David L. Weeks

Expression of the Helicobacter pylori ureI Gene Is Required for Acidic pH Activation of Cytoplasmic Urease

ABSTRACT ureI encodes an integral cytoplasmic membrane protein. It is present in the urease gene cluster of Helicobacter pylori and is essential for infection and acid survival, but its role is unknown. To determine the function of UreI protein, we producedH. pylori ureI deletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential. We also determinedureI expression, urease activity, and the effect of urea on membrane potential of several gastric and nongastricHelicobacter species. ureI was found to be present in the genome of the gastric Helicobacter species and absent in the nongastric Helicobacter species studied, as determined by PCR. Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric Helicobacterspecies. When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH. The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited. No acidic pH activation or urea induced membrane potential changes were found in the nongastric Helicobacter species. The ureI gene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.

Proteins Released by Helicobacter pylori In Vitro

Secretion of proteins by Helicobacter pylori may contribute to gastric inflammation and epithelial damage. An in vitro analysis was designed to identify proteins released by mechanisms other than nonspecific lysis. The radioactivity of proteins in the supernatant was compared with that of the intact organism by two-dimensional gel phosphorimaging following a 4-h pulse-chase. The ratio of the amount of UreB, a known cytoplasmic protein, in the supernatant to that in the pellet was found to be 0.25, and this was taken as an index of lysis during the experiments (n = 6). Ratios greater than that of UreB were used to distinguish proteins that were selectively released into the medium. Thus, proteins enriched more than 10-fold in the supernatant compared to UreB were identified by mass spectrometry. Sixteen such proteins were present in the supernatant: VacA; a conserved secreted protein (HP1286); putative peptidyl cis-trans isomerase (HP0175); six proteins encoded by HP0305, HP0231, HP0973, HP0721, HP0129, and HP0902; thioredoxin (HP1458); single-stranded-DNA-binding 12RNP2 precursor (HP0827); histone-like DNA-binding protein HU (HP0835); ribosomal protein L11 (HP1202); a putative outer membrane protein (HP1564); and outer membrane proteins Omp21 (HP0913) and Omp20 (HP0912). All except HP0902, thioredoxin, HP0827, HP0835, and HP1202 had a signal peptide. When nalidixic acid, a DNA synthesis inhibitor, was added to inhibit cell division but not protein synthesis, to decrease possible contamination due to outer membrane shedding, two outer membrane proteins (Omp21 and Omp20) disappeared from the supernatant, and the amount of VacA also decreased. Thus, 13 proteins were still enriched greater than 10-fold in the medium after nalidixic acid treatment, suggesting these were released specifically, possibly by secretion. These proteins may be implicated in H. pylori-induced effects on the gastric epithelium.

The control of gastric acid and Helicobacter pylori eradication

This review focuses on the gastric acid pump as a therapeutic target for the control of acid secretion in peptic ulcer and gastro‐oesophageal reflux disease. The mechanism of the proton pump inhibitors is discussed as well as their clinical use. The biology of Helicobacter pylori as a gastric denizen is then discussed, with special regard to its mechanisms of acid resistance. Here the properties of the products of the urease gene clusters, ureA, B and ureI, E, F, G and H are explored in order to explain the unique location of this pathogen. The dominant requirement for acid resistance is the presence of a proton gated urea transporter, UreI, which increases access of gastric juice urea to the intrabacterial urease 300‐fold. This enables rapid and continuous buffering of the bacterial periplasm to ≈ pH 6.0, allowing acid resistance and growth at acidic pH in the presence of 1 m M urea. A hypothesis for the basis of combination therapy for eradication is also presented.

Genes of Helicobacter pylori Regulated by Attachment to AGS Cells

ABSTRACT Reciprocal interactions between Helicobacter pylori and cells of the gastric epithelium to which it adheres may affect colonization. Changes in gene expression of H. pylori induced by adhesion to AGS gastric cancer cells by coculture were compared to changes in gene expression of H. pylori cultured without AGS cells by using cDNA filter macroarrays. Adhesion was quantitatively verified by confocal microscopy of green fluorescent protein-expressing bacteria. Four experiments showed that 22 and 21 H. pylori genes were consistently up- and down-regulated, respectively. The up-regulated genes included pathogenicity island, motility, outer membrane protein, and translational genes. The σ28 factor antagonist flgM, flgG, the stress response gene, flaA, omp11, and the superoxide dismutase gene (sodB) were down-regulated. The up-regulation of cag3, flgB, tonB, rho, and deaD was confirmed by quantitative PCR, and the up-regulation of lpxD, omp6, secG, fabH, HP1285, HP0222, and HP0836 was confirmed by reverse transcription (RT)-PCR. The down-regulation of flaA, sodB, and HP0874 was confirmed by quantitative PCR, and the down-regulation of omp11 was confirmed by RT-PCR. The alteration of gene expression in H. pylori after adhesion to gastric cells in vitro suggests that changes in motility, outer membrane composition, and stress responses, among other changes, may be involved in gastric colonization.

