Phenylphosphonate transport by Helicobacter pylori

BACKGROUND: Helicobacter pylori can utilize phenylphosphonate as a sole source of phosphorus, and it is able to transport the phosphonate N-phosphonoacetyl-L-aspartate. However, H. pylori does not have any genes homologous to those of the known pathways for phosphonate degradation in bacteria, indicating that it must have novel pathways for the transport and metabolism of phosphonates. METHODS: Phenylphosphonate transport by H. pylori was studied in strains LC20, J99 and N6 by the centrifugation through oil method using [(14)C]-labeled phenylphosphonate. RESULTS: The Michaelis constants of transport K(t) and V(max) for phenylphosphonate showed similar kinetics in the three strains. The Arrhenius plot for phenylphosphonate transport rates at permeant concentrations of 50 micromol/L was linear over the temperature range 10-40 degrees C with an activation energy of 3.5 kJ/mol, and a breakpoint between 5 and 10 degrees C. Transport rates increased with monovalent cation size. The effects of various inhibitors were investigated: iodoacetamide, amiloride, valinomycin, and nigericin reduced the rate of phenylphosphonate transport; sodium azide and sodium cyanide increased the transport rate; and monensin had no effect. CONCLUSIONS: The kinetics and properties of H. pylori phenylphosphonate transport were characterized, and the data suggested a carrier-mediated transport mechanism.     

Phosphonate catabolism by <Emphasis Type="Italic">Campylobacter</Emphasis> spp.

The catabolism of phosphonates (Phn) by Campylobacter spp. was investigated employing nuclear magnetic resonance spectroscopy and cell culture techniques. The bacteria were capable of cleaving the Phn bonds of different compounds, including -aminomethylphosphonate, phosphonoacetate and phenylphosphonate (PhePhn). The kinetic parameters of these activities were determined in vivo in intact cells and in situ in whole-cell lysates. Cleavage of Phn-bearing compounds was associated with the cell-wall and cytosolic fractions. Results from substrate competition experiments suggested that at least two enzyme activities appeared to be involved in the cleavage of carbon–phosphate (C–P) bonds. In silico analyses indicated that no genes orthologous to those encoding C–P bond-cleaving enzymes in other bacteria were present in the Campylobacter jejuni genome. In most bacteria studied, Phn catabolism is induced under conditions of phosphate limitation; however, in Campylobacter spp. these activities were expressed in cells grown in media rich in phosphate. In chemically defined media, PhePhn supported bacterial growth and proliferation at concentrations above 100 M in the absence of phosphate. Thus, Phn utilisation may be a survival mechanism of Campylobacter spp. in milieux lacking sufficient phosphate. The expression of these enzyme activities in media abundant in phosphate suggested also that they may have other physiological roles.     

Pyruvate metabolism in Helicobacter pylori

The metabolism of pyruvate by Helicobacter pylori was investigated employing one- and two-dimensional ¹H and ¹³C nuclear magnetic resonance spectroscopy. Generation of pyruvate from l-serine in incubations with whole cell lysates indicated the presence of serine dehydratase activity in the bacterium. Pyruvate was formed also in cell suspensions and lysates from phosphoenol pyruvate. Metabolically competent cells incubated aerobically with pyruvate yielded alanine, lactate, acetate, formate, and succinate. The production of alanine and lactate indicated the presence of alanine transaminase and lactate dehydrogenase activities, respectively. Accumulation of acetate and formate as metabolic products provided evidence for the existence of a mixed-acid fermentation pathway in the microorganism. Formation of succinate suggested the incorporation of the pyruvate carbon skeleton into the Kreb's cycle. Addition of pyruvate to various liquid culture media did not affect bacterial growth or loss of viability. The variety of products formed using pyruvate as the sole substrate showed the important role of this metabolite in the energy metabolism of H. pylori.     

