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Identifier

000403907

Title

Μελέτη των πρωτεϊνών του εκκριτικού συστήματος τύπου IV της Coxiella Burnetii

Alternative Title

Study of proteins of the secretion system type IV of Coxiella Burnetii

Author

Μαθιουδάκη, Ειρήνη Κ.

Thesis advisor

Τσιώτης, Γεώργιος

Reviewer

Γανωτάκης, Δημήτριος
Ψαρουλάκη, Άννα

Abstract

Coxiella Burnetii is an obligatory intracellular bacterial pathogen and is the causative agent of Q fever in human and other mammalian animals. Studies about Coxiella, have shown that, specific molecular machines contribute by transferring protein molecules, which interfere in the cellular processes of the host, resulting in the establishment of the infection. In that way, the bacteria manages its survival and proliferation inside the host cell. Such a molecular machine is the IVB secretion system (T4BSS), known as Dot/Icm system that transports proteins from the bacterium to the host cell. This secretion system has been studied with details in the bacterium Legionella pneumophilia and it was found to be composed of 27 proteins, 24 of which were present in C.burnetii strains. In this particular study, we examined four proteins from the secretion T4BSS system of C.burnetii, as well as the core subcomplex, composed of DotC, DotD, IcmG (DotF), IcmE (DotG) and IcmK (DotH). Proteins being studied are IcmK an outer membrane protein, the periplasmic protein IcmE , DotC and DotD proteins. First of all, the expression ability of the above proteins in E.Coli strains C43 (DE3) was tested by using pttQ18A2 and pttQ18C3 as plasmid vectors . The first plasmid vector contained the gene for each protein marked by the His- tag (six histidines residues) at the C-terminus, while the second vector contained as a tag, a small peptide of 8 amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) called strep-tag, also at the C- terminus of the protein. In this way, it was possible to isolate the respective protein by affinity chromatography and more particularly, by using a nickel and a streptavidin column for each of these cases respectively. Further purification of the target protein was achieved by Molecular Exclusion Chromatography. Successful isolation, purification and identification by mass spectrometry of the desired proteins, opens the way for their structural characterization by the method of Electron Microscopy and Crystallography. The study of the secreted proteins of pathogenic bacteria Coxiella Burnetii is very important to potential medical applications, provided that the specific protein molecules contribute to the growth of the bacterium inside the host cell, being target molecules for drug development.

Language

Greek

Subject

Affinity chromatography

Electrophoresis

IVB secretorion system

Mass spectrometry

Membrane proteins

Molecular exclusion chromatography

Q fever

Εκκριτικό σύστημα τύπου IVB

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