All posts by Postępy Mikrobiologii

Sekrecja pęcherzyków błonowych jako mechanizm promujący infekcje H. pylori

Secretion of outer membrane vesicles as a mechanism promoting H. pylori infections
P. Krzyżek
8 August 2017

1. Wstęp. 2. Sekrecja pęcherzyków błonowych u H. pylori. 3. Proteom pęcherzyków błonowych H. pylori. 4. Transport czynników wirulencji poprzez OMV. 4.1. Toksyna VacA. 4.2. Onkoproteina CagA. 4.3. Inne substancje. 5. Udział OMV w formowaniu biofilmu. 5.1. Funkcje biofilmu. 5.2. Zaangażowanie OMV w tworzenie biofilmu u bakterii. 5.3. Zaangażowanie OMV w tworzenie biofilmu u H. pylori. 5.4. Funkcja strukturalna zewnątrzkomórkowego DNA H. pylori. 6. Zewnątrzkomórkowe DNA jako nośnik informacji. 6.1. Wpływ na wirulencję. 6.2. Transformacja. 6.3. Naturalna kompetencja H. pylori. 7. Podsumowanie

Abstract: Helicobacter pylori commonly colonizes the human gastric mucosa. Infections with this microorganism can contribute to serious health consequences, such as peptic ulceration, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma. Chronic persistence of this bacteria in the host organism is probably strongly dependent on the secretion of outer membrane vesicles (OMV). These organelles are small, electron-dense, extracellular structures which are secreted in large amounts during stressful conditions, among others. H. pylori OMV mediate transfer of virulence factors such as toxins and immunomodulatory compounds. They contribute to avoiding a response from the host immune system and inducing chronic gastritis. OMV secretion also affects the formation of cell aggregates, microcolonies and biofilm matrix. Enhanced OMV production is connected to maintenance of direct contact through cell-cell and
cell-surface interactions. A key component of OMV, which determines their structural function, is extracellular DNA (eDNA) anchored to the surface of these organelles. eDNA associated with OMV additionally determines the genetic recombination in the process of horizontal gene transfer. H. pylori is naturally competent for genetic transformation and is constantly capable of DNA uptake from the environment. The natural competence state promotes the assimilation of eDNA anchored to the OMV surface. This is probably dependent on ComB and ComEC components, which are involved in the transformation process. For this reason, the OMV secretion mediates intensive exchange of genetic material, promotes adaptation to changing environmental conditions and enables persistent infecting of the gastric mucosa by H. pylori.

1. Introduction. 2. Secretion of outer membrane vesicles by H. pylori. 3. Proteome of H. pylori outer membrane vesicles. 4. Transport of virulence factors through OMV. 4.1. Toxin VacA. 4.2. Oncoprotein CagA. 4.3. Other substances. 5. OMV involvement in biofilm formation. 5.1. Functions of biofilm. 5.2. OMV influence on bacterial biofilm formation. 5.3. OMV influence on biofilm formation by H. pylori. 5.4. Structural function of H. pylori extracellular DNA. 6. Extracellular DNA as an information carrier. 6.1. Influence on virulence. 6.2. Transformation. 6.3. Natural competence of H. pylori. 7. Conclusions

Autor: Postępy Mikrobiologii
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Strategie badań tiolowych oksydoreduktaz

Strategies for the analysis of thioloxidorductases
E. K. Jagusztyn-Krynicka, A. M. Banaś, M. J. Grzeszczuk
8 August 2017

1. Wprowadzenie. 2. Analizy funkcjonowania białek Dsb in vivo. 2.1. Wyznaczanie stanu redoks białek. 2.2. Analiza fenotypowa zmutowanych szczepów. 3. Analizy funkcjonalne białek Dsb in vitro. 3.1. Test redukcji insuliny. 3.2 Określanie potencjału redoks. 3.3. Analiza aktywności oksydacyjnej i izomeryzacyjnej. 3.4. Określanie wartości pKa nukleofilowej cysteiny motywu CXXC. 3.5. Analizy oddziaływań pomiędzy DsbA a DsbB. 3.6. Struktury białek. 3.7. Identyfikacja substratów białek Dsb. 4. Podsumowanie

