Acid Acclimation Research Articles

Probing the Nickel Coordination Environment in Helicobacter Pylori NikR

Nickel homeostasis in gram-negative bacteria is often regulated at the transcriptional level by the nickel-dependent protein NikR, which belongs to the ribbon-helix-helix DNA-binding protein family. Generally, NikR is found as a homotetramer, with four nickel ions bound of the tetrameric interface at a metal binding domain (MBD). In Helicobacter pylori, NikR (HpNikR), unlike homologous NikR proteins, sits at a regulatory hub and has been found to have a variety of regulatory roles. HpNikR has been implicated in the regulation of proteins that function in acid acclimation, nickel uptake, virulence, and stress response. There are a number of questions about the nickel coordination environment in HpNikR due to a series of puzzling and sometimes contradictory structural and experimental results. Here we present results from complimentary computational and experimental techniques to help further resolve these questions. Specifically, we use molecular dynamics simulations and spectroscopic experiments to investigate the nickel coordination environment. We use circular dichroism to observe differences in the thermal stability of HpNikR depending on the nickel:protein stoichiometry, with stabilization increasing as nickel concentration increases. Stability of HpNikR is found to be pH dependent. Elucidation of details about nickel coordination in HpNikR will improve our understanding of the functional role of genetic regulatory pathways in H. pylori pathogenicity.

Gastric Infection by Helicobacter pylori

Helicobacter pylori infects half of the world's population and plays a causal role in ulcer disease and gastric cancer. This pathogenic neutralophile uniquely colonizes the acidic gastric milieu through the process of acid acclimation. Acid acclimation is the ability of the organism to maintain periplasmic pH near neutrality in an acidic environment to prevent a fall in cytoplasmic pH in order to maintain viability and growth in acid. Recently, due to an increase in antibiotic resistance, the rate of H. pylori eradication has fallen below 80% generating renewed interest in novel eradication regimens and targets. In this article, we review the gastric biology of H. pylori and acid acclimation, various detection procedures, antibiotic resistance and the role that gastric acidity plays in the susceptibility of the organism to antibiotics currently in use and propose several novel drug targets that would promote eradication in the absence of antibiotics.

Protein Trafficking Involving the HP0165/HP0166 Two-Component System (TCS) of Helicobacter pylori Plays an Important Role in Acid Acclimation

Protein Trafficking Involving the HP0165/HP0166 Two-Component System (TCS) of Helicobacter pylori Plays an Important Role in Acid Acclimation

Regulatory circuits inHelicobacter pylori: network motifs and regulators involved in metal-dependent responses

The ability of Helicobacter pylori, one of the most successful human bacterial pathogens, to colonize the acidic gastric niche persistently, depends on the proper homeostasis of intracellular metal ions, needed as cofactors of essential metallo-proteins involved in acid acclimation, respiration and detoxification. This fundamental task is controlled at the transcriptional level mainly by the regulators Fur and NikR, involved in iron homeostasis and nickel response, respectively. Herein, we review the molecular mechanisms that underlie the activity of these key pleiotropic regulators. In addition, we will focus on their involvement in the transcriptional regulatory network of the bacterium, pinpointing a surprising complexity of network motifs that interconnects them and their gene targets. These motifs appear to confer versatile dynamics of metal-dependent responses by extensive horizontal connections between the regulators and feedback control of metal-cofactor availability.

Detailed In Vivo Analysis of the Role of Helicobacter pylori Fur in Colonization and Disease

