The Helicobacter pylori UreI protein: role in adaptation to acidity and identification of residues essential for its activity and for acid activation.

Helicobacter pylori is a human gastric pathogen that survives the strong acidity of the stomach by virtue of its urease activity. This activity produces ammonia, which neutralizes the bacterial microenvironment. UreI, an inner membrane protein, is essential for resistance to low pH and for the gastric colonization of mice by H. pylori. In the heterologous Xenopus oocytes expression system, UreI behaves like an H+-gated urea channel, and His-123 was found to be important for low pH activation. We investigated the role of UreI directly in H. pylori and showed that, in the presence of urea, strains expressing wild-type UreI displayed very rapid stimulation of extracellular ammonia production upon exposure to pH

[UreI: a Helicobacter pylori protein essential for resistance to acidity and for the early steps of murine gastric mucosa infection]. / UreI: une protéine de Helicobacter pylori essentielle à la résistance à l'acidité et aux étapes précoces de l'infection de la muqueuse gastrique murine.

UNLABELLED: Helicobacter pylori (H. pylori) is a Gram negative microaerophilic bacteria whose only known niche is the human gastric mucosa. The presence of H. pylori is associated with various pathologies ranging from peptic ulcer disease to gastric carcinoma. H. pylori virulence is dependent on its exceptional ability to resist to the stomach acidity by hydrolyzing urea into ammonia. Survival of H. pylori to acidity in the presence of urea relies on the activity of a membrane protein, UreI. AIMS: We decided to better characterize the role of UreI (i) in vitro in ammonia production through the action of urease, and (ii) in vivo in the colonization of the gastric mucosa. METHODS: Ammonia production by a wild type strain of H. pylori or by a UreI-deficient strain was measured as a function of extracellular pH. In addition, the kinetics of elimination of a UreI-deficient mutant in vivo were realized in the mouse model for colonization. RESULTS: UreI was associated with an increase of ammonia production in acidic conditions in vitro and was necessary for the initial steps of the mouse stomach colonization. CONCLUSION: UreI thus behaves as a sensor of extracellular pH. This protein activates urease at acidic pH; thereby, it probably allows H. pylori to resist to acidity in vivo during the first steps of infection.

The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues.

Aliphatic amidases (EC 3.5.1.4) are enzymes catalysing the hydrolysis of short-chain amides to produce ammonia and the corresponding organic acid. Such an amidase, AmiE, has been detected previously in Helicobacter pylori. Analysis of the complete H. pylori genome sequence revealed the existence of a duplicated amidase gene that we named amiF. The corresponding AmiF protein is 34% identical to its AmiE paralogue. Because gene duplication is widely considered to be a fundamental process in the acquisition of novel enzymatic functions, we decided to study and compare the functions of the paralogous amidases of H. pylori. AmiE and AmiF proteins were overproduced in Escherichia coli and purified by a two-step chromatographic procedure. The two H. pylori amidases could be distinguished by different biochemical characteristics such as optimum pH or temperature. AmiE hydrolysed propionamide, acetamide and acrylamide and had no activity with formamide. AmiF presented an unexpected substrate specificity: it only hydrolysed formamide. AmiF is thus the first formamidase (EC 3.5.1.49) related to aliphatic amidases to be described. Cys-165 in AmiE and Cys-166 in AmiF were identified as residues essential for catalysis of the corresponding enzymes. H. pylori strains carrying single and double mutations of amiE and amiF were constructed. The substrate specificities of these enzymes were confirmed in H. pylori. Production of AmiE and AmiF proteins is dependent on the activity of other enzymes involved in the nitrogen metabolism of H. pylori (urease and arginase respectively). Our results strongly suggest that (i) the H. pylori paralogous amidases have evolved to achieve enzymatic specialization after ancestral gene duplication; and (ii) the production of these enzymes is regulated to maintain intracellular nitrogen balance in H. pylori.