Global and seasonal variation of marine phosphonate metabolism

Marine microbial communities rely on dissolved organic phosphorus (DOP) remineralisation to meet phosphorus (P) requirements. We extensively surveyed the genomic and metagenomic distribution of genes directing phosphonate biosynthesis, substrate-specific catabolism of 2-aminoethylphosphonate (2-AEP, the most abundant phosphonate in the marine environment), and broad-specificity catabolism of phosphonates by the C-P lyase (including methylphosphonate, a major source of methane). We developed comprehensive enzyme databases by curating publicly available sequences and then screened metagenomes from TARA Oceans and Munida Microbial Observatory Time Series (MOTS) to assess spatial and seasonal variation in phosphonate metabolism pathways. Phosphonate cycling genes were encoded in diverse gene clusters by 35 marine bacterial and archaeal classes. More than 65% of marine phosphonate cycling genes mapped to Proteobacteria with production demonstrating wider taxonomic diversity than catabolism. Hydrolysis of 2-AEP was the dominant phosphonate catabolism strategy, enabling microbes to assimilate carbon and nitrogen alongside P. Genes for broad-specificity catabolism by the C-P lyase were far less widespread, though enriched in the extremely P-deplete environment of the Mediterranean Sea. Phosphonate cycling genes were abundant in marine metagenomes, particularly from the mesopelagic zone and winter sampling dates. Disparity between prevalence of substrate-specific and broad-specificity catabolism may be due to higher resource expenditure from the cell to build and retain the C-P lyase. This study is the most comprehensive metagenomic survey of marine microbial phosphonate cycling to date and provides curated databases for 14 genes involved in phosphonate cycling.Subject terms: Water microbiology, Microbial ecology, Microbial biooceanography, Metagenomics     

Phosphate-limited ocean regions select for bacterial populations enriched in the carbon–phosphorus lyase pathway for phosphonate degradation

In tropical and subtropical oceanic surface waters phosphate scarcity can limit microbial productivity. However, these environments also have bioavailable forms of phosphorus incorporated into dissolved organic matter (DOM) that microbes with the necessary transport and hydrolysis metabolic pathways can access to supplement their phosphorus requirements. In this study we evaluated how the environment shapes the abundance and taxonomic distribution of the bacterial carbon–phosphorus (C–P) lyase pathway, an enzyme complex evolved to extract phosphate from phosphonates. Phosphonates are organophosphorus compounds characterized by a highly stable C–P bond and are enriched in marine DOM. Similar to other known bacterial adaptions to low phosphate environments, C–P lyase was found to become more prevalent as phosphate concentrations decreased. C–P lyase was particularly enriched in the Mediterranean Sea and North Atlantic Ocean, two regions that feature sustained periods of phosphate depletion. In these regions, C–P lyase was prevalent in several lineages of Alphaproteobacteria (Pelagibacter, SAR116, Roseobacter and Rhodospirillales), Gammaproteobacteria, and Actinobacteria. The global scope of this analysis supports previous studies that infer phosphonate catabolism via C–P lyase is an important adaptive strategy implemented by bacteria to alleviate phosphate limitation and expands the known geographic extent and taxonomic affiliation of this metabolic pathway in the ocean.     

The use of AlbuMAX II(®) as a blood or serum alternative for the culture of Helicobacter pylori

Background: Growth of Helicobacter pyloriin vitro depends on supplementation of the medium with blood or serum. However, these supplements often require frozen storage and can show batch‐to‐batch variation, resulting in differences in bacterial growth. In this study, we introduce the use of a commercially available, lipid‐rich supplement called AlbuMAX II^® (Gibco BRL, Grand Island, NY, USA) for use as a serum/blood replacement for H. pylori culture. Materials and Methods: The growth of H. pylori on solid and liquid media was examined by comparing growth after supplementation with horse blood, fetal calf serum, β‐cyclodextrin or AlbuMAX II^® (Gibco BRL). Human gastric adenocarcinoma (AGS) cellular responses to H. pylori were measured by NF‐κB luciferase assays and IL‐8 ELISA. Results: We show that the growth of H. pylori on both solid and liquid media containing AlbuMAX II^® (Gibco BRL) were comparable to levels obtained on blood agar or liquid media supplemented with serum. Growth was consistently higher in media supplemented with AlbuMAX II^® (Gibco BRL) than media containing β‐cyclodextrin. Furthermore, bacteria grown in AlbuMAX II^® (Gibco BRL) induced proinflammatory responses in AGS cells. Conclusions: AlbuMAX II^® (Gibco BRL) can be used as a serum/blood replacement for the cultivation of H. pylori in solid and liquid media. This medium could be useful for an improved understanding of H. pylori metabolism or for antigen production. Furthermore, AlbuMAX II^® (Gibco BRL) may be suitable for use in remote locations, particularly in areas where frozen storage of serum may be a problem.     

