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1.
Cell Commun Signal ; 19(1): 108, 2021 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-34742300

RESUMO

BACKGROUND: High temperature requirement A (HtrA) is an active serine protease secreted by the group-I carcinogen Helicobacter pylori (H. pylori). The human cell adhesion protein and tumor suppressor E-cadherin (hCdh1) expressed on the surface of gastric epithelial cells was identified as the first HtrA substrate. HtrA-mediated hCdh1 cleavage and subsequent disruption of intercellular adhesions are considered as important steps in H. pylori pathogenesis. In this study, we performed a proteomic profiling of H. pylori HtrA (HpHtrA) to decipher the complex mechanism of H. pylori interference with the epithelial barrier integrity. RESULTS: Using a proteomic approach we identified human desmoglein-2 (hDsg2), neuropilin-1, ephrin-B2, and semaphorin-4D as novel extracellular HpHtrA substrates and confirmed the well characterized target hCdh1. HpHtrA-mediated hDsg2 cleavage was further analyzed by in vitro cleavage assays using recombinant proteins. In infection experiments, we demonstrated hDsg2 shedding from H. pylori-colonized MKN28 and NCI-N87 cells independently of pathogen-induced matrix-metalloproteases or ADAM10 and ADAM17. CONCLUSIONS: Characterizing the substrate specificity of HpHtrA revealed efficient hDsg2 cleavage underlining the importance of HpHtrA in opening intercellular junctions. Video Abstract.

2.
Toxins (Basel) ; 13(9)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34564597

RESUMO

BACKGROUND: Helicobacter pylori (Hp) colonizes the human stomach and can induce gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. Clinical observations suggest a role for the Hp virulence factor cytotoxin-associated gene A (CagA) in pathogenesis. The pathogenic activity of CagA is partly regulated by tyrosine phosphorylation of C-terminal Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in host cells. However, CagA differs considerably in EPIYA motifs, whose functions have been well characterized in epithelial cells. Since CagA is fragmented in immune cells, different CagA variants may exhibit undetected functions in B cells. METHODS: B cells were infected with Hp isolates and isogenic mutants expressing different CagA EPIYA variants. CagA translocation and tyrosine phosphorylation were investigated by Western blotting. Apoptosis was analyzed by flow cytometry and metabolic activity was detected by an MTT assay. RESULTS: Isogenic CagA EPIYA variants are equally well translocated into B cells, followed by tyrosine phosphorylation and cleavage. B cell apoptosis was induced in a CagA-independent manner. However, variants containing at least one EPIYA-C motif affected metabolic activity independently of phosphorylation or multiplication of EPIYA-C motifs. CONCLUSIONS: The diverse structure of CagA regulates B cell physiology, whereas B cell survival is independent of CagA.

3.
Cell Commun Signal ; 18(1): 160, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33023610

RESUMO

BACKGROUND: Helicobacter pylori (H. pylori) is a gram-negative bacterium that chronically infects approximately 50% of the world's human population. While in most cases the infection remains asymptomatic, 10% of infected individuals develop gastric pathologies and 1-3% progress to gastric cancer. Although H. pylori induces severe inflammatory responses, the host's immune system fails to clear the pathogen and H. pylori can persist in the human stomach for decades. As suppressor of cytokine signaling (SOCS) proteins are important feedback regulators limiting inflammatory responses, we hypothesized that H. pylori could modulate the host's immune responses by inducing SOCS expression. METHODS: The phenotype of human monocyte-derived DCs (moDCs) infected with H. pylori was analyzed by flow cytometry and multiplex technology. SOCS expression levels were monitored by qPCR and signaling studies were conducted by means of Western blot. For functional studies, RNA interference-based silencing of SOCS1-3 and co-cultures with CD4+ T cells were performed. RESULTS: We show that H. pylori positive gastritis patients express significantly higher SOCS3, but not SOCS1 and SOCS2, levels compared to H. pylori negative patients. Moreover, infection of human moDCs with H. pylori rapidly induces SOCS3 expression, which requires the type IV secretion system (T4SS), release of TNFα, and signaling via the MAP kinase p38, but appears to be independent of TLR2, TLR4, MEK1/2 and STAT proteins. Silencing of SOCS3 expression in moDCs prior to H. pylori infection resulted in increased release of both pro- and anti-inflammatory cytokines, upregulation of PD-L1, and decreased T-cell proliferation. CONCLUSIONS: This study shows that H. pylori induces SOCS3 via an autocrine loop involving the T4SS and TNFα and p38 signaling. Moreover, we demonstrate that high levels of SOCS3 in DCs dampen PD-L1 expression on DCs, which in turn drives T-cell proliferation. Video Abstract.


