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OBJECTIVES: Antibiotic treatment is extremely stressful for bacteria and has profound effects on their viability. Such administration induces physiological changes in bacterial cells, with considerable impact on their genome structure that induces mutations throughout the entire genome. This study investigated drug resistance profiles and structural changes in the entire genome of uropathogenic Escherichia coli (UPEC) strains isolated from six adapted clones that had evolved under laboratory conditions. METHODS: Eight UPEC strains, including two parental strains and six adapted clones, with different fluoroquinolone resistance levels originally isolated from two patients were used. The minimum inhibitory concentration (MIC) of 28 different antibiotics including levofloxacin was determined for each of the eight strains. In addition, the effects of mutations acquired with increased drug resistance in the levofloxacin-resistant strains on expression of genes implicated to be involved in drug resistance were examined. RESULTS: Of the eight UPEC strains used to test the MIC of 28 different antibiotics, two highly fluoroquinolone-resistant strains showed increased MIC in association with many of the antibiotics. As drug resistance increased, some genes acquired mutations, including the transcriptional regulator acrR and DNA-binding transcriptional repressor marR. Two strain groups with genetically different backgrounds (GUC9 and GFCS1) commonly acquired mutations in acrR and marR. Notably, acquired mutations related to efflux pump upregulation also contributed to increases in MIC for various antibiotics other than fluoroquinolone. CONCLUSIONS: The present results obtained using strains with artificially acquired drug resistance clarify the underlying mechanism of resistance to fluoroquinolones and other types of antibiotics.
Assuntos
Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Humanos , Levofloxacino/farmacologia , Levofloxacino/uso terapêutico , Escherichia coli Uropatogênica/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Fluoroquinolonas/farmacologia , Fluoroquinolonas/uso terapêutico , Resistência a Múltiplos Medicamentos , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Farmacorresistência Bacteriana/genéticaRESUMO
Although serotyping is the most important method of identification of taxonomy in Salmonella, conventional serotype determination with a complete set of antisera is time consuming and laborious. Recently, rapid serotyping procedures with polymerase chain reaction (PCR) have been developed. In this study, we established a novel PCR-based rapid serotyping method that employs a unique target gene. Alignment study of Salmonella-specific gene (Salmonella enterotoxin [stn]) revealed a correlation between the stn gene sequence and the serotype of the organism. In 750 bp of stn gene, 55 nucleotides indicated single nucleotide polymorphism (SNP)-like polymorphism, and the correlation between the SNP-like polymorphism and the serotype of the organism suggests that SNP-like sequences in stn gene can serve as an index for serotyping. To develop a rapid serotyping method based on the SNP-like polymorphism, we selected serotype-associated 12 SNP-like sites in the stn gene and established a method based on high-resolution melting (HRM) and PCR, which identifies nucleotides at SNP-like sites within 1.5 h. This newly established rapid serotyping procedure (stn-HRM) could identify nine serotypes, including the frequently isolated serovar Enteritidis. These nine serotypes cover 64.3% of cases of Salmonella, as reported by the World Health Organization/Global Foodborne Infection Network (WHO/GFN) Country Databank from 2001 to 2010. In this study, we employed a unique target gene, stn, which is completely independent of the genes that were targeted in previously reported rapid serotyping procedures. Therefore, the results obtained by our newly developed stn-HRM procedure are independent of the results obtained by other procedures. Besides, stn-HRM can ensure accurate identification of the bacterial species as stn is a Salmonella-specific gene. It is expected that the combination of newly constructed stn-HRM and previously reported procedures could further improve the credibility of Salmonella isolate serotyping.