Enzyme activities associated with arabitol and mannitol biosynthesis and catabolism in Schizophyllum commune

Enzymes of polyol metabolism were studied in basidiospore germination of Schizophyllum commune during periods of in vivo arabitol and mannitol pool depletion (growth on glucose-asparagine) and during their subsequent synthesis (growth on acetate-NH ₄ ⁺ ). Optimal conditions for assays were established and specific activities of enzymes employing d-arabitol, d-mannitol, d-ribulose, d-fructose and d-xylulose as substrates were traced. Inquiries into the products formed during these reactions showed that d-ribulose generated arabitol while d-fructose produced mannitol with d-xylulose giving rise to xylitol. The dehydrogenase reactions were further investigated using polyacrylamide disc gel electrophoresis. Here was revealed the existence of at least two separate enzymatic activities pertaining to the catabolism of arabitol and mannitol. Also noted were the electrophoretic patterns when d-sorbitol, ribitol, xylitol and ethanol were used as substrates.     

Effects of medium composition on extracellular proteinase stability and yield in batch cultures of a Thermus sp.

Summary Thermus sp. Rt41A produces an extracellular proteinase that is produced concomitant with growth and l-glutamate catabolism. Calcium-chelating medium components were shown to decrease the half-life of the proteinase in growing cultures. Medium modifications avoiding these components resulted in an increase in the half-life and in the peak level of proteinase. By adding the inorganic phosphate requirement for growth in anabolic amounts to pH-controlled batch cultures, stability of the proteinase in the medium was greatly enhanced and there was consequent improvement in the total proteinase yield. This approach also allowed a balanced increase in substrate and phosphate concentrations to increase the cell and proteinase yield in batch culture in an almost stoichiometric manner.Offprint requests to: H. W. Morgan     

Ligninolytic activity of Phanerochaete chrysosporium: Physiology of suppression by NH 4 + and l-glutamate

Previous research showed that addition of nutrient nitrogen to ligninolytic (stationary, nitrogen-starved) cultures of the wood-decomposing basidiomycete Phanerochaete chrysosporium causes a suppression of lignin degradation. The present study examined early effects on nitrogen metabolism that followed addition of NH ₄ ⁺ and l-glutamate at concentrations that yield similar patterns of suppression. Both nitrogenous compounds were rapidly assimilated (>80% in 6 h). Both caused an initial 80% or greater increase in the intracellular glutamate pool and had similar effects in increasing the specific activities of NADP- and NAD-glutamate dehydrogenases and glutamine synthetase. Differences between the effects of added NH ₄ ⁺ and glutamate showed that suppression was not correlated with intracellular pools of arginine or glutamine, nor was the maintenance of an elevated glutamate pool required to maintain the suppressed state. While a portion of the initial glutamate suppression could be attributed to an effect on central carbon metabolism through glutamate catabolism by NAD-glutamate dehydrogenase, the long term suppression by glutamate and the suppression by NH ₄ ⁺ were more specific. Suppression by NH ₄ ⁺ or glutamate in the presence or absence of protein synthesis (cycloheximide) followed essentially identical kinetics during 12 h. These results indicate that nitrogen additions cause a biochemical repression of enzymes associated with lignin degradation. Results are consistent with the hypothesis that nitrogen metabolism via glutamate plays a role in initiation of repression.Non-Standard Abbreviations DMS 2,2-dimethylsuccinate - TCA trichloroacetic acid     

Activities of muscadine grape skin and quercetin against Helicobacter pylori infection in mice

Aims: To explore the preventative potential of muscadine grape skin (MGS) and the single flavonoid, quercetin, as an alternative means for ameliorating Helicobacter pylori infection and/or the H. pylori‐induced inflammatory response in mice. Methods and Results: The antimicrobial and anti‐inflammatory properties of MGS and quercetin, a major phenolic constituent, were evaluated against H. pylori in vitro and in vivo. The antimicrobial activity of quercetin was evaluated against 11 H. pylori strains in vitro with inhibition of all strains at 128–64 μg ml^?1. In vivo studies showed a moderate reduction in H. pylori counts following treatment with 5 and 10% MGS or quercetin (25 mg kg^?1 body weight) in addition to significantly reduced inflammatory cytokines (TNF‐α, IL‐1β and IFN‐γ) when compared with untreated mice. Conclusions: MGS and quercetin did not significantly reduce H. pylori growth in a mouse model. However, these products were effective in regulating the inflammatory response to H. pylori infection. Significance and Impact of the Study: Our results suggest that H. pylori infection may be reduced or prevented via the consumption of fruits rich in certain phenolic compounds (e.g. quercetin) such as muscadine grapes.     

