Vpliv proteina poliribonukleotid nukleotidiltransferaza bakterije Aggregatibacter actinomycetemcomitans na rast kvasovke Saccharomyces cerevisiae

Pinter, Matic (author), Butala, Matej (mentor), Petrovič, Uroš (comentor)

Parodontalne bolezni so skupina najbolj razširjenih obolenj ustne votline. Ena izmed invazivnejših oblik parodontalnih bolezni je agresivni parodontitis. Pri pacientih z agresivnim parodontitisom so določili izrazito povišano število bakterije Aggregatibacter actinomycetemcomitans (A. a.), ki s svojimi viruletnimi faktorji negativno vpliva na gostiteljsko tkivo. Z bioinformatskim orodji so nedavno ustvarili seznam proteinov seva A. a. D7S, ki naj bi interagirali z molekulami mRNA v človeških celicah. Iz pripravljenega seznama smo izbrali 6 proteinov (PNPaza, DnaK, F0F1 ATP sintaza, RPS1, TadG in ThrRS) in preverili njihov vpliv na rast kvasovke Saccharomyces cerevisiae, v katere smo predhodno transformirali pripravljene plazmidne kostrukte z genom za izbrani protein pod kontrolo galaktoznega promotorja. Ob prisotnosti galaktoze kot edinega vira sladkorja v gojišču, smo tako sprožili izražanje izbranih genov v kvasovki. Ugotovili smo, da sprožitev gena za PNPazo ali za različico tega proteina, ki ima na amino terminalnem koncu pripeto značko 3x-FLAG, inhibira rast kvasovke. Uspešno smo prepoznali tudi ključni aminokislinski ostanek proteina PNPaza, saj ob spremembi aminoksiline na mestu 133 varianta proteina ne deluje inhibitorno na rast kvasovke. Pripravili smo tudi plazmidni konstrukt za s 3x-FLAG tagom označenim proteinom PNPaza pod kontrolo nativnega promotorja bakterije A. a. Verjamemo, da bodo slednji konstrukt in v nalogi pridobljeni rezultati omogočili nadaljnje raziskave, ki bodo preučile vpliv proteina PNPaza na humane celice.

• Language: Slovenian
• Keywords: Parodontitis, Aggregatibacter actinomycetemcomitans, protein PNPaza, zunajcelični membranski vezikli, kvasovke
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2020
• PID: 20.500.12556/RUL-120296
• COBISS.SI-ID: 33663235
• Publication date in RUL: 18.09.2020

PINTER, Matic, 2020, Vpliv proteina poliribonukleotid nukleotidiltransferaza bakterije Aggregatibacter actinomycetemcomitans na rast kvasovke Saccharomyces cerevisiae [online]. Master’s thesis. [Accessed 27 March 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=120296

Secondary language

• Language: English
• Title: The Influence of bacterial Aggregatibacter actinomycetemcomitans polyribonucleotide nucleotidyltransferase protein on the growth of Saccharomyces cerevisiae

Abstract

Periodontal diseases are the group of the most common diseases of the oral cavity. One of the more invasive forms of periodontal disease is aggressive periodontitis. In patients with aggressive periodontitis, a significantly increased number of Aggregatibacter actinomycetemcomitans (A. a.) has been found, which has a negative effect on the host tissue with its virulence factors. Using bioinformatics tools, they recently compiled a list of A. a. D7S proteins that are believed to interact with mRNA molecules in human cells. From the prepared list we selected 6 proteins (PNPase, DnaK, F0F1 ATP synthase, RPS1, TadG and ThrRS) and tested their influence on Saccharomyces cerevisiae yeasts, into which we transformed plasmid constructs with genes for the selected protein. Expression of the selected protein was induced when yeast carrying such plasmid construct was grown in medium containing galactose as the sole carbon source. We found that the growth of transformants with induced PNPase gene expression was completely inhibited. The same result was achieved by testing the effect of PNPase to which the 3x-FLAG tail was attached, which will be useful for further research. We have also successfully identified a key catalytic residue in PNPase, that when mutated, precluded the PNPase inhibitory effect. In addition, we also prepared a plasmid construct enabling the 3x-FLAG tagged PNPase expression from its native promoter in A. a. We anticipate that the results obtained in this study will provide a solid basis for further research on the effect of A. a. PNPase on the human cells.

• Keywords: Periodontitis, Aggregatibacter actinomycetemcomitans, PNPase, outer membrane vesicles, yeast