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Enterotoxinas/classificação , Enterotoxinas/genética , Polimorfismo de Nucleotídeo Único , Salmonella/isolamento & purificação , Sorotipagem/métodos , Humanos , Reação em Cadeia da Polimerase , Salmonella/classificação , Salmonella/genética , SorogrupoRESUMO
Vibrio cholerae produced-Cholix toxin (Cholix) is a cytotoxin that ADP-ribosylates eukaryotic elongation factor 2, inhibiting protein synthesis, and inducing apoptosis. Here, we identified prohibitin (PHB) 1 and 2 as novel Cholix-interacting membrane proteins in immortalised human hepatocytes and HepG2 cells by Cholix immunoprecipitation assays. The expression level of PHB1 was decreased by Cholix after a 12hr incubation. Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage was significantly enhanced in PHB (PHB1 or PHB2) knockdown cells. In contrast, transiently overexpressed PHB in hepatocytes attenuated Cholix-induced Bax/Bak conformational changes and PARP cleavage. In addition, Cholix-induced reactive oxygen species production and accumulation of fragmented mitochondria were enhanced in PHB-knockdown cells. Furthermore, Cholix induced activation of Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which was enhanced in PHB-knockdown cells, followed by actin filament depolymerisation and accumulation of tubulin in the blebbing cells. Inhibition of ROCK1 by siRNA or its inhibitor suppressed Cholix-induced PARP cleavage and reactive oxygen species generation. Our findings identify PHB as a new protein that interacts with Cholix and is involved in Cholix-induced mitochondrial dysfunction and cytoskeletal rearrangement by ROCK1 activation during apoptosis.
Assuntos
Fatores de Ribosilação do ADP/química , Toxinas Bacterianas/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Interações Hospedeiro-Patógeno/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Vibrio cholerae/genética , ADP-Ribosilação , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/microbiologia , Citoesqueleto de Actina/ultraestrutura , Sequência de Aminoácidos , Apoptose/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular Transformada , Fator de Iniciação 2 em Eucariotos/genética , Regulação da Expressão Gênica , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/microbiologia , Hepatócitos/patologia , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , Mitocôndrias/ultraestrutura , Proibitinas , Ligação Proteica , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/deficiência , Transdução de Sinais , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidade , Virulência , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Ovarian follicular cysts are one of the most common causes of reproductive failure in mammals. A comparative gene expression approach may aid in elucidating the causes of ovarian cyst disease. In the present study, the differential display technique was used to identify mRNA sequences that accumulate preferentially in theca cells of bovine cystic follicles. Dedicator of cytokinesis 6 (Dock6) expression was observed in the theca cells of cystic follicles. Small interfering RNA (siRNA) knockdown of Dock6 increased progesterone (P4) production and StAR expression in theca cells of high-estrogen follicular cysts, but did not affect androstenedione (A4) production. We propose that Dock6 may be a marker associated with the development of follicular cysts. Additionally, Dock6 may be involved in the development of cystic follicles by suppressing P4 production rather than increasing A4 production in theca cells.
Assuntos
Estrogênios/biossíntese , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Folículo Ovariano/metabolismo , Progesterona/biossíntese , Animais , Sequência de Bases , Bovinos , Primers do DNA , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Folículo Ovariano/citologia , Interferência de RNA , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: Prior reports suggested that infection with Helicobacter pylori was associated with respiratory diseases; pathogenetic mechanisms however, were not defined. We tested the hypothesis that VacA, an exotoxin of H. pylori, a gastric pathogen, was aspirated into the lung and could stimulate secretion of inflammatory cytokines by lung epithelial cells. METHODS: The presence of VacA was determined by immunohistochemistry in surgical lung biopsy tissue samples from 72 patients with interstitial pneumonia. The effects of VacA on A549 human alveolar epithelial adenocarcinoma cells and normal human bronchial epithelial cells were determined. After incubation with VacA, the secretions of cytokines were measured by Multiplex Luminex(®) Assays. RESULTS: VacA was detected with anti-VacA antibodies in bronchial epithelial cells and alveolar epithelial cells from 10 of 72 patients with interstitial pneumonia. VacA was more prevalent in lungs of patients with collagen vascular disease-associated interstitial pneumonia than in those of patients with idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia and cryptogenic organizing pneumonia. Incubation of A549 cells and normal human bronchial epithelial cells with VacA for 24 h was cytotoxic, and resulted in vacuolation. VacA induced interleukin-8 production by A549 cells and normal human bronchial epithelial cells and interleukin-6 production by A549 cells. Based on multiplex screening, interleukin-8 and interleukin-6 were the primary secretory products induced by VacA. CONCLUSIONS: H. pylori VacA is present in human lung and can induce interleukin-8 and interleukin-6 production by human lung cells. VacA could have a role in the pathogenesis of respiratory diseases by its cytotoxic effects and by inducing the secretion of interleukin-8 and interleukin-6 by targeted airway epithelial cells.