[Bacteriology and pathogenicity of Helicobacter pylori]. / Bactériologie et pathogénicité d'Helicobacter pylori.

Helicobacter pylori is the prototype of bacteria belonging to a new genus, the Helicobacter genus. It is a gram-negative, highly motile and microaerophilic bacterium, with a spiral shape, that colonizes the human gastric mucosa and causes several gastroduodenal diseases. Pathogenicity of H. pylori relies upon its capacity to adapt to a hostile environment and to escape the host response. Resistance to acidity, motility, adhesion, molecular mimicry, resistance to phagocytosis, synthesis of a cytotoxin, induction of an inflammatory response are the major strategies developed by H. pylori to colonize persistently and damage gastric tissue.

The Helicobacter pylori UreI protein is not involved in urease activity but is essential for bacterial survival in vivo.

We produced defined isogenic Helicobacter pylori ureI mutants to investigate the function of UreI, the product of one of the genes of the urease cluster. The insertion of a cat cassette had a strong polar effect on the expression of the downstream urease genes, resulting in very weak urease activity. Urease activity, measured in vitro, was normal in a strain in which ureI was almost completely deleted and replaced with a nonpolar cassette. In contrast to previous reports, we thus found that the product of ureI was not necessary for the synthesis of active urease. Experiments with the mouse-adapted H. pylori SS1 strain carrying the nonpolar ureI deletion showed that UreI is essential for H. pylori survival in vivo and/or colonization of the mouse stomach. The replacement of ureI with the nonpolar cassette strongly reduced H. pylori survival in acidic conditions (1-h incubation in phosphate-buffered saline solution at pH 2.2) in the presence of 10 mM urea. UreI is predicted to be an integral membrane protein and may therefore be involved in a transport process essential for H. pylori survival in vivo.

Identification and characterization of an aliphatic amidase in Helicobacter pylori.

We report, for the first time, the presence in Helicobacter pylori of an aliphatic amidase that, like urease, contributes to ammonia production. Aliphatic amidases are cytoplasmic acylamide amidohydrolases (EC 3.5.1.4) hydrolysing short-chain aliphatic amides to produce ammonia and the corresponding organic acid. The finding of an aliphatic amidase in H. pylori was unexpected as this enzyme has only previously been described in bacteria of environmental (soil or water) origin. The H. pylori amidase gene amiE (1017 bp) was sequenced, and the deduced amino acid sequence of AmiE (37746Da) is very similar (75% identity) to the other two sequenced aliphatic amidases, one from Pseudomonas aeruginosa and one from Rhodococcus sp. R312. Amidase activity was measured as the release of ammonia by sonicated crude extracts from H. pylori strains and from recombinant Escherichia coli strains overproducing the H. pylori amidase. The substrate specificity was analysed with crude extracts from H. pylori cells grown in vitro; the best substrates were propionamide, acrylamide and acetamide. Polymerase chain reaction (PCR) amplification of an internal amiE sequence was obtained with each of 45 different H. pylori clinical isolates, suggesting that amidase is common to all H. pylori strains. A H. pylori mutant (N6-836) carrying an interrupted amiE gene was constructed by allelic exchange. No amidase activity could be detected in N6-836. In a N6-urease negative mutant, amidase activity was two- to threefold higher than in the parental strain N6. Crude extracts of strain N6 slowly hydrolysed formamide. This activity was affected in neither the amidase negative strain (N6-836) nor a double mutant strain deficient in both amidase and urease activities, suggesting the presence of an independent discrete formamidase in H. pylori. The existence of an aliphatic amidase, a correlation between the urease and amidase activities and the possible presence of a formamidase indicates that H. pylori has a large range of possibilities for intracellular ammonia production.

Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli.

We analysed the effects of the overproduction of parts or all of a multisubunit ATP-binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli. We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, sigma24, involved in the response to extracytoplasmic stresses. The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane. Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter. Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF. These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery. Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.