Dual roles of CagA protein in Helicobacterpylori-induced chronic gastritis in mice

Cytotoxin-associated gene A (CagA) acts directly on gastric epithelial cells. However, the roles of CagA in host adaptive immunity against Helicobacter pylori (H. pylori) infection are not fully understood. In this study, to investigate the roles of CagA in the development of H. pylori-induced chronic gastritis, we used an adoptive-transfer model in which spleen cells from C57BL/6 mice with or without H. pylori infection were transferred into RAG2^−/− mice, with gastric colonization of either CagA⁺H. pylori or CagA⁻H. pylori. Colonization of CagA⁺H. pylori but not CagA⁻H. pylori in the host gastric mucosa induced severe chronic gastritis in RAG2^−/− mice transferred with spleen cells from H. pylori-uninfected mice. In addition, when CagA⁺H. pylori-primed spleen cells were transferred into RAG2^−/− mice, CD4⁺ T cell infiltration in the host gastric mucosa were observed only in RAG2^−/− mice infected with CagA⁺H. pylori but not CagA⁻H. pylori, suggesting that colonization of CagA⁺H. pylori in the host gastric mucosa is essential for the migration of H. pylori-primed CD4⁺ T cells. On the other hand, transfer of CagA⁻H. pylori-primed spleen cells into CagA⁺H. pylori-infected RAG2^−/− mice induced more severe chronic gastritis with less Foxp3⁺ regulatory T-cell infiltration as compared to transfer of CagA⁺H. pylori-primed spleen cells. In conclusion, CagA in the stomach plays an important role in the migration of H. pylori-primed CD4⁺ T cells in the gastric mucosa, whereas CagA-dependent T-cell priming induces regulatory T-cell differentiation, suggesting dual roles for CagA in the pathophysiology of H. pylori-induced chronic gastritis.     

Interleukin-8 is the single most up-regulated gene in whole genome profiling of <Emphasis Type="Italic">H. pylori</Emphasis> exposed gastric epithelial cells

Background  

The association between Helicobacter pylori infection and upper gastrointestinal disease is well established. However, only a small fraction of H. pylori carriers develop disease, and there are great geographical differences in disease penetrance. The explanation to this enigma lies in the interaction between the bacterium and the host. H. pylori Outer Membrane Phospholipase A (OMPLA) has been suggested to play a role in the virulence of this bacterium. The aim of this study was to profile the most significant cellular pathways and biological processes affected in gastric epithelial cells during 24 h of H. pylori exposure, and to study the inflammatory response to OMPLA⁺ and OMPLA^- H. pylori variants.     

Ornithine dissimilation byTreponema denticola

Treponema denticola convertedl-ornithine, a product ofl-arginine catabolism, to putrescine via a decarboxylation reaction and to proline via a deamination reaction. Ornithine decarboxylation byT. denticola extracts was stimulated by pyridoxal 5′-phosphate. In the absence of pyridoxal 5′-phosphate, (NH₄)₂SO₄-fractionated extracts converted ornithine to proline and ammonia. This activity was not stimulated by α-keto acids, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide or ADP. Neither ornithine δ-transaminase (l-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13) nor Δ¹ reductase [l-proline: NAD(P) 5-oxidoreductase, EC 1.5.1.2.] activity was detectable in cell extracts. These results indicate that formation of proline from ornithine inT. denticola is catalyzed by an enzyme system analogous to the ornithine cyclase (deaminating) ofClostridium sporogenes. Exogenous ornithine inhibited the growth ofT. denticola. Thus, in addition to generating putrescine and proline, the ornithine dissimilatory pathways may serve to prevent accumulation of inhibitory concentrations of ornithine in the spirochete's environment.     

Catabolism of arginine byCarnobacterium spp. isolated from vacuum-packed sugar-salted fish

The ability ofCarnobacterium spp. originally isolated from vacuum-packed, sugar-salted fish to catabolize arginine was examined. All strains were able to produce citrulline, ornithine, and NH₃ from arginine, presumably by the arginine deiminase pathway. The metabolism of arginine was concurrent with acid production from glucose for one strain ofCarnobacterium sp. but delayed for one strain ofCarnobacterium piscicola. The arginine catabolism was not inhibited in the presence of 2% glucose for three strains of carnobacteria during growth in test broth and/or shrimp extract. Growth as well as arginine catabolism was delayed for two strains of carnobacteria by lowering the temperature from 9°C to 4°C. A similar result was obtained by incubating one strain ofC. piscicola in CO₂. None of the compoundsl-citrulline,l-ornithine hydrochloride, and (NH₄)₂SO₄ had any effect on growth or arginine catabolism of this strain. Neither did pH of the medium affect the time for initiation of arginine catabolism.     