Assuntos
Sistemas de Secreção Bacterianos , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Helicobacter pylori/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antígenos de Bactérias/metabolismo , Antígeno B7-H1/metabolismo , Proteínas de Bactérias/metabolismo , Proliferação de Células , Quimiocinas/metabolismo , Retroalimentação Fisiológica , Infecções por Helicobacter/metabolismo , Humanos , Janus Quinases/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Monócitos/metabolismo , Mutação/genética , Fosforilação , Transdução de Sinais , Receptores Toll-Like/metabolismo
4.
Biochemistry ; 59(39): 3772-3781, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32936629

RESUMO

Naturally occurring membranolytic antimicrobial peptides (AMPs) are rarely cell-type selective and highly potent at the same time. Template-based peptide design can be used to generate AMPs with improved properties de novo. Following this approach, 18 linear peptides were obtained by computationally morphing the natural AMP Aurein 2.2d2 GLFDIVKKVVGALG into the synthetic model AMP KLLKLLKKLLKLLK. Eleven of the 18 chimeric designs inhibited the growth of Staphylococcus aureus, and six peptides were tested and found to be active against one resistant pathogenic strain or more. One of the peptides was broadly active against bacterial and fungal pathogens without exhibiting toxicity to certain human cell lines. Solution nuclear magnetic resonance and molecular dynamics simulation suggested an oblique-oriented membrane insertion mechanism of this helical de novo peptide. Temperature-resolved circular dichroism spectroscopy pointed to conformational flexibility as an essential feature of cell-type selective AMPs.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Sequência de Aminoácidos , Desenho de Fármacos , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento
5.
Microorganisms ; 8(9)2020 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-32878302

RESUMO

Persistent infections with the human pathogen Helicobacter pylori (H. pylori) have been closely associated with the induction and progression of a wide range of gastric disorders, including acute and chronic gastritis, ulceration in the stomach and duodenum, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma. The pathogenesis of H. pylori is determined by a complicated network of manifold mechanisms of pathogen-host interactions, which involves a coordinated interplay of H. pylori pathogenicity and virulence factors with host cells. While these molecular and cellular mechanisms have been intensively investigated to date, the knowledge about outer membrane vesicles (OMVs) derived from H. pylori and their implication in bacterial pathogenesis is not well developed. In this review, we summarize the current knowledge on H. pylori-derived OMVs.

6.
Pathog Dis ; 78(7)2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866262

RESUMO

Persistent infections with the bacterial group-I carcinogen Helicobacter pylori (H. pylori) have been associated with a broad range of gastric disorders, including gastritis, ulceration, gastric cancer or mucosa-associated lymphoid tissue (MALT) lymphoma. Pathogenesis of H. pylori requires a balance between immune tolerance and defense. Although H. pylori induces inflammatory responses, the immune system cannot eliminate the pathogen. The detailed molecular mechanisms of how H. pylori interferes with cells of the immune system, in particular infiltrated B cells, are not well investigated. Previously, it was shown that the bacterial effector and oncoprotein cytotoxin-associated gene A (CagA) is delivered into B cells followed by its tyrosine-phosphorylation. To investigate the functional consequences in B cells colonized by CagA-positive H. pylori, we analyzed the global transcriptome of H. pylori-infected Mec-1 cells by RNA sequencing. We found 889 differentially expressed genes (DEGs) and validated JUN, FOSL2, HSPA1B, SRC, CXCR3, TLR-4, TNF-α, CXCL8, CCL2, CCL4, MHC class I and MHC class II molecules by qPCR, western blot, flow cytometry and ELISA assays. The H. pylori-specific mRNA expression signature reveals a downregulation of inflammation- and migration-associated genes, whereas central signal transduction regulators of cell survival and death are upregulated.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/fisiologia , Interações Hospedeiro-Patógeno/genética , Transcriptoma , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Infecções por Helicobacter/complicações , Infecções por Helicobacter/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Linfoma de Zona Marginal Tipo Células B/etiologia , Reprodutibilidade dos Testes , Neoplasias Gástricas/etiologia
7.
Sci Rep ; 10(1): 10563, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601479