Helicobacter pylori persistently colonizes the harsh and dynamic environment of the stomach in over one-half of the world's population and has been identified as a causal agent in a spectrum of pathologies that range from gastritis to invasive adenocarcinoma. The ferric uptake regulator (Fur) is one of the few regulatory proteins that has been identified in H. pylori. Fur regulates genes important for acid acclimation and oxidative stress and has been shown to be important for colonization of H. pylori in both murine and Mongolian gerbil models of infection. To more thoroughly define the role of Fur in vivo, we conducted an extensive temporal analysis of the location of, competitive ability of, and resultant pathology induced by a Deltafur strain in the Mongolian gerbil model of infection and compared the results to results for its wild-type parent. We found that at the earliest time points postinfection, significantly more Deltafur bacteria than wild-type bacteria were recovered. However, this trend was reversed by day 3, when there was significantly increased recovery of the wild-type strain. The increased recovery of the Deltafur strain at 1 day postinfection reflected increased recovery from both the corpus and the antrum of the stomach. When the wild-type strain was allowed to colonize first, the Deltafur strain was unable to compete for colonization at any time postinfection. However, when the Deltafur strain was allowed to colonize first, the wild type efficiently outcompeted the Deltafur strain only at early times postinfection. Finally, we demonstrated that there was a delay in the development and severity of inflammation and pathology of the Deltafur strain in the gastric mucosa even after comparable levels of colonization occurred. Together, these data indicate that H. pylori Fur is most important at early stages of infection and illustrate the importance of the ability of H. pylori to adapt to its constantly fluctuating environment when it is establishing infection, inflammation, and disease.

Gastric infection by Helicobacter pylori

Helicobacter pylori infection causes chronic active gastritis, ulcer disease, and gastric cancer. Current eradication regimens use a proton pump inhibitor (PPI) and two antibiotics. Triple therapy now has a success rate less than 80%, below the cutoff for efficacious eradication. Antibiotic resistance, inconsistent acid control by PPIs, and poor patient compliance contribute to the failure rate. H. pylori is a neutralophile that has developed special acid acclimation mechanisms to colonize its acidic gastric niche. Identifying the components of these mechanisms will provide novel bactericidal drug targets. Alternatively, better 24-hour acid control would increase the efficacy of antibiotics, leading to dual therapy with improved PPIs and amoxicillin. Studies of acid acclimation by H. pylori have identified several potential eradication targets including UreI, alpha-carbonic anhydrase, and a two-component system. Continuing improvement of PPIs has led to the development of at least three candidate drugs with improved 24-hour acid control.

Cloning, polymorphism, and inhibition of β-carbonic anhydrase of Helicobacter pylori

Carbonic anhydrase (CA) catalyzes the reversible hydration of CO(2) to bicarbonate and a proton, and alpha-class CA has been reported to facilitate the acid acclimation of Helicobacter pylori (hpalphaCA). The purpose of this study was to characterize the beta-class CA of H. pylori (hpbetaCA) and elucidate the role of this enzyme as a possible drug target for eradication therapy. We isolated DNA clones of independent H. pylori strains obtained from patients with gastritis (n = 15), gastric ulcer (n = 6), or gastric cancer (n = 16), and then studied genetic polymorphisms. In addition, the susceptibility of H. pylori to sulpiride, an antiulcer drug and efficient inhibitor of both hpalphaCA and hpbetaCA, was studied with an in vitro killing assay. DNA sequences of all 37 hpbetaCA clones encoded a 221 amino acid polypeptide with a variety of polymorphisms (57 types of amino acid substitution at 48 residue positions). There was no polymorphism functionally relevant to the gastric lesion type. One strain included unique residues that were not seen in the other 36 clones from Japanese patients but which were found in a strain obtained from the United Kingdom. Sulpiride had killing effects at concentrations greater than 200 microg/ml for H. pylori, including strains resistant to clarithromycin, metronidazole, or ampicillin. Helicobacter pylori might have evolved independently in the Caucasian and Japanese populations. Dual inhibition of alpha-and beta-class CAs could be applied as alternative therapy for eradication of H. pylori.