A Thin‐Layer Liquid Culture Technique for the Growth of Helicobacter pylori

Background and Aims: Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin‐layer liquid culture technique for the growth of H. pylori. Methods: A thin‐layer liquid culture system was established by adding liquid media to a 90‐mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Results: Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI‐1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum‐free RPMI‐1640 supported the growth of H. pylori when supplemented with dimethyl‐β‐cyclodextrin (200 μg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD₆₀₀ with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD₆₀₀ contained 1.3 ± 0.1 × 10⁹ CFU/mL. γ‐Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin‐layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Conclusions: Thin‐layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.     

Functional elucidation of hypothetical proteins for their indispensable roles toward drug designing targets from Helicobacter pylori strain HPAG1

Helicobacter pylori is a flagellated and slow growing gram-negative bacterium that persistently infects about half of the entire world population. In present study, we examined the proteome of H. pylori strain HPAG1 for identification of key uncharacterized proteins toward their novel regulatory functions. The complete proteome of this strain consists of 1539 proteins, out of which 520 proteins are annotated as hypothetical. Based on the functional motifs in their primary sequences, we were able to classify 254 of these hypothetical proteins into 6 functional categories. Further, KEGG database was used to find the roles of these hypothetical proteins in several pathways and structural prediction was done by homology modeling methods. Thirty-three of these hypothetical proteins were found to have strong association in various pathways including signaling and defense mechanisms. We noted that 27 of these proteins are specific to H. pylori and can be selected for drug designing targets, based on their virulence and regulatory role. We were able to successfully model the 3D structures of three of these proteins: YP_626977.1, YP_626786.1, and YP_628146.1. The stability of these proteins was also validated using molecular dynamics simulations, and their possible role in the regulation of different pathways was explained. These novel annotations may contribute to the understanding of disease mechanism at molecular level and provide novel potential targets for designing new drugs against H. pylori strain HPAG1.     

Interactions between Helicobacter pylori infection and host metabolic homeostasis: A comprehensive review

The microbiota actively and extensively participates in the regulation of human metabolism, playing a crucial role in the development of metabolic diseases. Helicobacter pylori (H. pylori), when colonizing gastric epithelial cells, not only induces local tissue inflammation or malignant transformation but also leads to systemic and partial changes in host metabolism. These shifts can be mediated through direct contact, toxic components, or indirect immune responses. Consequently, they influence various molecular metabolic events that impact nutritional status and iron absorption in the host. Unraveling the intricate and diverse molecular interaction links between H. pylori and human metabolism modulation is essential for understanding pathogenesis mechanisms and developing targeted treatments for related diseases. However, significant challenges persist in comprehensively understanding the complex association networks among H. pylori itself, the infected host's status, the host microbiome, and the immune response. Previous metabolomics research has indicated that H. pylori infection and eradication may selectively shape the metabolite and microbial profiles of gastric lesions. Yet, it remains largely unknown how these diverse metabolic pathways, including isovaleric acid, cholesterol, fatty acids, and phospholipids, specifically modulate gastric carcinogenesis or affect the host's serum metabolism, consequently leading to the development of metabolic-associated diseases. The direct contribution of H. pylori to metabolisms still lacks conclusive evidence. In this review, we summarize recent advances in clinical evidence highlighting associations between chronic H. pylori infection and metabolic diseases, as well as its potential molecular regulatory patterns.     

A Novel Mechanism for Resistance to the Antimetabolite N-Phosphonoacetyl-l-Aspartate by Helicobacter pylori