Sites of pH regulation of the urea channel of Helicobacter pylori

Helicobacter pylori (Hp) and Streptococcus salivarius (Ss) require intrabacterial urease for acid resistance and express a urea channel, UreI. The presence of UreI was shown to increase urea permeability ≈ 300‐fold over that of a non‐polar ureI deletion mutant. Expression of SsUreI in Xenopus oocytes increased urea uptake pH independently, whereas HpUreI shows an acidic pH dependence, half‐maximal at pH 6.0. Mutagenesis of all histidines, aspartates, glutamates and the lysine in the periplasmic domain of HpUreI showed that His‐123, His‐131, Asp‐129, Asp‐140, Glu‐138 and Lys‐132 in the second periplasmic loop (PL2) and His‐193 in the C‐terminus (Ct) were important for activation of transport. With the exception of a lysine that was shown to substitute for His‐193 in HpUreI, these charged amino acids are absent in SsUreI. A chimera in which PL1 of HpUreI was replaced by PL1 of SsUreI retained activity at acidic pH and gained partial activity at neutral pH. Exchange of PL2 inactivated transport, whereas exchange of Ct had no effect. Chimeras, in which either PL1 or PL2 of HpUreI replaced those of SsUreI, retained wild‐type transport, but replacement of the Ct or both loops inactivated transport. PL1 appears to be important for restricting transport through HpUreI at neutral pH, whereas protonation of three histidines in PL2 and Ct and the presence of three dicarboxylic amino acids in PL2 appears to be necessary to activate HpUreI at acidic pH.

Specific identification of three low molecular weight membrane-associated antigens of Helicobacter pylori

A large number of Helicobacter pylori proteins are antigenic, but antibodies to these proteins persist in spite of the eradication of the infection.

Does OxyVita, a new-generation hemoglobin-based oxygen carrier, or oxyglobin acutely interfere with coagulation compared with normal saline or 6% hetastarch? An ex vivo thromboelastography study.

OBJECTIVES Because hetastarches have deleterious effects on coagulation that increase with molecular weight (MWt), risk of coagulopathy associated with a high MWt hemoglobin-based oxygen carrier (HBOC) was studied. DESIGN Preliminary laboratory study of donor blood using thromboelastography (TEG). SETTING University laboratory. PARTICIPANTS Volunteer donor blood. INTERVENTIONS Experiments simulated hemodilution during clinical resuscitation of hemorrhagic shock with varying doses of HBOCs. Coagulopathy related to 1:11, 1:5, 1:2, or 1:1 dilution of whole blood with normal saline, 6% hetastarch (670 kilodaltons [kD]), hemoglobin glutamer-200 (HBOC-200, 200 kD), or OxyVita (OXYVITA Inc, New Windsor, NY) (a new-generation, zero-link polymerized bovine hemoglobin-based oxygen carrier, 33 megadaltons) were analyzed. MEASUREMENTS AND MAIN RESULTS At 2 lower levels of hemodilution, hetastarch, HBOC-200, and OxyVita produced equivalent reductions in maximum clot strength (TEG-MA and TEG-G) that reached statistical significance compared with whole blood and normal saline. At 2 higher dilutions, OxyVita and HBOC-200 impaired maximum clot strength compared with whole blood, normal saline, and hetastarch. Dilution with hetastarch had a greater effect on clot propagation (K and alpha) than either HBOC. CONCLUSIONS OxyVita and HBOC-200, HBOCs with different MWt, had similar effects on coagulation as measured by TEG. The impairment of coagulation by HBOCs and hetastarch occurred at doses corresponding to 12 mL/kg or a blood volume replacement of 17%. The use of HBOCs at doses corresponding to 23 mL/kg or a blood volume replacement of 33% significantly decreased coagulation to levels associated with increased clinical bleeding in this preliminary study. Minimal coagulopathic effects are expected with use of OxyVita at the manufacturers anticipated effective dose of 10 g or 2 to 3 mL/kg.