RESUMO

Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Bactérias/metabolismo , Caderinas/metabolismo , Cobre/metabolismo , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Chaperonas Moleculares/metabolismo , Peptídeos/metabolismo , Proteômica/métodos , Serina Endopeptidases/metabolismo , Serina Proteases/metabolismo , Zinco/metabolismo
8.
Int J Mol Sci ; 21(11)2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32486097

RESUMO

Helicobacter pylori (H. pylori) is a stomach pathogen that persistently colonizes the gastric mucosa, often leading to chronic inflammation and gastric pathologies. Although infection with H. pylori is the primary risk factor for gastric cancer, the underlying mechanisms of pathogen persistence and consequential chronic inflammation are still not well understood. Conventional dendritic cells (cDCs), which are among the first immune cells to encounter H. pylori in the gastric lining, and the cytokines and chemokines they secrete, contribute to both acute and chronic inflammation. Therefore, this study aimed to unravel the contributions of specific signaling pathways within human CD1c+ cDCs (cDC2s) to the composition of secreted cytokines and chemokines in H. pylori infection. Here, we show that the type IV secretion system (T4SS) plays only a minor role in H. pylori-induced activation of cDC2s. In contrast, Toll-like receptor 4 (TLR4) signaling drives the secretion of inflammatory mediators, including IL-12 and IL-18, while signaling via TLR10 attenuates the release of IL-1ß and other inflammatory cytokines upon H. pylori infection. The TLR2 pathway significantly blocks the release of CXCL1 and CXCL8, while it promotes the secretion of TNFα and GM-CSF. Taken together, these results highlight how specific TLR-signaling pathways in human cDC2s shape the H. pylori-induced cytokine and chemokine milieu, which plays a pivotal role in the onset of an effective immune response.


Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Receptor 10 Toll-Like/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Antígenos CD1/metabolismo , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori , Humanos , Inflamação , Mediadores da Inflamação/metabolismo , Leucócitos Mononucleares/citologia , Transdução de Sinais , Neoplasias Gástricas/microbiologia
9.
Nanoscale ; 12(3): 2154-2155, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31912840

RESUMO

Correction for 'Nanoparticle binding attenuates the pathobiology of gastric cancer-associated Helicobacter pylori' by Dana Westmeier et al., Nanoscale, 2018, 10, 1453-1463.

10.
BMC Microbiol ; 19(1): 255, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31726993

RESUMO

BACKGROUND: High temperature requirement A (HtrA) is a widely expressed chaperone and serine protease in bacteria. HtrA proteases assemble and hydrolyze misfolded proteins to enhance bacterial survival under stress conditions. Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that induces listeriosis in humans. In previous studies, it was shown that deletion of htrA in the genome of L. monocytogenes increased the susceptibility to cellular stress and attenuated virulence. However, expression and protease activity of listerial HtrA (LmHtrA) were never analyzed in detail. RESULTS: In this study, we cloned LmHtrA wildtype (LmHtrAwt) and generated a proteolytic inactive LmHtrASA mutant. Recombinant LmHtrAwt and LmHtrASA were purified and the proteolytic activity was analyzed in casein zymography and in vitro cleavage assays. LmHtrA activity could be efficiently blocked by a small molecule inhibitor targeting bacterial HtrA proteases. The expression of LmHtrA was enhanced in the stationary growth phase of L. monocytogenes and significantly contributed to bacterial survival at high temperatures. CONCLUSIONS: Our data show that LmHtrA is a highly active caseinolytic protease and provide a deeper insight into the function and mechanism, which could lead to medical and biotechnological applications in the future.


Assuntos
Caseínas/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microbiologia de Alimentos , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/química , Resposta ao Choque Térmico , Listeria monocytogenes/patogenicidade , Viabilidade Microbiana , Dobramento de Proteína , Multimerização Proteica , Proteólise , Regulação para Cima
11.
Toxins (Basel) ; 11(10)2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614680

RESUMO

Helicobacter pylori (H. pylori) has been identified as a leading cause of gastric cancer, which is one of the most frequent and malignant types of tumor. It is characterized by its rapid progression, distant metastases, and resistance to conventional chemotherapy. A number of receptor tyrosine kinases and non-receptor tyrosine kinases have been implicated in H. pylori-mediated pathogenesis and tumorigenesis. In this review, recent findings of deregulated EGFR, c-Met, JAK, FAK, Src, and c-Abl and their functions in H. pylori pathogenesis are summarized.