Assuntos
Proteínas de Bactérias/metabolismo , Helicobacter pylori/metabolismo , Pulmão/microbiologia , Adulto , Idoso , Proteínas de Bactérias/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/biossíntese , Feminino , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Humanos , Pulmão/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Analyses of efflux pumps overexpression and mutations in quinolone resistance determining region (QRDR) in early stage of development of resistance to fluoroquinolones (FQs) are valuable to discuss countermeasures against them. We induced levofloxacin (LVFX)-resistant strains from susceptible uropathogenic Escherichia coli in vitro to analyze the mechanisms of development of FQs-resistance. METHODS: 89 strains were exposed to discontinuous elevation of LVFX dose, and mRNA level of efflux pumps and their regulators as well as mutations developed in QRDR of LVFX-resistant strains were analyzed. RESULTS: In 5 strains, a stepwise increase in MIC to LVFX (up to >128 µg/ml)was observed. Compared to the parent strains, additional mutations in QRDR were observed in the strains developing high MIC. Remarkable increase of marA expression was observed even in the early stage of LVFX-resistance development, and it lasted until high-level resistance was developed. On the other hand, moderate increase in acrB expression but only low increase in yhiU, yhiV, mdfA, tolC and sdiA were observed. CONCLUSIONS: These results suggested that marA expression is a sensitive marker for early detection of development of LVFX-resistance.
Assuntos
Antibacterianos/farmacologia , Proteínas de Ligação a DNA/genética , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/genética , Fluoroquinolonas/farmacologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/genética , Proteínas de Ligação a DNA/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Humanos , Testes de Sensibilidade Microbiana , MutaçãoRESUMO
In food hygiene, the surveillance of foodborne pathogens in wild animals is indispensable because we cannot control hygienic status of them. Yezo sika deer (Cervus nippon yesoensis), which are found only on the island of Hokkaido, Japan, are the most common game animal in the country. In this study, we analyzed the incidence of Shiga toxin-producing Escherichia coli (STEC) in Yezo sika deer hunted in the Tokachi sub-prefecture, which is one of the densest zones for the sub-species. Real-time polymerase chain reaction testing detected STEC in 18.3% of fecal samples (59/323) collected from deer hunted between 2016 and 2017, whereas no Shigella and Salmonella markers were detected. No correlation was found between STEC detection from fecal samples and characteristics of carcasses, such as hunting area, age, and fascioliasis. From 59 STEC-positive fecal samples, we isolated 37 STEC strains, including 34 O- and H-genotyped strains, in which 16 different serogroups were detected. Genetic analysis revealed that our isolates included various stx gene types (stx1+/stx2-, stx1+/stx2+, and stx1-/stx2+) and carried eae. This study demonstrated that STEC strains with various features colonized the Yezo sika deer, similar to other subspecies of sika deer. We conclude that continuous surveillance activity is important to monitor the suitability of game animals as a food source and to assess the validity of the food safety management system for game meat production.