Contrast-enhanced immunoelectron microscopy for Helicobacter pylori

Since a method of contrast enhancement for immunoelectron microscopy has not been available in bacteriology, the morphological localization of proteins of Helicobacter pylori is not well known. In this report, we established a method of contrast enhancement in immunoelectron microscopy in this organism. Immunostained ultrathin sections are stained with a mixture of alcian blue and osmium tetroxide prior to staining with uranyl acetate. This method of staining provided good contrast enhancement of the bacterial cell wall and membrane without any loss of immunolabeled gold particles on the ultrathin section.

Methemoglobin effects on coagulation: a dose-response study with HBOC-200 (Oxyglobin) in a thrombelastogram model.

OBJECTIVES Because oxidation affects platelet and coagulation factors, hemoglobin auto-oxidation in HBOCs results in the transformation to methemoglobin, which may have additive adverse effects on coagulation. The risk of coagulopathy after different dilutions of HBOC-200 with low and high methemoglobin concentrations was studied. DESIGN A laboratory study on donor blood using thromboelastography (TEG; Haemoscope, Niles, IL). SETTING A university laboratory. PARTICIPANTS Volunteer donor blood. INTERVENTIONS Blood samples simulated hemodilution during clinical resuscitation of hemorrhagic shock with varying doses of HBOC-200 (Oxyglobin; Biopure Corp, Cambridge, MA). Coagulopathy related to 1:11, 1:5, 1:2, and 1:1 dilution of whole blood with HBOC-200 high methemoglobin concentrations (65%) and HBOC-200 low methemoglobin concentrations (1%) were analyzed. MEASUREMENTS AND MAIN RESULTS Analysis of fixed effects of dilution on coagulation showed that the progressive dilution of HBOC-200 (low methemoglobin) and HBOC-200 (high methemoglobin) produced significant prolongation in reaction time (R) and clot propagation (K) and significant decreases in clot kinetics (alpha) and clot strength (MA and G). Analysis of fixed effects of treatment group on coagulation showed that clot propagation (K, alpha) and clot strength (MA and G) are significantly different in HBOC-200 (high methemoglobin) compared with HBOC-200 (low methemoglobin). CONCLUSIONS High methemoglobin concentrations in HBOC-200 cause additive coagulation impairment that likely results from the effects of oxidative substances on platelet function and coagulation proteins. Oxidative products adversely react with coagulation factors and modify redox-sensitive sites in the platelets. Therefore, if methemoglobinemia occurs as a result of HBOC administration and if the levels are significantly elevated (greater than 10%), impairment of coagulation is possible.

Does Hextend Impair Coagulation Compared to 6% Hetastarch? An Ex Vivo Thromboelastography Study

The objective of this study was to determine if coagulation is different between 6% hetastarch in normal saline (NS) and 6% hetastarch in lactated Ringers solution (LR), with use of an ex vivo thromboelastography (TEG) model with healthy donated volunteer blood. We simulated hemodilution that occurs during clinical resuscitation of hemorrhagic or hypovolemic shock, using healthy human donor whole blood (WB) ex vivo. Coagulopathy related to low, medium, high, or very high dilution of WB with NS or a high-molecular-weight hetastarch-based plasma expander, 6% hetastarch in NS (HSNS) or 6% hetastarch in lactated Ringers [Hextend (HSLR)], was analyzed by thromboelastography (TEG). No changes were noted in the TEG profile of undiluted WB controls during the 6-hour period of use (P > 0.95). Dilution with HSNS and HSLR significantly impaired coagulation compared to both WB control and NS. Progressive dilution with NS impaired coagulation but to a lesser extent than colloids (P < 0.01). Low dilution of blood with NS increased clot strength by 12% (not significant; P = 0.097). We conclude that WB containing citrate obtained from healthy donors for TEG analysis yields reproducible data over a minimum of 6 hours. Either hetastarch, when present at concentrations comparable to the manufacturers maximum recommended dose of 20 mL/kg (equivalent to the high dilution used in these experiments), decreases clot tensile strength to levels associated with an increased risk of bleeding. Substitution of lactated Ringers for NS in 6% hetastarch appears to offer no advantage in avoiding hemostatic compromise in an in vitro model.