Assuntos
Infecções por Helicobacter/enzimologia , Helicobacter pylori , Proteínas Tirosina Quinases/metabolismo , Neoplasias Gástricas/enzimologia , Animais , Infecções por Helicobacter/complicações , Humanos , Neoplasias Gástricas/etiologia
12.
Cell Commun Signal ; 17(1): 10, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30704478

RESUMO

BACKGROUND: Deregulated c-Abl activity has been intensively studied in a variety of solid tumors and leukemia. The class-I carcinogen Helicobacter pylori (Hp) activates the non-receptor tyrosine kinase c-Abl to phosphorylate the oncoprotein cytotoxin-associated gene A (CagA). The role of c-Abl in CagA-dependent pathways is well established; however, the knowledge of CagA-independent c-Abl processes is scarce. METHODS: c-Abl phosphorylation and localization were analyzed by immunostaining and immunofluorescence. Interaction partners were identified by tandem-affinity purification. Cell elongation and migration were analyzed in transwell-filter experiments. Apoptosis and cell survival were examined by FACS analyses and MTT assays. In mice experiments and human biopsies, the involvement of c-Abl in Hp pathogenesis was investigated. RESULTS: Here, we investigated the activity and subcellular localization of c-Abl in vitro and in vivo and unraveled the contribution of c-Abl in CagA-dependent and -independent pathways to gastric Hp pathogenesis. We report a novel mechanism and identified strong c-Abl threonine 735 phosphorylation (pAblT735) mediated by the type-IV secretion system (T4SS) effector D-glycero-ß-D-manno-heptose-1,7-bisphosphate (ßHBP) and protein kinase C (PKC) as a new c-Abl kinase. pAblT735 interacted with 14-3-3 proteins, which caused cytoplasmic retention of c-Abl, where it potentiated Hp-mediated cell elongation and migration. Further, the nuclear exclusion of pAblT735 attenuated caspase-8 and caspase-9-dependent apoptosis. Importantly, in human patients suffering from Hp-mediated gastritis c-Abl expression and pAblT735 phosphorylation were drastically enhanced as compared to type C gastritis patients or healthy individuals. Pharmacological inhibition using the selective c-Abl kinase inhibitor Gleevec confirmed that c-Abl plays an important role in Hp pathogenesis in a murine in vivo model. CONCLUSIONS: In this study, we identified a novel regulatory mechanism in Hp-infected gastric epithelial cells by which Hp determines the subcellular localization of activated c-Abl to control Hp-mediated EMT-like processes while decreasing cell death.


Assuntos
Apoptose , Movimento Celular , Helicobacter pylori/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Linhagem Celular Tumoral , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Humanos , Modelos Biológicos , Fosforilação , Fosfotreonina/metabolismo , Fosfotirosina/metabolismo , Proteína Quinase C/metabolismo , Transporte Proteico
13.
Nanoscale ; 10(3): 1453-1463, 2018 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-29303193

RESUMO

Enteric bacteria may cause severe diseases, including gastric cancer-associated Helicobacter pylori. Their infection paths overlap with the oro-gastrointestinal uptake route for nanoparticles, increasingly occurring during environmental or consumer/medical exposure. By comprehensive independent analytical methods, such as live cell fluorescence, electron as well as atomic force microscopy and elemental analysis, we show that a wide array of nanoparticles (NPs) but not microparticles form complexes with H. pylori and enteric pathogens without the need for specific functionalization. The NP-assembly that occurred rapidly was not influenced by variations in physiological temperature, though affected by the NPs' physico-chemical characteristics. Improved binding was observed for small NPs with a negative surface charge, whereas binding could be reduced by surface 'stealth' modifications. Employing human gastric epithelial cells and 3D-organoid models of the stomach, we show that NP-coating did not inhibit H. pylori's cellular attachment. However, even the assembly of non-bactericidal silica NPs attenuated H. pylori infection by reducing CagA phosphorylation, cytoskeletal rearrangement, and IL-8 secretion. Here we demonstrate that NP binding to enteric bacteria may impact their pathobiology which could be further exploited to rationally modulate the (patho)biology of microbes by nanomaterials.


