangement is required for urea to pass through the pore see Supplementary Discussion and Supplementary Fig. During transition through C P , urea was found to be sandwiched between the aromatic Phe84 on one side and Leu6 and Val9 on the opposite side, with the Trp indole donating a hydrogen bond Fig.

Conduction events are indicated in green. Unlike for C P , no structural rearrangement was observed for C C over the course of the simulations see Supplementary Fig.

This reorientation of Trp is periodic and occurs on the timescale of tens to hundreds of nanoseconds. C C has a higher free energy barrier than C P for urea, and passage requires sequential hydrogen bond formation and breakage with key conserved residues, suggesting that it forms the selectivity filter.

During passage through C C , the urea carbonyl first receives a hydrogen bond from the hydroxyl of Tyr88, which is later lost during transport across the constriction. Immediately, on the other side of the barrier the side chain carbonyl of protonated Glu offers a readily available hydrogen bond acceptor for one urea amino group Fig.

The simulations reveal that the hydrogen bond from Tyr88 is required for guiding urea into and through C C , which explains why the Y88F mutant was found to be non-functional in oocytes 5 see also Supplementary Discussion and Supplementary Fig.

Hydrogen bonding to Glu might be important for allowing urea to break its bond with Tyr88 and hop over the constriction barrier; this is supported by the observation that the EQ mutant is also functional 5. After passage through C C , water rapidly releases urea from the cytoplasmic vestibule to complete the urea transport process. The vestibules of the Hp UreI channels were initially empty, but rapidly filled with a large number of water molecules during equilibration.

Over the course of the simulations, water hydrates all parts of the pore including the two constriction sites see Fig. Remarkably, protomers were found to efficiently conduct water at single-channel rates of 2.

Periodic rotation of Trp frequently changes the cross section of the cytoplasmic constriction C C , modulating the rate water conduction through the channel see Fig.

Both values are independent of the direction of water flux. The seal of the closed state is formed by Tyr88 and Trp, with the latter acting as the gating residue. The average rate of water transport from fitting to the dotted lines is 4. This is approximately four orders of magnitude higher than the rate of urea transport. The transitions between low and high conducting states are visible as slight changes in the slope for both upward and downward conduction b. Conductance is equal in both directions, resulting in a zero net water transport through the channel pore in the absence of an osmotic gradient.

This suggests that, although hydration of urea does not seem to be essential for urea transport per se , it might be important for urea transport through the Hp UreI channels. Although water flux through the channel is high, any significant conduction of protons through Hp UreI would almost certainly be lethal to H.

A polar cytoplasmic vestibule probably serves to prevent urea moving backwards through the channel after having traversed C C , while the hydrophobic lining of the periplasmic vestibule almost certainly serves to discourage ions to enter the channel.

These findings suggest that the periplasmic vestibule and constrictions are too hydrophobic to allow ions to pass through the channel. S1 To check the retrograde pathway of the hydrolysis products, we performed equilibrium simulations in the presence of NH 3 and CO 2.

Both solutes were found to preferentially go through the bulk bilayer and the central lipid plug, and the rate in both cases is much higher than the urea flux rate, suggesting no particular molecular mechanism is required to provide efficient efflux of buffering products to the periplasm.

The permeation rate for CO 2 is 4. This result agrees with previous molecular dynamics simulations that demonstrated that both CO 2 and NH 3 can easily cross membranes of different compositions and do not require specific proteins for transport Unbiased microsecond equilibrium molecular dynamics simulations of Hp UreI have been used to study the mechanism of urea conduction at atomic detail see Fig.

The hexameric assembly was found to be critical to preserve the structural integrity of the channels in the membrane Supplementary Fig. This unusual arrangement might serve to ensure a tight seal between Hp UreI and the cytoplasmic urease to avoid toxic alkalinization of the cytoplasm by the NH 3 produced by urea hydrolysis Supplementary Fig.

The arrangement might also play a role in the pH gating of the channel by periplasmic loop folding cooperatively or onto neighboring channels. The simulations reveal that this low affinity is due to a combination of dehydration and confinement of urea in the Hp UreI channel.

Water molecules lost from the bulk water hydration shell cannot be fully replaced by contacts with pore-lining side chains because of a lack of sufficient hydrogen bonding partners, resulting in an energetic penalty associated with urea dehydration.

