Komparativní proteomová analýza mikrobů Francisella tularensis s identifikací jedinečných proteinů hmotnostní spektrometrií

Mgr. Martin Hubálek, Ph.D.

Doctoral thesis

Comparative proteome analysis of Francisella tularensis with identification of unique proteins by mass spectrometry

Abstract:

The main goals of the work presented were to investigate: i) subspecies variations of pathogenic bacterium Francisella tularensis on proteome level; ii) the possible mechanisms of Francisella tularensis pathogenesis and iii) the possibilities to use proteomics techniques for bacterial identification and typing. The initial experiments were aimed at mapping the complex protein mixture of whole cell lysate by two-dimensional electrophoresis (2-DE). The platform of 2-DE database has been established and interactive web based data are available (http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/ind ex.cgi). Once the reliable and reproducible system of 2-DE separation was achieved the follow-up experiments that utilize the platform were initiated. Comparative analyses of three Francisella tularensis subspecies was started soon after the first successful 2-DE experiments and resulted in identification of 2 DE spots corresponding to proteins with different expression patterns in the three subspecies. Soon, the necessity to fractionate the protein content was clear, especially in case of membrane proteins. An improved 2-DE sample preparation procedure that yielded different protein profile as compare to whole cell lysate was developed and employed. Many lipoproteins were identified, including TUL4, one of the proteins that are used for identification of Francisella tularensis through PCR. Other identified lipoproteins, such as FTT1103, showed an influence on virulence of the bacterium in following experiments. In parallel the gel-free method based on two dimensional liquid chromatography and tandem mass spectrometry was developed. The method yielded higher percentage of proteins with predicted transmembrane domains. However, the overall combined number of detected membrane proteins from genome of F. tularensis was still rather low. IglC, hypothetical 23 kDa protein, was found among the proteins that showed differences between the subspecies. There were several spot detected for this protein within one strain and the spot profile differs between the subspecies. Mass spectrometry revealed two amino acid substitutions that confirmed the result of genome sequencing and can explain the different spot profile among the subspecies. The intraspecies difference is most probably related to the different level of deamidation, but this has to be corroborated by further experiments. The substitution of the amino acids between the subspecies was shown to be capable of utilization for subspecies discrimination by mass spectrometry. It supports the idea of using mass spectrometry as an accurate and sensitive method for bacterial typing.

Kľúčové slová

komparativní proteomová analýza, hmotnostní spektrometrie, Francisella tularensis

Jazyk práce: Czech

Datum vytvoření / odevzdání či podání práce: 21. 11. 2007

Obhajoba závěrečné práce

• Obhajoba proběhla 26. 11. 2007
• Vedúci: prof. RNDr. Igor Kučera, DrSc.
• Oponent: prof. Mgr. Jan Preisler, Ph.D., doc. RNDr. Ivan Rychlík, Ph.D.

Masaryk University

Faculty of Science

Doctoral programme / odbor:
Biochemistry (4-years) / Biochemistry