Assuntos
Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidade , Nanopartículas/metabolismo , Neoplasias Gástricas/microbiologia , Aderência Bacteriana , Células Epiteliais/microbiologia , Mucosa Gástrica/citologia , Humanos , Organoides/microbiologia , Dióxido de Silício
14.
ACS Chem Biol ; 12(9): 2254-2259, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28763193

RESUMO

Certain cationic peptides interact with biological membranes. These often-complex interactions can result in peptide targeting to the membrane, or in membrane permeation, rupture, and cell lysis. We investigated the relationship between the structural features of membrane-active peptides and these effects, to better understand these processes. To this end, we employed a computational method for morphing a membranolytic antimicrobial peptide into a nonmembranolytic mitochondrial targeting peptide by "directed simulated evolution." The results obtained demonstrate that superficially subtle sequence modifications can strongly affect the peptides' membranolytic and membrane-targeting abilities. Spectroscopic and computational analyses suggest that N- and C-terminal structural flexibility plays a crucial role in determining the mode of peptide-membrane interaction.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Lipossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Sequência de Aminoácidos , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Células HeLa , Humanos , Mitocôndrias/metabolismo , Modelos Moleculares , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/crescimento & desenvolvimento
15.
Small ; 13(40)2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28799716

RESUMO

Specific interactions of peptides with lipid membranes are essential for cellular communication and constitute a central aspect of the innate host defense against pathogens. A computational method for generating innovative membrane-pore-forming peptides inspired by natural templates is presented. Peptide representation in terms of sequence- and topology-dependent hydrophobic moments is introduced. This design concept proves to be appropriate for the de novo generation of first-in-class membrane-active peptides with the anticipated mode of action. The designed peptides outperform the natural template in terms of their antibacterial activity. They form a kinked helical structure and self-assemble in the membrane by an entropy-driven mechanism to form dynamically growing pores that are dependent on the lipid composition. The results of this study demonstrate the unique potential of natural template-based peptide design for chemical biology and medicinal chemistry.


Assuntos
Peptídeos/química , Peptídeos Catiônicos Antimicrobianos/química , Biologia Computacional , Descoberta de Drogas
16.
Int J Mol Sci ; 18(7)2017 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-28677627

RESUMO

Birch pollen allergy is highly prevalent, with up to 100 million reported cases worldwide. Proteases in such allergen sources have been suggested to contribute to primary sensitisation and exacerbation of allergic disorders. Until now the protease content of Betula verrucosa, a birch species endemic to the northern hemisphere has not been studied in detail. Hence, we aim to identify and characterise pollen and bacteria-derived proteases found within birch pollen. The pollen transcriptome was constructed via de novo transcriptome sequencing and analysis of the proteome was achieved via mass spectrometry; a cross-comparison of the two databases was then performed. A total of 42 individual proteases were identified at the proteomic level. Further clustering of proteases into their distinct catalytic classes revealed serine, cysteine, aspartic, threonine, and metallo-proteases. Further to this, protease activity of the pollen was quantified using a fluorescently-labelled casein substrate protease assay, as 0.61 ng/mg of pollen. A large number of bacterial strains were isolated from freshly collected birch pollen and zymographic gels with gelatinase and casein, enabled visualisation of proteolytic activity of the pollen and the collected bacterial strains. We report the successful discovery of pollen and bacteria-derived proteases of Betula verrucosa.


Assuntos
Betula/enzimologia , Peptídeo Hidrolases/análise , Pólen/enzimologia , Alérgenos/análise , Alérgenos/imunologia , Betula/genética , Perfilação da Expressão Gênica , Humanos , Extratos Vegetais , Proteínas de Plantas/análise , Proteínas de Plantas/imunologia , Pólen/microbiologia , Proteólise , Proteoma , Proteômica/métodos , Rinite Alérgica Sazonal/imunologia , Transcriptoma
17.
Oxid Med Cell Longev ; 2017: 1320241, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28744336

RESUMO

Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas ("intestinal" and "diffuse"), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection.


Assuntos
Complexo I de Transporte de Elétrons/biossíntese , Gastrite/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Infecções por Helicobacter/enzimologia , Helicobacter pylori , Proteínas de Neoplasias/biossíntese , Fosforilação Oxidativa , Neoplasias Gástricas/enzimologia , Feminino , Gastrite/patologia , Infecções por Helicobacter/patologia , Humanos , Masculino , Neoplasias Gástricas/patologia
18.
Toxins (Basel) ; 9(4)2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28398251

