Extragastric Manifestations of Helicobacter pylori Infection: Other Helicobacters

The finding that Helicobacter pylori is the main cause of gastritis and peptic ulcer disease has opened a new era in the gastrointestinal world. Today there is evidence that H. pylori may also play a role in different nongastric diseases, opening the new "extragastric manifestations of H. pylori infection" field. Concerning this, several studies have been published in the last year. The most convincing data arise from those investigating idiopathic thrombocytopenic purpura and sideropenic anemia, while there is also an increasing evidence for a possible association with atherosclerotic disease. Furthermore, the discovery of a number of other novel Helicobacter species has stimulated the research in different extragastric diseases, in which an infectious hypothesis is plausible. In particular, several species have been studied for a potential role in different liver and intestinal diseases with interesting findings.     

Trans-Translation in Helicobacter pylori: Essentiality of Ribosome Rescue and Requirement of Protein Tagging for Stress Resistance and Competence

Background

The ubiquitous bacterial trans-translation is one of the most studied quality control mechanisms. Trans-translation requires two specific factors, a small RNA SsrA (tmRNA) and a protein co-factor SmpB, to promote the release of ribosomes stalled on defective mRNAs and to add a specific tag sequence to aberrant polypeptides to direct them to degradation pathways. Helicobacter pylori is a pathogen persistently colonizing a hostile niche, the stomach of humans.

Principal Findings

We investigated the role of trans-translation in this bacterium well fitted to resist stressful conditions and found that both smpB and ssrA were essential genes. Five mutant versions of ssrA were generated in H. pylori in order to investigate the function of trans-translation in this organism. Mutation of the resume codon that allows the switch of template of the ribosome required for its release was essential in vivo, however a mutant in which this codon was followed by stop codons interrupting the tag sequence was viable. Therefore one round of translation is sufficient to promote the rescue of stalled ribosomes. A mutant expressing a truncated SsrA tag was viable in H. pylori, but affected in competence and tolerance to both oxidative and antibiotic stresses. This demonstrates that control of protein degradation through trans-translation is by itself central in the management of stress conditions and of competence and supports a regulatory role of trans-translation-dependent protein tagging. In addition, the expression of smpB and ssrA was found to be induced upon acid exposure of H. pylori.

Conclusions

We conclude to a central role of trans-translation in H. pylori both for ribosome rescue possibly due to more severe stalling and for protein degradation to recover from stress conditions frequently encountered in the gastric environment. Finally, the essential trans-translation machinery of H. pylori is an excellent specific target for the development of novel antibiotics.     

Peptides with indirect in vivo activity against an intracellular pathogen: selective lysis of infected macrophages.

A collection of natural peptides, simplified analogs of natural peptides, de novo amphipathic peptides and de novo amphipathic peptides composed of 50-80% alpha,alpha-dialkylated glycines (alpha,alpha-Dags) were synthesized on solid-phase resin as the C-terminus amides using N-alpha-fluorenylmethyloxycarbonyl protection. The synthesis of the peptides rich in alpha,alpha-Dags used acid fluoride coupling methods. The peptides show antimicrobial activity against Escherichia coli and Staphylococcus aureus but no direct antimicrobial activity against Brucella abortus at 100 microm in vitro. However, in vivo treatment with several of these peptides results in significant reductions of B. abortus in chronically infected immune BALB/c mice relative to infected control animals. The chronically infected mice were susceptible to peptide toxicity at much lower peptide doses than control animals. The highest nonlethal dose for infected mice was only 25 microg for melittin, whereas 500 microg doses were nonlethal for many of the other peptides. Several of the alpha,alpha-Dag-rich peptides selectively destroy B. abortus-infected murine macrophages in vitro. Thus, these peptides apparently reduce the bacterial load in vivo by destroying a portion of the infected macrophages and exposing the sequestered bacteria to the immune response in the mice.     

