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The role and the fate of the extracellular DNA (eDNA) associated with the Outer Membrane Vesicles (OMVs) produced by Helicobacter pylori
Università degli studi di Chieti
Department of Pharmacy Via dei Vestini, 31 66100 Chieti Italy
Prof.ssa Amelia Cataldi
Prof.ssa Rossella Grande
Type of Research Project
- Basic science
What is the background of the project?
Membrane vesicles (MVs) are spherical, phospholipidic bilayered structures which bleb from Gram-positive and Gram-negative bacteria during their growth. They are involved in several mechanisms such as horizontal gene transfer, microbial communication, biofilm formation, resistance to antibiotics, as the delivery of virulence factors as well as antimicrobial activity and immunomodulation. The MVs function and cargo strictly depend on the bacterial species and culture conditions as well as on the extracellular environment. MVs can transport a wide variety of components including nucleic acids, proteins and polysaccharides (Brown et al., 2015; Schwechheimer and Kuehn, 2015). Helicobacter pylori produces Outer Membrane Vesicles (OMVs) in the planktonic and biofilm phenotypes. Such structures are involved in direct cell-cell binding suggesting that OMVs serve as a potentially novel gastric cell colonization factor of this microorganism (Yonezawa et al., 2009). Recently, it has also been demonstrated that extracellular DNA (eDNA) is associated with the OMVs surface, suggesting a possible structural role (Grande et al., 2015).
What is the aim of the project?
The aim of the present project is an in-depth characterization of the eDNA associated with OMVs through: 1. The quantification and the molecular analysis of the eDNA associated with OMVs produced by H. pylori grown at sub-inhibitory concentration of antibiotics. 2. The sequencing of the eDNA associated with OMVs .
What techniques and methods are used?
- Preparation of bacterial culture media and cultivation of Helicobacter pylori - Development of the biofilm of Helicobacter pylori - Biofilm characterization by live/dead assay and Confocal Laser scanning Microscopy (CLSM) - Isolation of Outer Membrane Vesicles (OMVs) by filtration and ultracentrifugation - eDNA detection and quantification by PicoGreen assay and Flow Cytometry - eDNA extraction, analysis and sequencing by molecular methods. - Analysis of the received data
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks of the student will be performed on his/her own
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
- The student will be involved in the following tasks: - Preparation of bacterial culture media - Cultivation of microorganisms - Development of the microbial biofilm - Biofilm characterization by live/dead assay and analysis by fluorescence microscopy - Isolation of Outer Membrane Vesicles (OMVs) by filtration and ultracentrifugation - eDNA detection and quantification by PicoGreen assay - eDNA extraction - eDNA analysis by PCR (polymerase chain reaction)
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
A theoretical teaching as well as scientific publications regarding the project will be provided to the student.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Subjects passed: Microbiology and Biological Sciences
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Students in biomedical fields
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