Abstract: Bacterial Dsb (disulfide bond) enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between thiol groups of cysteine residues. This process is critical for the correct folding and structural stability of many secreted and membrane proteins. The rapidly expanding number of sequenced bacterial genomes has revealed the enormous diversity among bacterial Dsb systems. While the Escherichia coli oxidative protein folding has been studied in great details, the mechanism of the Dsb systems functioning in other bacteria are rather poorly understood. Herein, we present the current methodology, both in vivo and in vitro experimental techniques, which allow us to understand the functioning of the Dsb proteins and has broaden our knowledge in the field of biochemistry and microbiology of this posttranslational protein modification. Many bacterial virulence factors are extracytoplasmic Dsb-dependent proteins. Thus, this system plays an important role in bacterial pathogenesis and the proteins of the Dsb network represent possible targets for new drugs.

1. Introduction. 2. Analysis of the Dsb functioning in vivo. 2.1. Determination of the in vivo redox state. 2.2. Phenotypic assay of the mutated strains. 3. Analysis of the Dsb functioning in vitro. 3.1. Insulin reduction assay. 3.2. Determination of the redox potential. 3.3. Assay of the oxidative and isomerase activity. 3.4. Determination of the pKa value of the cysteine residue 3.5. Determination of the interaction between DsbA and DsbB. 3.6. Protein structures. 3.7. Searching for Dsb protein substrates. 4. Conclusions

Autor: Postępy Mikrobiologii
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Wrażliwość krętków Borrelia burgdorferi sensu lato na antybiotyki in vitro

Susceptibility of spirochetes Borrelia burgdorferi sensu lato to antibiotics in vitro
T. Chmielewski, S. Tylewska-Wierzbanowska
8 August 2017

1. Wstęp. 2. Hodowla i wzrost Borrelia burgdorferi sensu lato w warunkach in vitro. 3. Wrażliwość na antybiotyki Borrelia burgdorferi in vitro. 4. Podsumowanie

Abstract: Empiric therapy has been applied in the treatment of Lyme disease. This therapy is selected following the sensitivity analysis of the proposed drug in all species of bacteria which can cause a similar type of infection and on the basis of the clinical efficacy of antibiotic treatment. Established schemes based on data collected from many centers in the world, including type of antibiotic, dose and duration of his administration, and the stage and form of Lyme disease have been created. Number of in vitro methods of spirochetes susceptibility to antibiotics has been also developed. Unfortunately, the lack of standardization often makes it impossible to compare the results of MIC and MBC. Furthermore, little is known about the interactions of the various antimicrobial substances and spirochetes. There is a need for testing of clinical strains isolated from patients after treatment, which would explain the problems associated with “refractory” cases of Lyme disease. The paper presents the research on the antibiotic-spirochete interactions observed in vitro.

1. Introduction. 2. In vitro culture and growth of Borrelia burgdorferi sensu lato. 3. In vitro susceptibility of Borrelia burgdorferi sensu stricto strains to antimicrobial agents. 4. Summary

Autor: Postępy Mikrobiologii
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Genetyczne metody różnicowania mikroorganizmów w systemie gleba – roślina

Genetic differentiation methods of microorganisms in the soil – plant system
M. Łyszcz, A. Gałązka
7 August 2017

1. Wstęp. 2. Elektroforeza w gradiencie czynnika denaturującego DGGE, elektroforeza w gradiencie temperatury TGGE. 3. Polimorfizm konformacji pojedynczej nici DNA SSCP. 4. Łańcuchowa reakcja polimerazy w czasie rzeczywistym (Real Time PCR). 5. Podsumowanie

Abstract: Biodiversity is a key concept in finding important features of new microorganisms. Microorganisms play an important role in the soil ecosystem and participate, among others, in such processes as the maintenance of soil structure, humification, release of organic compounds, disposal of pollutants and transformation of organic matter. The maintenance of competent state of soil microbial communities, i.e. the appropriate microorganism count, activity and diversity, is a necessary condition for the functioning of a highly complex system such as the soil. Phyllosphere bacteria have the potential to influence plant biogeography and ecosystem function through their influence on plant performance under different environmental conditions, but the drivers of variation in leaf-associated bacterial biodiversity among host plants are not well understood. Hence, undoubtedly, an important research aspect is the selection and development of indicators to evaluate microbial biodiversity of the soil and plant phyllosphere. In this publication, selected molecular methods used for the diversity assessment of microorganisms have been presented.

