Héctor Toledo

An Exported Rhodanese-Like Protein Is Induced during Growth of Acidithiobacillus ferrooxidans in Metal Sulfides and Different Sulfur Compounds

ABSTRACT By proteomic analysis we found a 21-kDa protein (P21) from Acidithiobacillus ferrooxidans ATCC 19859 whose synthesis was greatly increased by growth of the bacteria in pyrite, thiosulfate, elemental sulfur, CuS, and ZnS and was almost completely repressed by growth in ferrous iron. After we determined the N-terminal amino acid sequence of P21, we used the available preliminary genomic sequence of A. ferrooxidans ATCC 23270 to isolate the DNA region containing the p21 gene. The nucleotide sequence of this DNA fragment contained a putative open reading frame (ORF) coding for a 23-kDa protein. This difference in size was due to the presence of a putative signal peptide in the ORF coding for P21. When p21 was cloned and overexpressed in Escherichia coli, the signal peptide was removed, resulting in a mature protein with a molecular mass of 21 kDa and a calculated isoelectric point of 9.18. P21 exhibited 27% identity and 42% similarity to the Deinococcus radiodurans thiosulfate-sulfur transferase (rhodanese; EC 2.8.1.1) and similar values in relation to other rhodaneses, conserving structural domains and an active site with a cysteine, both characteristic of this family of proteins. However, the purified recombinant P21 protein did not show rhodanese activity. Unlike cytoplasmic rhodaneses, P21 was located in the periphery of A. ferrooxidans cells, as determined by immunocytochemical analysis, and was regulated depending on the oxidizable substrate. The genomic context around gene p21 contained other ORFs corresponding to proteins such as thioredoxins and sulfate-thiosulfate binding proteins, clearly suggesting the involvement of P21 in inorganic sulfur metabolism in A. ferrooxidans.

Novel genotypes in Helicobacter pylori involving domain V of the 23S rRNA gene

Background:  Helicobacter pylori is a pathogenic bacterium that infects a half of the human population. In Chile, between 55% and 79% of people are colonized by H. pylori. At present, therapeutic strategies to eradicate the bacterium depend on the knowledge of its resistance to antibiotics. The clarithromycin resistance in H. pylori is associated with point mutations in the 23S rRNA. This study analyzes 23S rRNA gene mutations and minimum inhibitory concentration (MIC) for clarithromycin in H. pylori isolates from patients of the metropolitan region of Chile.

Tetracycline resistance in Chilean clinical isolates of Helicobacter pylori

OBJECTIVES Since high-level tetracycline resistance in Helicobacter pylori has been associated with a AGA926-928-->TTC substitution in the 16S rRNA genes rrnA/B, the aim of the study was to screen for tetracycline resistance in H. pylori clinical isolates obtained from Santiago, Chile by using a recently reported molecular assay. METHODS A PCR-restriction fragment length polymorphism (PCR-RFLP) assay of the conserved 535 bp region of the H. pylori 16S rRNA genes rrnA/B (between nucleotides 710 and 1245) using HinfI was followed by DNA sequencing of the same fragment obtained from tetracycline-resistant H. pylori clinical isolates. RESULTS The PCR-RFLP assay revealed that the tetracycline-resistant H. pylori isolates lacked the AGA926-928-->TTC substitution. In contrast, DNA sequencing of the 535 bp PCR fragment from 11 tetracycline-resistant H. pylori Chilean clinical isolates showed an association of low-level tetracycline resistance with 1 bp (A928C) or 2 bp (AG926-927-->GT and/or A926G/A928C) substitutions in both 16S rRNA genes. CONCLUSIONS The PCR-RFLP (HinfI) assay alone is unreliable for the detection of tetracycline resistance in Chilean clinical isolates of H. pylori. To that end, it must be complemented by sequencing of the 535 bp PCR fragment.

Helicobacter pylori–Induced Loss of Survivin and Gastric Cell Viability Is Attributable to Secreted Bacterial Gamma-Glutamyl Transpeptidase Activity

Helicobacter pylori is the etiologic agent of a series of gastric pathologies that may culminate in the development of gastric adenocarcinoma. An initial step in this process is the loss of glandular structures in the gastric mucosa, presumably as the consequence of increased apoptosis and reduced cellular regeneration, which may be attributed to the combination of several bacterial and host factors and to an unfavorable proinflammatory environment. In a previous study, we showed that survivin, a member of the inhibitor of apoptosis protein family, is expressed in the normal human gastric mucosa and that its levels decrease in the mucosa of infected patients and in gastric cells exposed in culture to the bacteria, coincident with increased cell death in the latter case. We investigated the bacterial factors responsible for loss of survivin in gastric cells exposed to H. pylori. The results of this study indicated that the loss of survivin due to H. pylori infection involves proteasome-mediated degradation of the protein. Studies with isogenic mutants deficient in either CagA, VacA, lipopolysaccharide, or gamma-glutamyl transpeptidase (GGT) implicated the latter in H. pylori-induced loss of survivin and cell viability. Moreover, experiments with the GGT inhibitor 6-diazo-5-oxo-l-norleucine and purified recombinant GGT protein indicated that secreted bacterial GGT activity was required and sufficient to induce these effects.