The pH-Responsive Regulon of HP0244 (FlgS), the Cytoplasmic Histidine Kinase of Helicobacter pylori

Helicobacter pylori colonizes the acidic gastric environment, in contrast to all other neutralophiles, whose acid resistance and tolerance responses allow only gastric transit. This acid adaptation is dependent on regulation of gene expression in response to pH changes in the periplasm and cytoplasm. The cytoplasmic histidine kinase, HP0244, which until now was thought only to regulate flagellar gene expression via its cognate response regulator, HP0703, was found to generate a response to declining medium pH. Although not required for survival at pH 4.5, HP0244 is required for survival at pH 2.5 with 10 mM urea after 30 min. Transcriptional profiling of a HP0244 deletion mutant grown at pH 7.4 confirmed the contribution of HP0244 to sigma(54) activation via HP0703 to coordinate flagellar biosynthesis by a pH-independent regulon that includes 14 flagellar genes. Microarray analysis of cells grown at pH 4.5 without urea revealed an additional 22 genes, including 4 acid acclimation genes (ureA, ureB, ureI, and amiE) that are positively regulated by HP0244. Additionally, 86 differentially expressed genes, including 3 acid acclimation genes (ureF, rocF [arginase], and ansB [asparaginase]), were found in cells grown at pH 2.5 with 30 mM urea. Hence, HP0244 has, in addition to the pH-independent flagellar regulon, a pH-dependent regulon, which allows adaptation to a wider range of environmental acid conditions. An acid survival study using an HP0703 mutant and an electrophoretic mobility shift assay with in vitro-phosphorylated HP0703 showed that HP0703 does not contribute to acid survival and does not bind to the promoter regions of several genes in the HP0244 pH-dependent regulon, suggesting that there is a pathway outside the HP0703 regulon which transduces the acid-responsive signal sensed by HP0244.

The α and β Classes Carbonic Anhydrases from Helicobacter pylori as Novel Drug Targets

Helicobacter pylori (H. pylori) successfully resides in the human stomach in highly acidic conditions, causing a variety of gastroduodenal lesions, including gastric ulcer, gastric cancer and MALT lymphoma. For acid acclimation of H. pylori, two types of enzymes, urease and carbonic anhydrase (CA), play a central role. They cooperatively function to maintain neutral pH in the bacterial cytoplasm and periplasm. The genome project of H. pylori identified two different classes of CA with different subcellular localization: a periplasmic alpha-class CA (hp alphaCA) and a cytoplasmic beta-class CA (hp betaCA). These two CAs are catalytically efficient with almost identical activity to that of the human isoform CA I for the CO(2) hydration reaction, and highly inhibited by many sulfonamides/sulfamates, including acetazolamide, ethoxzolamide, topiramate and sulpiride, all clinically used drugs. Furthermore, certain CA inhibitors, such as acetazolamide and methazolamide, were shown to inhibit the bacterial growth in vitro. Since the efficacy of eradication therapies currently employed has been decreasing due to drug resistance and side effects of the commonly used drugs, the dual inhibition of alpha- and/or beta-CAs of H. pylori could be applied as an alternative therapy in patients with H. pylori infection or for the prevention of gastroduodenal diseases provoked by this widespread pathogen.

Gene expressionin vivoshows thatHelicobacter pyloricolonizes an acidic niche on the gastric surface

Helicobacter pylori is a gastric-dwelling pathogen responsible, with acid secretion, for peptic ulcer and a 20-fold increase in the risk of gastric cancer. Several transcriptomes have been described after short-term exposure to acidity in vitro, but there are no data identifying the effects of chronic gastric exposure on bacterial gene expression. Comparison of the in vivo to the in vitro transcriptome at pH 7.4 identified several groups of genes of known function that increased expression >2-fold, and three of these respond both to acidity in vitro and to gastric infection. Almost all known acid acclimation genes are highly up-regulated. These include ureA, ureB, and rocF and the pH-gated urea channel, ureI. There is also up-regulation of two groups of motility and chemotaxis genes and for pathogenicity island genes, especially cagA, a predictor for pathogenicity. Most of these genes interact with HP0166, the response element of the pH-sensing two-component histidine kinase, HP0165/HP0166, ArsRS. Based on the pH profile of survival of ureI deletion mutants in vitro and their inability to survive in gastric acidity, the habitat of the organism at the gastric surface is acidic with a pH < or = 4.0. Hence, the pH of the habitat of H. pylori on the surface of the stomach largely determines the regulation of these specific groups of genes.

