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1.
Cell Chem Biol ; 30(5): 499-512.e5, 2023 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-37100053

RESUMO

Respiratory complex I is a multicomponent enzyme conserved between eukaryotic cells and many bacteria, which couples oxidation of electron donors and quinone reduction with proton pumping. Here, we report that protein transport via the Cag type IV secretion system, a major virulence factor of the Gram-negative bacterial pathogen Helicobacter pylori, is efficiently impeded by respiratory inhibition. Mitochondrial complex I inhibitors, including well-established insecticidal compounds, selectively kill H. pylori, while other Gram-negative or Gram-positive bacteria, such as the close relative Campylobacter jejuni or representative gut microbiota species, are not affected. Using a combination of different phenotypic assays, selection of resistance-inducing mutations, and molecular modeling approaches, we demonstrate that the unique composition of the H. pylori complex I quinone-binding pocket is the basis for this hypersensitivity. Comprehensive targeted mutagenesis and compound optimization studies highlight the potential to develop complex I inhibitors as narrow-spectrum antimicrobial agents against this pathogen.


Assuntos
Helicobacter pylori , Humanos , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Mutagênese , Mutação , Oxirredução , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
2.
Mol Microbiol ; 116(3): 841-860, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34164854

RESUMO

Helicobacter pylori displays a worldwide infection rate of about 50%. The Gram-negative bacterium is the main reason for gastric cancer and other severe diseases. Despite considerable knowledge about the metabolic inventory of H. pylori, carbon fluxes through the citrate cycle (TCA cycle) remained enigmatic. In this study, different 13 C-labeled substrates were supplied as carbon sources to H. pylori during microaerophilic growth in a complex medium. After growth, 13 C-excess and 13 C-distribution were determined in multiple metabolites using GC-MS analysis. [U-13 C6 ]Glucose was efficiently converted into glyceraldehyde but only less into TCA cycle-related metabolites. In contrast, [U-13 C5 ]glutamate, [U-13 C4 ]succinate, and [U-13 C4 ]aspartate were incorporated at high levels into intermediates of the TCA cycle. The comparative analysis of the 13 C-distributions indicated an adaptive TCA cycle fully operating in the closed oxidative direction with rapid equilibrium fluxes between oxaloacetate-succinate and α-ketoglutarate-citrate. 13 C-Profiles of the four-carbon intermediates in the TCA cycle, especially of malate, together with the observation of an isocitrate lyase activity by in vitro assays, suggested carbon fluxes via a glyoxylate bypass. In conjunction with the lack of enzymes for anaplerotic CO2 fixation, the glyoxylate bypass could be relevant to fill up the TCA cycle with carbon atoms derived from acetyl-CoA.


Assuntos
Aminoácidos/metabolismo , Ciclo do Carbono , Carbono/metabolismo , Ácido Cítrico/metabolismo , Glucose/metabolismo , Helicobacter pylori/metabolismo , Acetilcoenzima A/metabolismo , Ácido Aspártico/metabolismo , Metabolismo dos Carboidratos , Ciclo do Ácido Cítrico , Ácido Glutâmico/metabolismo , Gliceraldeído/metabolismo , Glioxilatos/metabolismo , Infecções por Helicobacter/microbiologia , Humanos , Malatos/metabolismo , Redes e Vias Metabólicas , Ácido Succínico/metabolismo
3.
J Pharm Sci ; 110(3): 1302-1309, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33253724

RESUMO

Infections with Helicobacter pylori are a global challenge. Currently, H. pylori infections are treated systemically, but the eradication rates of the different therapy regimens are declining due to the growing number of bacterial strains resistant to major antibiotics. Here, we present a strategy for the local eradication of H. pylori by the use of Penicillin G sodium (PGS). In vitro experiments revealed that PGS shows high antibiotic activity against resistant strains of Helicobacter pylori with a minimum inhibitory concentration (MIC) of 0.125 µg/ml. In order to provide luminal concentrations above the MIC for longer periods of time, an extended release tablet was developed. Alkalizers were included to prevent acidic degradation of PGS within the tablet matrix. Out of the tested alkalizers MgO, l-Lysine, NaHCO3, and Na2CO3 NaHCO3 provided the strongest rise in pH inside the hydrated matrix when tested in simulated gastric fluid. Better PGS stability can mainly reasoned from that, addition of MgO resulted in high pH values within the matrix, causing basic degradation of PGS. This work is a first step towards the use of extended release tablets containing PGS for the local treatment of H. pylori as a safe and cost-effective alternative to common systemic treatment regimens.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Helicobacter/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana
4.
Front Cell Infect Microbiol ; 10: 602958, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33392108