The mechanism of resistance to N-phosphonoacetyl-l-aspartate (PALA), a potent inhibitor of aspartate carbamoyltransferase (which catalyzes the first committed step of de novo pyrimidine biosynthesis), in Helicobacter pylori was investigated. At a 1 mM concentration, PALA had no effects on the growth and viability of H. pylori. The inhibitor was taken up by H. pylori cells and the transport was saturable, with a K_m of 14.8 mM and a V_max of 19.1 nmol min⁻¹ μl of cell water⁻¹. By ³¹P nuclear magnetic resonance (NMR) spectroscopy, both PALA and phosphonoacetate were shown to have been metabolized in all isolates of H. pylori studied. A main metabolic end product was identified as inorganic phosphate, suggesting the presence of an enzyme activity which cleaved the carbon-phosphorus (C-P) bonds. The kinetics of phosphonate group cleavage was saturable, and there was no evidence for substrate inhibition at higher concentrations of either compound. C-P bond cleavage activity was temperature dependent, and the activity was lost in the presence of the metal chelator EDTA. Other cleavages of PALA were observed by ¹H NMR spectroscopy, with succinate and malate released as main products. These metabolic products were also formed when N-acetyl-l-aspartate was incubated with H. pylori lysates, suggesting the action of an aspartase. Studies of the cellular location of these enzymes revealed that the C-P bond cleavage activity was localized in the soluble fraction and that the aspartase activity appeared in the membrane-associated fraction. The results suggested that the two H. pylori enzymes transformed the inhibitor into noncytotoxic products, thus providing the bacterium with a mechanism of resistance to PALA toxicity which appears to be unique.Helicobacter pylori has been established as the causative agent of chronic gastritis and a significant proportion of duodenal and gastric ulcers (14). Recently, the World Health Organization classified H. pylori as a group 1 carcinogen, owing to its role in the development of gastric cancer (10). The failure of some regimens in the treatment of H. pylori infection has motivated work in our laboratory directed at characterizing the physiology of the bacterium, with the aim of discovering potential sites for therapeutic intervention, including nucleotide biosynthetic pathways (24, 25).Earlier studies on the uptake of nucleotide precursors by H. pylori showed that there was relatively little acquisition of pyrimidine nucleotide precursors by the salvage of preformed bases and nucleosides (24). Uracil, a commonly salvaged pyrimidine base, is also not required for the growth of this bacterium (34), suggesting that the majority of its pyrimidine nucleotides are synthesized through the de novo pathway. In contrast, humans can utilize the de novo or salvage pathway for the synthesis of pyrimidine nucleotides. Inhibitors of H. pylori de novo pyrimidine biosynthesis may therefore be potentially effective therapeutic drugs, as the host could still efficiently acquire its nucleotide requirements by salvage. This potential was demonstrated earlier by the finding that the inhibition of de novo pyrimidine biosynthesis at the second enzyme of this pathway, dihydroorotase, resulted in the killing of H. pylori cells (35).Aspartate carbamoyltransferase (ACTase) (EC 2.1.3.2) catalyzes the first committed step in the de novo formation of pyrimidine nucleotides and is a key regulatory enzyme in bacteria (8). N-Phosphonoacetyl-l-aspartate (PALA) is a synthetic, transition state bisubstrate analogue of the intermediate of the ACTase-catalyzed reaction (5). PALA belongs to a group of organophosphorus compounds known as phosphonates, characterized by their extremely stable carbon-phosphorus (C-P) bond in place of the more common carbon-oxygen-phosphorus ester bond (39), which confers on them the advantage of inherent stability. Natural phosphonates are found in phosphonolipids, glycolipids, glycoproteins, and polysaccharides of many different organisms. PALA and other synthetic phosphonates have been produced for use as herbicides, antibacterial agents (1, 28), and even as agents of chemical warfare (38).PALA is a potent inhibitor of the ACTase-catalyzed reaction in a range of prokaryotic and eukaryotic organisms, including Escherichia coli (5), Pyrococcus abyssi (33), and Leishmania donovani (29), and in mammalian cells (36). Owing to its stability and toxic effects on a key regulatory enzyme, PALA has been employed as an antitumor agent to inhibit the growth of rapidly proliferating cancer cells (9, 36). The inhibitor was also suggested as a possible antimetabolite for the protozoan pathogen L. donovani due to its cytotoxic effects on this organism (29). However, we have not found any detailed studies investigating the effects of PALA on the viability of bacterial cells. Recent results indicated that PALA is a potent inhibitor of ACTase activity in H. pylori, with 50% inhibition of enzyme activity observed at 0.1 μM PALA, and that PALA binds to the enzyme over 2,500 times more tightly than carbamoyl phosphate (3). This finding suggested that ACTase in H. pylori was a potential target for therapeutic intervention. However, initial results in our laboratory showed that PALA did not have inhibitory effects on the growth and viability of the bacterium.The aim of this work was to elucidate the mechanism(s) for H. pylori resistance to the potentially toxic effects of PALA. The effects on growth and viability, the transport of the inhibitor into whole cells, and the metabolic fate of this compound inside the cell were investigated by radiotracer analyses and nuclear magnetic resonance (NMR) spectroscopy.     

Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence

Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini-blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active β-lactamase fusion proteins (BlaM⁺) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line Katoill. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild-type strain.