The HP0165-HP0166 Two-Component System (ArsRS) Regulates Acid-Induced Expression of HP1186 α-Carbonic Anhydrase in Helicobacter pylori by Activating the pH-Dependent Promoter

The periplasmic alpha-carbonic anhydrase of Helicobacter pylori is essential for buffering the periplasm at acidic pH. This enzyme is an integral component of the acid acclimation response that allows this neutralophile to colonize the stomach. Transcription of the HP1186 alpha-carbonic anhydrase gene is upregulated in response to low environmental pH. A binding site for the HP0166 response regulator (ArsR) has been identified in the promoter region of the HP1186 gene. To investigate the mechanism that regulates the expression of HP1186 in response to low pH and the role of the HP0165-HP0166 two-component system (ArsRS) in this acid-inducible regulation, Northern blot analysis was performed with RNAs isolated from two different wild-type H. pylori strains (26695 and 43504) and mutants with HP0165 histidine kinase (ArsS) deletions, after exposure to either neutral pH or low pH (pH 4.5). ArsS-dependent upregulation of HP1186 alpha-carbonic anhydrase in response to low pH was found in both strains. Western blot analysis of H. pylori membrane proteins confirmed the regulatory role of ArsS in HP1186 expression in response to low pH. Analysis of the HP1186 promoter region revealed two possible transcription start points (TSP1 and TSP2) located 43 and 11 bp 5' of the ATG start codon, respectively, suggesting that there are two promoters transcribing the HP1186 gene. Quantitative primer extension analysis showed that the promoter from TSP1 (43 bp 5' of the ATG start codon) is a pH-dependent promoter and is regulated by ArsRS in combating environmental acidity, whereas the promoter from TSP2 may be responsible for control of the basal transcription of HP1186 alpha-carbonic anhydrase.

Urea Transport in Bacteria: Acid Acclimation by Gastric Helicobacter spp

Urea transporters in bacteria are relatively rare. There are three classes, the ABC transporters such as those expressed by cyanobacteria and Corynebacterium glutamicum, the Yut protein expressed by Yersinia spp and the UreI expressed by gastric Helicobacter spp. This review focuses largely on the UreI proton-gated channel that is part of the acid acclimation mechanism essential for gastric colonization by the latter. UreI is a six-transmembrane polytopic integral membrane protein, N and C termini periplasmic, and is expressed in all gastric Helicobacter spp that have been studied but also in Helicobacter hepaticus and Streptococcus salivarius. The first two are proton-gated, the latter is pH insensitive. Site-directed mutagenesis and chimeric constructs have identified histidines and dicarboxylic amino acids in the second periplasmic loop of H. pylori and the first loop of H. hepaticus UreI and the C terminus of both as involved in a hydrogen-bonding dependence of proton gating, with the membrane domain in these but not in the UreI of S. salivarius responding to the periplasmic conformational changes. UreI and urease are essential for gastric colonization and urease associates with UreI during acid exposure, facilitating activation of the UreA and UreB apoenzyme complex by Ni2+ insertion by the UreF-UreH and UreE-UreG assembly proteins. Transcriptome analysis of acid responses of H. pylori also identified a cytoplasmic and periplasmic carbonic anhydrase as responding specifically to changes in periplasmic pH and these have been shown to be essential also for acid acclimation. The finding also of upregulation of the two-component histidine kinase HP0165 and its response element HP0166, illustrates the complexity of the acid acclimation processes involved in gastric colonization by this pathogen.