In addition, the simulations show that urea, which has a liquid state dipole moment of 6—8 D 11 , preferentially orients inside the channel during transport, with the amino groups pointing away from the central lipid plug see Fig. The net alignment of urea in the channel, observed in the simulations, matches the electrostatic charge distribution on the periplasmic channel surface, previously derived by electrostatic potential calculations 5 , with the carbonyl group pointing towards a positive protein surface facing the central plug and the amino groups facing towards a negative protein surface directed towards the bulk lipids in the bilayer.

The surface charge arrangement might serve to correctly pre-orient urea molecules for efficient transport through the selectivity filter further inside the channel pore. A total of 15 urea transport events were captured in the simulations of wild-type Hp UreI. This result prompted us to experimentally determine the urea conduction rates in H.

Nevertheless, both experimental and simulation results confirm that the urea conduction rate is much lower than that of typical channel proteins. One reason might be that high conducting channels typically conduct smaller entities, such as water or monatomic ions.

Indeed, the rate of water transport through Hp UreI was found to be much higher, equivalent to that of aquaporins see below. This suggests that Hp UreI is a much more efficient channel at low urea concentration gradients, which may be one explanation why H. Another explanation might be that UTs generally do not show pH gating, essential for H. The Hp UreI crystal structure revealed two constrictions in the channel, which open transiently during the simulations to allow passage of urea.

Even in their open states, the constrictions remain largely hydrophobic and narrow Figure 7. Together with the observation that the C P is more hydrophobic that is, offers less hydrogen bonding partners than C C, this suggests that C P and C C probably function as proton rejection and urea selectivity filters, respectively.

During transport through the adjacent constriction sites C P periplasmic and C C cytoplasmic , urea is sandwiched between highly conserved residues, while simultaneously hydrogen bonding to orthogonally oriented sidechains. C P is much more hydrophobic, probably to reject protons, while C C selects for urea-like molecules. Although the channel architecture is completely different, a somewhat analogous local arrangement was observed in the UT channels, where urea was found to be sandwiched between two Phe sidechains with a Glu residue accepting hydrogen bonds from the urea amino groups 6.

The constriction closes again shortly afterwards with the Trp orientation returning to that observed in the crystal structure. Although UTs possess an entirely different architecture compared with Hp UreI, the free energy profile for transport of urea through the pore is similar, with two closely spaced barriers near the center of each channel Fig.

The UT profile is at least partly due to the two-fold pseudo symmetry of the long, narrow UT pore, explaining the equal height of both barriers. Hp UreI displays no such symmetry. While both the Hp UreI and UT free energy profiles are primarily the result of the cost of urea desolvation in the channel, the level of hydration is very different.

In the Hp UreI channel urea is always hydrated by at least four water molecules, while the maximum observed in the UT channel was less than three 7. This explains why UT channels have higher transport barriers. Several aquaglyceroporins, which can carry a range of solutes other than water across membranes, also conduct urea. This suggests that sufficient space and a high level of hydration are key requirements for urea conduction, irrespective of the pore chemistry.

The simulations identify a sequential interaction of urea with highly conserved residues that determine selectivity and control urea flux through the channel and are located at the cytoplasmic constriction C C.

These rotations are transient and occur on the timescale of tens to hundreds of nanoseconds, necessitating microsecond simulations. In the open configuration, Trp acts as a lever that pins urea against a hydrophobic wall formed by the highly conserved residues Leu6, Val9, and Leu13 Fig. Therefore, the weak affinities of the mutants mainly resulted from the mutation of amino acids rather than structural changes.

This confirmed that the predicted interfaces were credible. Among these residues, the mutation of D68 caused the greatest attenuation of interaction affinity by dramatically decreasing the association combined with an increase of the dissociation rate.

This suggested that D68 plays an important role in the interaction process. Further analysis showed that substitution of E25 or K26 by alanine could dramatically speed up both the association and dissociation rate.

Considering that these two residues are located at the forefront when UreA enters the cavity of Hsp60 Figure 4C , it is speculated that mutation of these residues to alanine would reduce the resistance of the association and speed up binding.

This study clarifies the interaction between urease, an important pathogenic factor of H. The residues playing key roles in the interaction between UreA and Hsp60 were further studied here. These results lay a foundation for revealing the assembly and maturation mechanism of H. HZ, XJ wrote the manuscript. All authors approved the final manuscript.

The open access publication fee will also from National Natural Science Foundation of China The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Peng Wang Shandong University, China for providing help with molecular docking and structural analysis. Thank MA. Austin, J. Structural comparison of urease and a GroEL analog from Helicobacter pylori.

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