Assuntos
Cervos , Escherichia coli Shiga Toxigênica , Animais , Fezes , Japão/epidemiologia , PrevalênciaRESUMO
Two novel probiotic strains of lactic acid bacteria were successfully isolated from the raw milk of dairy Japanese-Saanen goats. Selection criteria for positive candidates were grown on de Man-Rogosa-Sharpe or M17 selective medium at 30, 35, or 42 °C anaerobically, and characterized based on Gram reaction, catalase test, and tolerance to low pH and bile salts. Among the 101 isolated positive candidates, two strains, YM2-1 and YM2-3, were selected and identified as Lacticaseibacillus rhamnosus using 16S rDNA sequence similarity. Culture supernatants of the two strains exhibited antipathogenic activity against Salmonella enterica subsp. enterica serovar. Typhimurium, Shigella sonnei, methicillin-resistant Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli O157. The antipathogenic activities were retained to some extent after neutralization, indicating the presence of antipathogenic substances other than organic acids in the culture supernatants. The two strains were sensitive with coincidental minimum inhibition concentrations (indicated in the parentheses hereafter) to ampicillin (0.25 µg/mL), chloramphenicol (4 µg/mL), gentamycin (4 µg/mL), kanamycin (64 µg/mL), streptomycin (16 µg/mL), and tetracycline (4 µg/mL). Furthermore, the two strains were resistant to clindamycin (16 µg/mL) and erythromycin (4 µg/mL). In addition, both YM2-1 and YM2-3 strains showed less unfavorable activities, including bile acid bioconversion, carcinogenic-related enzymes, mucin degradation, plasminogen activation, and hemolysis, than the detection limits of in vitro evaluation methods used in this study. In summary, L. rhamnosus YM2-1 and YM2-3 are highly safe and promising probiotic strains applicable in the dairy industry, and were first isolated from the raw milk of Japanese-Saanen goats.
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Aims: In this study, we focused on the fat ratio within psoas muscle (FRPM) and sought to clarify the impact of FRPM on overall survival (OS) in stage IV gastric cancer (GC) patients undergoing systemic chemotherapy (n = 79, median age = 69 years, 59 males). Methods: The median FRPM was 1.67 %. Forty patients with FRPM ≥1.67 % were defined as the FRPM-high group, and the remaining 39 patients was defined as the FRPM-low group. The median PMI in male and female patients was 4.35 cm2/m2 and 2.88 cm2/m2. Thirty male patients with PMI ≥4.35 cm2/m2 and 10 female patients with PMI ≥2.88 cm2/m2 was defined as the PMI-high group, and the remaining 39 patients was defined as the PMI-low group. Results: The 1-, 2- and 3- year cumulative OS rate for all cases was 70.8%, 24.3% and 14.6%. The proportion of ECOG-PS 2 or 3 in patients with FRPM-high and FRPM-low was 17.5% (7/40) and 2.6% (1/39). The 1-, 2- and 3- year cumulative OS rate in patients with FRPM-high and FRPM-low was 67.3%, 14.3% and 7.6% in the FRPM-high group and 74.8%, 40.5% and 32.4% in the FRPM-low group (P = 0.0341). The 1-, 2- and 3- year cumulative OS rate in patients with PMI-high and PMI-low was 86.7%, 40.4% and 30.0% in the PMI-high group and 55.8%, 12.8% and 6.4% in the PMI-low group (P < 0.0001). In the multivariate analysis of factors associated with OS, PMI (P = 0.0047) and FRPM (P = 0.0019) were independent predictors for the OS. Conclusion: Higher FRPM can be associated with decreased physical activity, and not only skeletal muscle mass but also skeletal muscle function can be an essential prognostic factor in stage IV GC patients undergoing systemic chemotherapy.
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We sought to clarify the relevance in the neutrophil to lymphocyte ratio (NLR) and the SARC-F score in patients with gastrointestinal diseases (G-Ds, n = 672, median age = 73 years). Univariate and multivariate analysis for the SARC-F score were performed. Advanced malignancy was identified in 162 patients (24.1%). The median of NLR for all cases was 2.65. The median of NLR in ECOG-PS 0 (n = 436), 1 (n = 128), 2 (n = 49) and 3 or 4 (n = 59) was 2.26, 2.97, 4.41 and 5.99 (overall p < 0.0001). NLR had a significant correlation with the SARC-F score (r = 0.54, p < 0.0001). The median of NLR in the SARC-F score ≥4 (recommended value for sarcopenia, n = 84) and <4 (n = 588) was 5.87 and 2.48 (p < 0.0001). In all subgroup analyses, similar trends were seen. In the multivariate analysis, ECOG-PS (p < 0.0001) and NLR (p < 0.0001) were independent factors, while age had a trend for significance (p = 0.0686). In conclusion, we would like to emphasize the usefulness of NLR, a simple marker assessed only by blood tests, in predicting the possibility for sarcopenia by the SARC-F in G-Ds.