A thirty-year war against Helicobacter pylori: a personal reflection

The author personal results include therapeutic trials, the first Hungarian monograph on H. pylori, meta-analysis of the national and European eradication results and the first quality of life study in H. pylori-related functional dyspepsia. Less known are the interactions between H. pylori, carbonic anhydrase (CA) and its inhibitors. Gastric CA was described in 1939 by Horace W. Davenport. With the availability of acetazolamide in 1952 it was shown that it inhibits acid secretion in humans and dogs. Ioan Puca, from Szilágysomlyó, Romania, observed first, that in patients with chronic pulmonary obstructive disease, acetazolamide lead to a rapid healing of peptic ulcers. He patented in 1971 a drug containing acetazolamide + an electrolyte mixture named Ulcosilvanil and marketed it in Romania. Attempts to marketing it in Western countries were not successful; however, some trials were conducted in Hungary. Even the drug obtained high endoscopic healing and low recurrence rates of the peptic ulcers, the side effects and later, the development of H2 blockers and proton pump inhibitors led to its withdrawal in 1996. The author joined Puca's team between 1976–1990. To our greatest surprise, Sven Lindskog showed in 2002 that H. pylori expresses CA, which is inhibited by acetazolamide. Further, G. Sachs showed that CA is essential for the acid acclimation of H. pylori, while D. Y. Graham studied the effect of acetazolamide on H. pylori infection in vivo. These findings could explain the favorable effect of acetazolamide on peptic ulcer healing and the low recurrence rates found by us once upon a time. CA might either be a novel pharmacologic target, if specific inhibitors of the bacterial enzyme will be synthesized, or a potential antigen for vaccine development. For those presented above, the author considers that his fight against H. pylori lasted for thirty years: unfortunately, in the first 15 years, the enemy was not recognized.

Acid Acclimation byHelicobacter pylori

Helicobacter pylori is a Gram-negative neutralophile associated with peptic ulcers and gastric cancer. It has a unique ability to colonize the human stomach by acid acclimation. It uses the pH-gated urea channel, UreI, to enhance urea access to intrabacterial urease and a membrane-anchored periplasmic carbonic anhydrase to regulate periplasmic pH to approximately 6.1 in acidic media, whereas other neutralophiles cannot regulate periplasmic pH and thus only transit the stomach.

Identification of genes expressed in response to acid stress in Synechocystis sp. PCC 6803 using DNA microarrays

Plant cells are always exposed to various environmental stresses such as high light, low temperature and acid rain, and thus have to respond in order to survive these stresses. Although some mechanisms of responses to high light and low temperature etc., have been clarified, there is little information about the acclimation process to acid stress. In this study, the gene expression changes of Synechocystis sp. PCC 6803 in response to acid stress were examined using DNA microarrays (CyanoCHIP). We compared gene expression profiles of the cells treated at pH 8 (control) and pH 3 for 0.5, 1, 2 or 4 h. As a result, we found that 32 genes were upregulated by more than 3-fold, and 29 genes were downregulated by at least 3-fold after the acid treatment. Among these upregulated genes, expressions of slr0967 and sll0939 kept-increasing until 4 h under the acid stress and increased by 7 to 16-fold after the 4 h treatment. This suggests that the products of these two genes play important roles in the acid acclimation process.

The Periplasmic α-Carbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation

The role of the periplasmic alpha-carbonic anhydrase (alpha-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since alpha-CA catalyzes the conversion of CO2 to HCO3-, the role of CO2 in periplasmic buffering was studied using an alpha-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that alpha-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the alpha-CA knockout. In the mutant or in the presence of acetazolamide, there was an approximately 3 log10 decrease in acid survival. In acid, absence of alpha-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the alpha-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of alpha-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3- by the periplasmic alpha-CA.

Cryopreservation of in vitro sugar beet shoot tips using the encapsulation-dehydration technique: influence of abscisic acid and cold acclimation.

It has been previously shown that shoot tips of in vitro plantlets of sugar beet (Beta vulgaris L. clone SES1) can be cryopreserved using the encapsulation-dehydration technique (survival rate of 37% after freezing). This article reports the influence of abscisic acid (ABA) and cold acclimation on survival after cryopreservation. When ABA was added to the multiplication medium of the plants, the survival rate of shoot tips after cryopreservation was not increased (45%). After cold acclimation of the plants, their growth pattern differed (plants became apically dominant) and the survival rate of the shoot tips after cryopreservation clearly increased (70% survival and 50% plant regeneration after freezing). This improved protocol was successfully applied to three other clones.

Detection of Two Membrane Polypeptides Induced by Abscisic Acid and Cold Acclimation: Possible Role in Freezing Tolerance

Detection of Two Membrane Polypeptides Induced by Abscisic Acid and Cold Acclimation: Possible Role in Freezing Tolerance