1. Introduction. 2. Denaturing Gradient Gel Electrophoresis DGGE, Temperature Gradient Gel Electrophoresis TGGE, 3. SSCP – single strand conformation polymorphism. 4. Real-Time Quantitative PCR. 5. Summary

Autor: Postępy Mikrobiologii
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Metody genotypowe i fenotypowe wykorzystywane w typowaniu drobnoustrojów do celów epidemiologicznych

The application of genotyping and phenotyping techniques for epidemiological analysis of microorganisms
M. Brzozowski, P. Kwiatkowski, D. Kosik-Bogacka, J. Jursa-Kulesza
7 August 2017

1. Wstęp. 2. Metody fenotypowe. 2.1. Biotypowanie. 2.2. Typowanie fagowe. 2.3. Analiza profili lekowrażliwości. 2.4. Metody analizy białek. 2.5. Spektroskopia mas. 3. Metody genotypowe. 3.1. Genotypowanie bez wykorzystania sekwencjonowania. 3.1.1. REA-PFGE. 3.1.2. RFLP i PCR-RFLP. 3.1.3. AFLP. 3.1.4. RAPD. 3.1.5. Mikromacierze (CHIP DNA). 3.1.6. MLVA. 3.2. Metody genotypowe wykorzystujące sekwencjonowanie. 3.2.1. Technologie sekwencjonowania. 3.2.2. MLST i SLST. 3.2.3. WGS – wgSNP, cgMLST, wgMLST. 3.2.4. Zalety i wady WGS. 4. Popularność metod typowania bakterii w badaniach biomedycznych na podstawie analizy bazy PubMed. 5. Podsumowanie

Abstract: The research on similarity between bacteria in outbreak investigations enables the identification of bacterial strain responsible for infections, their source and modes of transmission. These investigations are also necessary for the analysis of spreading of bacteria, not only locally, e.g. in a hospital in a specific country, but also internationally and globally. Therefore, it is of great importance to have the most up to date knowledge regarding different methods used in bacterial typing. This review discusses and compares methods facilitating bacterial typing at a strain level. Phenotyping methods analysed in this article are: Biotyping, Antimicrobial Susceptibility Typing, Phage Typing and protein-based methods. Genotyping techniques reviewed in this article are based on digestion of genomic DNA, methods using amplification of DNA, and based on sequencing DNA. This would include Multilocus Sequence Typing (MLST) and Whole Genome Sequencing (WGS). Methods used in identification of bacterial strains are being constantly improved, and gaining more in depth knowledge and familiarising with their effectiveness enables better analysis and control of epidemiological situation e.g. in hospitals.

1. Introduction. 2. Phenotyping methods. 2.1. Biotyping. 2.2. Phage typing. 2.3. Antimicrobial susceptibility typing. 2.4. Protein-based methods. 2.5. Mass spectrometry. 3. Genotyping methods. 3.1. Genotyping without DNA sequencing. 3.1.1. REA-PFGE. 3.1.2. RFLP and PCR-RFLP. 3.1.3. AFLP. 3.1.4. RAPD. 3.1.5. Microarrays. 3.1.6. MLVA. 3.2. Genotyping using DNA sequencing 3.2.1. Sequencing technologies. 3.2.2. MLST and SLST. 3.2.3. WGS – wgSNP, cgMLST, wgMLST. 3.2.4. Advantages and disadvantages of WGS. 4. Popularity of typing methods in biomedical research – PubMed database analysis. 5. Conclusions

Autor: Postępy Mikrobiologii
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Advancements of Microbiology
Volume 63 (2024): Issue 3

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PTM

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