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BACKGROUND/AIM: To prospectively evaluate the efficacy and safety of the BNT162b2 vaccine in solid cancer patients undergoing systemic chemotherapy (n=63). PATIENTS AND METHODS: COVID-19 anti-spike protein antibody levels were measured before the first BNT162b2 vaccination, just before the second BNT162b2 vaccination, one month after the second BNT162b2 vaccination, and 3 months after the second BNT162b2 vaccination. Anti-spike protein antibody seropositivity was set at ≥0.8 U/ml. RESULTS: Colorectal cancer was the most commonly observed primary disease (36.5%). ECOG-PS 0 was observed in the majority (52.4%) of patients. The overall response rate and the median (range) anti-spike protein antibody levels in the whole cohort at 3 months after the second BNT162b2 vaccination were 98.4% (62/63) and 206 (0.4-3,813) U/ml. None of the patients required postponement or discontinuation of systemic chemotherapy because of an adverse reaction. CONCLUSION: The BNT162b vaccine in solid cancer patients undergoing systemic chemotherapy is effective and safe.
Assuntos
COVID-19 , Neoplasias , Vacinas , Humanos , Estudos Prospectivos , Vacina BNT162 , COVID-19/prevenção & controle , Neoplasias/tratamento farmacológico , Vacinas/uso terapêutico , Anticorpos AntiviraisRESUMO
Gangliosides are target receptors for bacterial entry, yet those present in human milk exhibit a protective role against bacterial infection. Here, we show that treatment with ganglioside mixture at a concentration of 100 microg/mL resulted in significant inhibition of the vacuole formation activity of Helicobacter pylori vacuolating cytotoxin (VacA) in gastric epithelial cancer AZ-521 cells. All gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1b) examined showed good neutralizing capacity against VacA. A pull-down assay was performed using lyso-GM1 coupled to Sepharose as the tagged polysaccharide polymer to capture VacA from H. pylori culture supernatant. GM1-VacA complexes were successfully precipitated, suggesting that GM1 binds directly to VacA. The hydrodynamic binding of lyso-GM1 and VacA measured by fluorescence correlation spectroscopy had a K(d) value of 190 nM. VacA also bound to lyso-GM1 at pH 2 corresponding to the physiological pH of human stomach. Collectively, these results showed that direct binding of H. pylori VacA to free gangliosides neutralizes the toxin activity of VacA. These findings offer an alternative insight into the role of gangliosides in VacA toxicity and the pathogenesis of H. pylori.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/toxicidade , Gangliosídeos/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Gangliosídeos/farmacologia , Humanos , Espectrometria de FluorescênciaRESUMO
The emergence of drug-resistant uropathogenic Escherichia coli (UPEC) has hampered antibiotic therapy for urinary tract infections. To elucidate the resistance mechanisms of UPEC, we performed whole-genome sequencing of eight UPEC strains with different fluoroquinolone resistance levels. Here, we report our sequencing data, providing a valuable resource for understanding such mechanisms.
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Safety and probiotic characteristics such as antimicrobial activities of three Lactobacillus rhamnosus strains, FSMM15, FSMM22 and FSMM26, previously isolated as potential probiotics from fermented mare's milk were investigated. The three FSMM strains were susceptible to ampicillin, gentamycin, kanamycin, streptomycin, tetracycline and chloramphenicol, whereas they were resistant to erythromycin (minimal inhibitory concentration (MIC) = 4-8 µg/mL) and clindamycin (MIC = 4 µg/mL); bioconversion of bile salts, hemolytic activity and mucin degradation activity were negative; enzymatic activities of α-chymotrypsin and ß-glucosidase were detected, but those of α-galactosidase, ß-glucuronidase and N-acetyl-ß-glucosaminidase, were undetectable. Among the strains, strain FSMM15 was chosen as a safer probiotic candidate due mainly to the lack of plasminogen binding ability. Despite lower acid production of strain FSMM15 than others, its cell-free culture supernatant inhibited growths of Salmonella Typhimurium LT-2, Shigella sonnei, Listeria monocytogenes, and Escherichia coli O157 with comparable levels of ampicillin, suggesting a favorable aspect of strain FSMM15 as a probiotic strain.