Helicobacter pylori—Induced Loss of the Inhibitor-of-Apoptosis Protein Survivin Is Linked to Gastritis and Death of Human Gastric Cells

Helicobacter pylori infects the human stomach and modifies signaling pathways that affect gastric epithelial cell proliferation and viability. Chronic exposure to this pathogen contributes to the onset of gastric atrophy, an early event in the genesis of gastric cancer associated with H. pylori infection. Susceptibility to H. pylori-induced cell death ultimately depends on the presence of protective host cell factors. Although expression of the inhibitor-of-apoptosis protein survivin in adults is frequently linked to the development of cancer, evidence indicating that the protein is present in normal gastric mucosa is also available. Thus, we investigated in human gastric tissue samples and cell lines whether H. pylori infection is linked to loss of survivin and increased cell death. Our results show that infection with H. pylori decreased survivin protein levels in the mucosa of patients with gastritis. Furthermore, survivin down-regulation correlated with apoptosis and loss of cell viability in gastrointestinal cells cocultured with different H. pylori strains. Finally, overexpression of survivin in human gastric cells was sufficient to reduce cell death after infection. Taken together, these findings implicate survivin as an important survival factor in the gastric mucosa of humans.

TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility

Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility.

Overview on chemotaxis and acid resistance in Helicobacter pylori

429Corresponding author: H. Toledo. Programa de Biologia Celular y Molecular, ICBM, Facultad de Medicina, Universidad deChile, Casilla 70086, Santiago-7, Chile. Tel: (562) 678 6053 - Fax: (562) 735 5580 - e-mail: htoledo@machi.med.uchile.clReceived: April 2, 2003. In revised form: September 3, 2003. Accepted: September 17, 2003.

Dietary supplementation with selenomethylselenocysteine produces a differential proteomic response

Organic forms of selenium offer important health benefits including cancer prevention. Selenium intake has been traditionally quantified as glutathioneperoxidase activity or selenium concentration in blood or tissues. However, these indexes do not reflect organic selenium intake. Effect of dietary supplementation of rats with selenomethylselenocysteine on the blood plasma proteome was investigated in order to detect protein abundance differences between experimental (supranutritional selenium supplementation) and control [minimum selenium dose and sodium selenate instead of selenomethylselenocysteine (SeMSeCys)] groups. Four experimental groups and six control groups consisting of six rats each were fed with base diet supplemented with SeMSeCys or sodium selenate in different concentrations for different periods of time. A proteomic approach, comprising two-dimensional gel electrophoresis and mass spectrometry, was used to assess protein abundance in blood plasma. Statistically significant differences in the abundance of some proteins were detected in all the experimental groups. Four proteins were found to increase their abundance in response to the experimental conditions: apolipoprotein E, haptoglobin and alpha-1-antitrypsin abundance was related to the extent of supplementation period and transthyretin in response to SeMSeCys dose. Apolipoprotein E and transthyretin were not differentially expressed when diets were supplemented with sodium selenate instead of SeMSeCys. We postulate that these proteins are potential biomarkers of chemoprotective selenium intake.

The DNA-Binding Protein HU has a Regulatory Role in the Acid Stress Response Mechanism in Helicobacter pylori

Bacterial genomes are compacted by association with histone‐like proteins to form a complex known as bacterial chromatin. The histone‐like protein HU is capable of binding and bending the DNA molecule, a function related to compaction, protection, and regulation of gene expression. In Helicobacter pylori, HU is the only histone‐like protein described so far. Proteomic analysis from our laboratory showed that this protein is overexpressed under acidic stress.

tlpA gene expression is required for arginine and bicarbonate chemotaxis in Helicobacter pylori

About half of the human population is infected with Helicobacter pylori, a bacterium causing gastritis, peptic ulcer and progression to gastric cancer. Chemotaxis and flagellar motility are required for colonization and persistence of H. pylori in the gastric mucus layer. It is not completely clear which chemical gradients are used by H. pylori to maintain its position. TlpA, a chemotaxis receptor for arginine/ bicarbonate, has been identified. This study aimed to find out whether tlpA gene expression is required for the chemotactic response to arginine/bicarbonate. Wild-type motile H. pylori ATCC 700392 and H. pylori ATCC 43504, a strain having an interrupted tlpA gene, were used. Also, a tlpA-knockout mutant of H. pylori 700392 (H. pylori 700-tlpA::cat) was produced by homologous recombination. Expression of tlpA was assessed by a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay. Chemotaxis was measured as a Relative Chemotaxis Response (RCR) by a modified capillary assay. H. pylori 700392 presented chemotaxis to arginine and sodium bicarbonate. H. pylori 700-tlpA::cat showed neither tlpA gene expression nor chemotaxis towards arginine and bicarbonate. Besides confirming that TlpA is a chemotactic receptor for arginine/bicarbonate in H. pylori, this study showed that tlpA gene expression is required for arginine/bicarbonate chemotaxis.