RESUMO

Persistent infections with the human pathogen and class-I carcinogen Helicobacter pylori (H. pylori) are closely associated with the development of acute and chronic gastritis, ulceration, gastric adenocarcinoma and lymphoma of the mucosa-associated lymphoid tissue (MALT) system. Disruption and depolarization of the epithelium is a hallmark of H. pylori-associated disorders and requires extensive modulation of epithelial cell surface structures. Hence, the complex network of controlled proteolysis which facilitates tissue homeostasis in healthy individuals is deregulated and crucially contributes to the induction and progression of gastric cancer through processing of extracellular matrix (ECM) proteins, cell surface receptors, membrane-bound cytokines, and lateral adhesion molecules. Here, we summarize the recent reports on mechanisms how H. pylori utilizes a variety of extracellular proteases, involving the proteases Hp0169 and high temperature requirement A (HtrA) of bacterial origin, and host matrix-metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs) and tissue inhibitors of metalloproteinases (TIMPs). H. pylori-regulated proteases represent predictive biomarkers and attractive targets for therapeutic interventions in gastric cancer.


Assuntos
Infecções por Helicobacter/complicações , Helicobacter pylori/metabolismo , Peptídeo Hidrolases/metabolismo , Neoplasias Gástricas/etiologia , Animais , Humanos , Mucosa Intestinal , Proteólise
19.
Mol Inform ; 36(1-2)2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28124834

RESUMO

We present a "deep" network architecture for chemical data analysis and classification together with a prospective proof-of-concept application. The model features a self-organizing map (SOM) as the input layer of a feedforward neural network. The SOM converts molecular descriptors to a two-dimensional image for further processing. We implemented lateral neuron inhibition for contrast enhancement. The model achieved improved classification accuracy and predictive robustness compared to feedforward network classifiers lacking the SOM layer. By nonlinear dimensionality reduction the networks extracted meaningful chemical features from the data and outperformed linear principal component analysis (PCA). The learning machine was trained on the sequence-length independent recognition of antibacterial peptides and correctly predicted the killing activity of a synthetic test peptide against Staphylococcus aureus in an in vitro experiment.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Aprendizado de Máquina , Peptídeos Catiônicos Antimicrobianos/farmacologia , Análise de Componente Principal , Staphylococcus aureus/efeitos dos fármacos
20.
Cell Commun Signal ; 14(1): 30, 2016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27931258

RESUMO

BACKGROUND: The serine proteases HtrA/DegP secreted by the human gastrointestinal pathogens Helicobacter pylori (H. pylori) and Campylobacter jejuni (C. jejuni) cleave the mammalian cell adhesion protein E-cadherin to open intercellular adhesions. A wide range of bacteria also expresses the HtrA/DegP homologs DegQ and/or DegS, which significantly differ in structure and function. METHODS: E-cadherin shedding was investigated in infection experiments with the Gram-negative pathogens H. pylori, enteropathogenic Escherichia coli (EPEC), Salmonella enterica subsp. Enterica (S. Typhimurium), Yersinia enterocolitica (Y. enterocolitica), and Proteus mirabilis (P. mirabilis), which express different combinations of HtrAs. Annotated wild-type htrA/degP, degQ and degS genes were cloned and proteolytically inactive mutants were generated by a serine-to-alanine exchange in the active center. All HtrA variants were overexpressed and purified to compare their proteolytic activities in casein zymography and in vitro E-cadherin cleavage experiments. RESULTS: Infection of epithelial cells resulted in a strong E-cadherin ectodomain shedding as reflected by the loss of full length E-cadherin in whole cell lysates and formation of the soluble 90 kDa extracellular domain of E-cadherin (NTF) in the supernatants of infected cells. Importantly, comparing the caseinolytic and E-cadherin cleavage activities of HtrA/DegP, DegQ and DegS proteins revealed that DegP and DegQ homologs from H. pylori, S. Typhimurium, Y. enterocolitica, EPEC and P. mirabilis, but not activated DegS, cleaved E-cadherin as a substrate in vitro. CONCLUSIONS: These data indicate that E-cadherin cleavage is confined to HtrA/DegP and DegQ proteins representing an important prevalent step in bacterial pathogenesis.


Assuntos
Caderinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/fisiologia , Infecções por Bactérias Gram-Negativas/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Periplásmicas/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Escherichia coli Enteropatogênica/enzimologia , Escherichia coli Enteropatogênica/fisiologia , Proteínas de Escherichia coli/química , Bactérias Gram-Negativas/química , Infecções por Bactérias Gram-Negativas/patologia , Proteínas de Choque Térmico/química , Humanos , Proteínas Periplásmicas/química , Proteólise , Alinhamento de Sequência , Serina Endopeptidases/química
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