Assuntos
Antibiose , Produtos Fermentados do Leite/microbiologia , Inocuidade dos Alimentos , Lacticaseibacillus rhamnosus/isolamento & purificação , Lacticaseibacillus rhamnosus/fisiologia , Probióticos , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Resistência Microbiana a Medicamentos , Feminino , Cavalos , Lacticaseibacillus rhamnosus/efeitos dos fármacosRESUMO
As cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae O1 or O139 infection, detection of CT is an important biomarker for diagnosis of the disease. The procedure for pathogenicity analysis of V. cholerae isolates must be carefully developed for the reason that the amount of CT produced by V. cholerae varies according to the medium used and culture conditions (i.e. temperature and aeration status) applied. Here we describe a reproducible rapid method for analysis of CT production by toxigenic V. cholerae with an immunochromatographic test strip that can detect as low as 10 ng/mL of purified recombinant CT.
Assuntos
Toxina da Cólera/análise , Vibrio cholerae/metabolismo , Genes Bacterianos/genética , Reação em Cadeia da Polimerase , Vibrio cholerae/genética , Microbiologia da ÁguaRESUMO
The nuclear-encoded protein RPS14 (ribosomal protein S14) of rice mitochondria is synthesized in the cytosol as a polyprotein consisting of a large N-terminal domain comprising preSDHB (succinate dehydrogenase B precursor) and the C-terminal RPS14. After the preSDHB-RPS14 polyprotein is transported into the mitochondrial matrix, the protein is processed into three peptides: the N-terminal prepeptide, the SDHB domain and the C-terminal mature RPS14. Here we report that the general MPP (mitochondrial processing peptidase) plays an essential role in processing of the polyprotein. Purified yeast MPP cleaved both the N-terminal presequence and the connector region between SDHB and RPS14. Moreover, the connector region was processed more rapidly than the presequence. When the site of cleavage between SDHB and RPS14 was determined, it was located in an MPP processing motif that has also been shown to be present in the N-terminal presequence. Mutational analyses around the cleavage site in the connector region suggested that MPP interacts with multiple sites in the region, possibly in a similar manner to the interaction with the N-terminal presequence. In addition, MPP preferentially recognized the unfolded structure of preSDHB-RPS14. In mitochondria, MPP may recognize the stretched polyprotein during passage of the precursor through the translocational apparatus in the inner membrane, and cleave the connecting region between the SDHB and RPS14 domains even before processing of the presequence.
Assuntos
Núcleo Celular/genética , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Poliproteínas/metabolismo , Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/enzimologia , Metaloendopeptidases/genética , Mitocôndrias/metabolismo , Mutação/genética , Oryza , Poliproteínas/química , Poliproteínas/genética , Dobramento de Proteína , Precursores de Proteínas/química , Precursores de Proteínas/genética , Transporte Proteico , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Succinato Desidrogenase/química , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Peptidase de Processamento MitocondrialRESUMO
Helicobacter pylori, a major cause of gastroduodenal diseases, produces vacuolating cytotoxin (VacA) and cytotoxin-associated gene A (CagA), which seem to be involved in virulence. VacA exhibits pleiotropic actions in gastroduodenal disorders via its specific receptors. Recently, we found that VacA induced the phosphorylation of cellular Src kinase (Src) at Tyr418 in AZ-521 cells. Silencing of receptor protein tyrosine phosphatase (RPTP)α, a VacA receptor, reduced VacA-induced Src phosphorylation. Src is responsible for tyrosine phosphorylation of CagA at its Glu-Pro-Ile-Tyr-Ala (EPIYA) variant C (EPIYA-C) motif in Helicobacter pylori-infected gastric epithelial cells, resulting in binding of CagA to SHP-2 phosphatase. Challenging AZ-521 cells with wild-type H. pylori induced phosphorylation of CagA, but this did not occur when challenged with a vacA gene-disrupted mutant strain. CagA phosphorylation was observed in cells infected with a vacA gene-disrupted mutant strain after addition of purified VacA, suggesting that VacA is required for H. pylori-induced CagA phosphorylation. Following siRNA-mediated RPTPα knockdown in AZ-521 cells, infection with wild-type H. pylori and treatment with VacA did not induce CagA phosphorylation. Taken together, these results support our conclusion that VacA mediates CagA phosphorylation through RPTPα in AZ-521 cells. These data indicate the possibility that Src phosphorylation induced by VacA is mediated through RPTPα, resulting in activation of Src, leading to CagA phosphorylation at Tyr972 in AZ-521 cells.
