Fachbereich Veterinärmedizin



    Proteinbiochemische Charakterisierung von Antigenen pathogener Prototheken-Spezies (2016)

    Irrgang, Alexandra (WE 10)
    Berlin: Mensch und Buch Verlag, 2016 — IV, 79 Seiten
    ISBN: 978-3-86387-731-6
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000102497
    Institut für Tier- und Umwelthygiene

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35
    14169 Berlin
    +49 30 838 51845

    Abstract / Zusammenfassung

    Prototheca spp. are unicellular, saprophytically living algae. Although these microorganisms belong to the phylogenetic group of the green algae, they are colorless due to the absence of chlorophyll. Therefore their metabolism is characterized as obligate heterotrophic. Macroscopically they bear a likeness to yeasts of the genus Candida but the algae can be differentiated from them easily under the microscope because they show no budding. Prototheca reproduce asexually by the formation of endogenous daughter cells. They are of medical and veterinary interest as some of the Prototheca species are associated with infections in humans and animals, known as protothecosis. They fulfill all qualifications to be highly pathogenic: a strong and resilient cell wall, resistance against physical, chemical and mechanical stress, formation of a resting cell stage and limited treatment options. However, protothecosis is a relatively rare disease. The pathogenic agent, the pathogenicity as well as the localization of the infection vary between the different host species. Protothecosis may occur as local infection for example as cutaneous manifestation but can run a disseminated course. There is little known about the underlying mechanisms of pathogenicity. To lay the basis of understanding the course of infection, the aim of this thesis was to determine immunoreactive proteins of Prototheca zopfii GT2 by western blot analysis and identify those using MALDI TOF MS. In the first part of this work, western blot analysis was used to identify immunodominant protein spots using sera of experimentally infected rabbits. With these 24 immunoreactive proteins of P. zopfii GT2 were detected, from which 15 proteins could be identified by MALDI TOF MS. Most of these proteins were proteins of regulatory functions and enzymes of the metabolism. Some of them - malate dehydrogenase (MDH), heat shock proteins 70 (Hsp70), elongation factor 1-􀄮􀀃 􀀋EF-􀀔􀄮) and 14-3-3 protein – are known as immunoreactive proteins from other eukaryotic pathogens. Further crossreactivity of the sera with proteins from P. zopfii GT1 and P. blaschkeae was tested with a high success rate. 48 of these crossreactive proteins could be determined using MALDI TOF MS, too. ATPase, MDH, EF- 􀀔􀄮 and Hsp70 were identified from western blot analysis of all tested strains. In the second part of this PhD thesis, experiments with sera from naturally infected dogs were performed as above. The canine sera display a marked crossreactivity between different species and genotypes, too. Taken together 198 proteins spots were analyzed by MALDI TOF MS from which 86 were identified. Comparable with the leporine sera, the main part of the immunoreactive proteins obtained with canine infection sera were proteins of regulation, replication, protein expression and metabolic enzymes. It is noticeable that MDH, Hsp70, EF-􀀔􀄮 and 14-3-3 protein were also found as immunoreactive proteins. These are involved in pathogenicity of the eukaryotic pathogens Anisakis sp., Schistosoma sp. and Cryptococcus sp. Further glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triosephosphate isomerase, enolase and succinyl-CoA synthetase could be determined as antigenic proteins. These so-called housekeeping enzymes are of special functions as surface-associated virulence factors in a number of pathogens. In this thesis, for the first time, the presence of GAPDH on the surface of Prototheca spp. was demonstrated using FACS analysis. It was shown that GAPDH is expressed on the surface of P. blaschkeae. This implies the assumption that GAPDH could be involved in the pathogenicity of P. blaschkeae causing bovine mastitis. It is also possible that P. zopfii GT2 disposes of other surface associated metabolic enzymes than GAPDH. It is strongly recommend that additional investigations be carried out to confirm the expression of these identified enzymes on the surface of Prototheca cells and ascertain their role in the infection process.