Comparative sialomics between hard and soft ticks: implications for the evolution of blood-feeding behavior

Ticks evolved various mechanisms to modulate their host's hemostatic and immune defenses. Differences in the anti-hemostatic repertoires suggest that hard and soft ticks evolved anti-hemostatic mechanisms independently, but raise questions on the conservation of salivary gland proteins in the ancestral tick lineage. To address this issue, the sialome (salivary gland secretory proteome) from the soft tick, Argas monolakensis, was determined by proteomic analysis and cDNA library construction of salivary glands from fed and unfed adult female ticks. The sialome is composed of approximately 130 secretory proteins of which the most abundant protein folds are the lipocalin, BTSP, BPTI and metalloprotease families which also comprise the most abundant proteins found in the salivary glands. Comparative analysis indicates that the major protein families are conserved in hard and soft ticks. Phylogenetic analysis shows, however, that most gene duplications are lineage specific, indicating that the protein families analyzed possibly evolved most of their functions after divergence of the two major tick families. In conclusion, the ancestral tick may have possessed a simple (few members for each family), but diverse (many different protein families) salivary gland protein domain repertoire.     

Probing conformational changes in orphan nuclear receptor: the NGFI-B intermediate is a partially unfolded dimer

Human nerve growth factor-induced B (NGFI-B) is a member of the NR4A subfamily of orphan nuclear receptors (NRs). Lacking identified ligands, orphan NRs show particular co-regulator proteins binding properties, different from other NRs, and they might have a non-classical quaternary organization. A body of evidence suggests that NRs recognition of and binding to ligands, DNA, homo- and heterodimerization partners and co-regulator proteins involve significant conformational changes of the NR ligand-binding domains (LBDs). To shed light on largely unknown biophysical properties of NGFI-B, here we studied structural organization and unfolding properties of NGFI-B ligand (like)-binding domain induced by chemical perturbation. Our results show that NGFI-B LBD undergoes a two-state guanidine hydrochloride (GndHCl) induced denaturation, as judged by changes in the alpha-helical content of the protein monitored by circular dichroism spectroscopy (CD). In contrast, changes in the tertiary structure of NGFI-B LBD, reported by intrinsic fluorescence, reveal a clear intermediate state. Additionally, SAXS results demonstrate that the intermediate observed by intrinsic fluorescence is a partially folded homodimeric structure, which further unfolds without dissociation at higher GndHCl concentrations. This partially unfolded dimeric assembly of NGFI-B LBD might resemble an intermediate that this domain access momentarily in the native state upon interactions with functional partners.     

Distribution of ectonucleotidases in the rodent brain revisited

Nucleotides comprise a major class of signaling molecules in the nervous system. They can be released from nerve cells, glial cells, and vascular cells where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and also adenosine is controlled and modulated by cell-surface-located enzymes (ectonucleotidases) that hydrolyze the nucleotide to the respective nucleoside. Extracellular hydrolysis of nucleotide ligands involves a considerable number of enzymes with differing catalytic properties differentially affecting the nucleotide signaling pathway. It is therefore important to investigate which type of ectonucleotidase(s) contributes to the control of nucleotide signaling in distinct cellular and physiological settings. By using a classical enzyme histochemical approach and employing various substrates, inhibitors, and knockout animals, we provide, for the first time, a comparative analysis of the overall distribution of catalytic activities reflecting four ectonucleotidase families: ecto-5′-nucleotidase, alkaline phosphatases, ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), and ectonucleotide pyrophyphatases/phosphodiesterases (E-NPPs). We place into perspective the earlier literature and provide novel evidence for a parenchymal localization of tissue non-specific alkaline phosphatase, E-NPPs, and E-NTPDases in the mouse brain. In addition, we specify the location of ectonucleotidases within the brain vasculature. Most notably, brain vessels do not express ecto-5′-nucleotidase. The preponderance of individual enzymes differs considerably between brain locations. The contribution of all types of ectonucleotidases thus needs to be considered in physiological and pharmacological studies of purinergic signaling in the brain. This work was supported by the Deutsche Forschungsgemeinschaft (140/17-3).     

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56-66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei -- the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.     

Biotransformation of Biphenyl by Paecilomyces lilacinus and Characterization of Ring Cleavage Products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4′-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

Dehalogenation of Chlorinated Hydroxybiphenyls by Fungal Laccase

We have investigated the transformation of chlorinated hydroxybiphenyls by laccase produced by Pycnoporus cinnabarinus. The compounds used were transformed to sparingly water-soluble colored precipitates which were identified by gas chromatography-mass spectrometry as oligomerization products of the chlorinated hydroxybiphenyls. During oligomerization of 2-hydroxy-5-chlorobiphenyl and 3-chloro-4-hydroxybiphenyl, dechlorinated C—C-linked dimers were formed, demonstrating the dehalogenation ability of laccase. In addition to these nonhalogenated dimers, both monohalogenated and dihalogenated dimers were identified.