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Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Neoplasias Duodenais/metabolismo , Neoplasias Duodenais/microbiologia , Helicobacter pylori/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Linhagem Celular Tumoral , Neoplasias Duodenais/patologia , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Humanos , Imunoprecipitação , Modelos Biológicos , Fosforilação , Fosfotirosina/metabolismo , RNA Interferente Pequeno/metabolismo , Transfecção , Quinases da Família src/metabolismoRESUMO
Our studies were undertaken to develop new insights into the function of the Salmonella Stn protein. An analysis of total cell membrane protein fraction suggested the possibility that Stn associates with OmpA. This possibility was confirmed by immunogold labeling using anti-OmpA antibody and far-western blotting. From these results, we conclude that Stn regulates membrane composition and integrity in Salmonella.
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Membrana Celular/química , Membrana Celular/metabolismo , Enterotoxinas/metabolismo , Salmonella/citologia , Salmonella/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Western Blotting , Enterotoxinas/isolamento & purificação , Imuno-Histoquímica , Microscopia Eletrônica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Salmonella/ultraestruturaRESUMO
Detection of Shiga toxins (Stx) is important for accurate diagnosis of Enterohemorrhagic Escherichia coli infection. In this study, we quantitatively analyzed Stx protein in nine patients' stool during an outbreak that occurred in Japan. Highly sensitive immunoassay (bead enzyme-linked immunosorbent assay (bead-ELISA)) revealed that the concentrations of toxins in stool of patients ranged from 0.71 to 10.44 ng/mL for Stx1 and 2.75 to 51.61 ng/mL for Stx2. To our knowledge, this is the first report that reveals the range of Stx protein concentrations in human stools.
Assuntos
Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Fezes/química , Toxinas Shiga/análise , Adolescente , Adulto , Criança , Pré-Escolar , Surtos de Doenças , Ensaio de Imunoadsorção Enzimática , Infecções por Escherichia coli/epidemiologia , Feminino , Humanos , Lactente , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Toxinas Shiga/genética , Adulto JovemRESUMO
Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Most H. pylori strains secrete VacA into the extracellular space. After exposure of VacA to acidic or basic pH, re-oligomerized VacA (mainly 6 monomeric units) at neutral pH is more toxic. Although the mechanisms have not been defined, VacA induces multiple effects on epithelial and lymphatic cells, i.e., vacuolation with alterations of endo-lysosomal function, anion-selective channel formation, mitochondrial damage, and the inhibition of primary human CD4+ cell proliferation. VacA binds to two types of receptor-like protein tyrosine phosphatases (RPTP), RPTPalpha and RPTPbeta, on the surface of target cells. Oral administration of VacA to wild-type mice, but not to RPTPbeta KO mice, results in gastric ulcers, suggesting that RPTPbeta is essential for intoxication of gastric tissue by VacA. As the potential roles of VacA as a ligand for RPTPalpha and RPTPbeta are only poor understood, further studies are needed to determine the importance of VacA in the pathogenisis of disease due to H. pylori infection.