RESUMO

Type IV secretion systems are protein secretion machineries that are frequently used by pathogenic bacteria to inject their virulence factors into target cells of their respective hosts. In the case of the human gastric pathogen Helicobacter pylori, the cytotoxin-associated gene (Cag) type IV secretion system is considered a major cause for severe disease, such as gastric cancer, and thus constitutes an attractive target for specific treatment options against H. pylori infections. Here, we have used a Cag type IV secretion reporter assay for screening a repurposing compound library for inhibitors targeting this system. We found that the antitumor agent cisplatin, a platinum coordination complex that kills target cells by formation of DNA crosslinks, is a potent inhibitor of the Cag type IV secretion system. Strikingly, we found that this inhibitory activity of cisplatin depends on a ligand exchange reaction which incorporates a solvent molecule (dimethylsulfoxide) into the complex, a modification which is known to be deleterious for DNA crosslinking, and for its anticancer activity. We extended our analysis to several analogous platinum complexes containing N-heterocyclic carbene, as well as DMSO or other ligands, and found varying inhibitory activities toward the Cag system which were not congruent with their DNA-binding properties, suggesting that protein interactions may cause the inhibitory effect. Inhibition experiments under varying conditions revealed effects on adherence and bacterial viability as well, and showed that the type IV secretion-inhibitory capacity of platinum complexes can be inactivated by sulfur-containing reagents and in complex bacterial growth media. Taken together, our results demonstrate DNA binding-independent inhibitory effects of cisplatin and other platinum complexes against different H. pylori processes including type IV secretion.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Antígenos de Bactérias , Proteínas de Bactérias/genética , Cisplatino/farmacologia , Humanos , Platina , Sistemas de Secreção Tipo IV
5.
PLoS One ; 11(5): e0154643, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27138472

RESUMO

The Dsb protein family is responsible for introducing disulfide bonds into nascent proteins in prokaryotes, stabilizing the structure of many proteins. Helicobacter pylori HP0231 is a Dsb-like protein, shown to catalyze disulfide bond formation and to participate in redox homeostasis. Notably, many H. pylori virulence factors are stabilized by the formation of disulfide bonds. By employing H. pylori HP0231 deficient strains we analyzed the effect of lack of this bacterial protein on the functionality of virulence factors containing putative disulfide bonds. The lack of H. pylori HP0231 impaired CagA translocation into gastric epithelial cells and reduced VacA-induced cellular vacuolation. Moreover, H. pylori HP0231 deficient bacteria were not able to colonize the gastric mucosa of mice, probably due to compromised motility. Together, our data demonstrate an essential function for H. pylori HP0231 in gastric colonization and proper function of bacterial virulence factors related to gastric pathology.


Assuntos
Helicobacter pylori/patogenicidade , Estômago/microbiologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Helicobacter pylori/metabolismo , Helicobacter pylori/fisiologia , Transporte Proteico , Vacúolos/microbiologia , Virulência
6.
Mol Microbiol ; 100(1): 188-203, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26713727

RESUMO

Bacterial type IV secretion systems are protein transporters with a remarkable diversity of substrates and substrate recognition mechanisms. Type IV-secreted proteins often contain C-terminal secretion signals, but may also require other regions for recognition as secretory substrates, or for full secretion efficiency. For example, type IV secretion of CagA, a major pathogenicity factor of the human gastric pathogen Helicobacter pylori, depends on a C-terminal signal and on N-terminal protein regions. To examine the involvement of individual CagA regions for type IV secretion efficiency, we have established and evaluated a ß-lactamase-dependent reporter system which allows quantitative determination of translocation into host cells. For validation, we used this reporter system to obtain quantitative data for type IV secretion of CagA variants with sequential C-terminal truncations. Alanine-scanning mutagenesis of the CagA C-terminus revealed that none of the characteristic charged residues in this region is necessary for type IV secretion. Translocation rates measured for CagA variants with N-terminal deletions show that CagA does not have an N-terminal signal sequence, but requires its N-terminal domain for efficient secretion. Finally, we provide evidence that only newly synthesized CagA protein is translocated, supporting a model in which type IV secretion is coupled to protein biosynthesis.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Helicobacter pylori/fisiologia , Sistemas de Secreção Tipo IV , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Transporte Biológico , Ativação Enzimática , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Deleção de Sequência , beta-Lactamases/metabolismo
7.
FEMS Microbiol Lett ; 355(2): 131-41, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24801548

RESUMO

The ubiquitous pathogen Listeria monocytogenes lives either saprophytically in the environment or within cells in a vertebrate host, thus adapting its lifestyle to its ecological niche. Growth experiments at 24 and 37 °C (environmental and host temperature) with ammonium or glutamine as nitrogen sources revealed that ammonium is the preferred nitrogen source of L. monocytogenes. Reduced growth on glutamine is more obvious at 24 °C. Global transcriptional microarray analyses showed that the most striking difference in temperature-dependent transcription was observed for central nitrogen metabolism genes, glnR (glutamine synthetase repressor GlnR), glnA (glutamine synthetase GlnA), amtB (ammonium transporter AmtB), glnK (PII regulatory protein GlnK), and gdh (glutamate dehydrogenase) when cells were grown on glutamine. When grown on ammonium, both at 24 and 37 °C, the transcriptional level of these genes resembles that of cells grown with glutamine at 37 °C. Electrophoretic mobility shift assay studies and qPCR analyses in the wild-type L. monocytogenes and the deletion mutant L. monocytogenes ∆glnR revealed that the transcriptional regulator GlnR is directly involved in temperature- and nitrogen source-dependent regulation of the respective genes. Glutamine, a metabolite known to influence GlnR activity, seems unlikely to be the (sole) intracellular signal mediating this temperature-and nitrogen source-dependent metabolic adaptation.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Nitrogênio/química , Temperatura , Proteínas de Bactérias/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
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