Interakcia hostitel´- patogén u vybraných mykotických infekcií

Mgr. Alena VRÁBLÍKOVÁ

Doctoral thesis

Interaction of Host-pathogen in Selected Mycotic Infections

Abstract:

Táto práca je zameraná na metódy izolácie RNA a cDNA z vláknitej huby Trichophyton mentagrophytes a na analýzu profilu exprésie cystein dioxygenázy na mRNA a proteínovej úrovni. CDO bola amplifikovaná pomocou PCR s použitím primerov navrhnutých na základe vysokej sekvenčnej podobnosti CDO popísaných u iných húb. PCR produkt bol klonovaný a sekvenovaný. His-tag obsahujúci rekombinantný proteín bol pripravený v expresnom systéme Escherichia coli. Proteín bol afinitne purifikovaný a identifikovaný pomocou hmotnostnej spektrometrie MALDI-TOF. Enzymatická aktivita bola stanovená monitorovaním produkcie cystein sulfinátu pomocou ESI-MS. Kultivačné podmienky výrazne ovplyvňujú enzymatickú aktivitu CDO. Všetky CDO, produkované za natívnych podmienok boli enzymaticky aktívne. Transkripčná aktivita Cdo mRNA bola monitorovaná pomocou RT Real-Time PCR s použitím -aktínu ako normalizačného konštitutívne exprimovaného génu. Doplnenie kultivačného média o keratín (ľudské vlasy) alebo L-cystín zvýšilo normalizovanú hladinu Cdo mRNA približne osem krát. Detailná analýza efektu L-cystínu na aktivitu CDO na úrovni mRNA (monitorovanú pomocou Real-Time PCR) a na proteínovej úrovni (monitorovanú pomocou SDS-PAGE a Western Blot s použitím afinitne purifikovanej sérovej protilátky špecifickej k CDO, pripravenej imunizáciou myší pomocou His-tag obsahujúceho rekombinantného CDO proteínu) odhalila, že u T. mentagrophytes je Cdo mRNA konštantne exprimovaná na nízkej hladine a CDO proteíny sú po translácii rýchlo degradované. V neprítomnosti L-cystínu môže byť translácia blokovaná. U T. mentagrophytes sa uplatňuje regulácia CDO aktivity na úrovni transkripcie i posttransalčných úprav.

Abstract:

This work is focused on the methods of isolation of RNA and cDNA from the fungus Trichophyton mentagrophytes and the analysis of the expression profile of cysteine dioxygenase at mRNA and protein levels. CDO was amplified by PCR using the primer pair designed on the basis of high sequence conservancy of CDO described in other fungi. PCR product was cloned into plasmid vector and sequenced. Escherichia coli expression system was used for production of recombinant His-tagged CDO protein . Protein was affinity purified and identity was confirmed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF). Enzymatic activity was assayed by monitoring the cysteine sulphinate production using Electrospray Mass Spectrometry (ESI-MS). Cultivation conditions of CDO-expressing E. coli markedly influenced CDO enzymatic activity. All CDO purified from E. coli under native conditoins were confirmed to be enzymatically active. The transcriptional activity of Cdo mRNA was monitored by RT Real-Time PCR using -actin as a housekeeping gene. Supplementation of cultivation medium with human hairs or L-cystine increases normalized Cdo mRNA level for about eight times. The detailed analysis of the effect of L-cystine on the activity of CDO at mRNA level (monitored by Real-Time PCR) and protein level (monitored by SDS-PAGE and western blot with the use of the antibody affinity purified from the serum of mice imunized by recombinant His-tag CDO protein ) revealed that in T. mentagrophytes the Cdo mRNA is constantly expressed at low level and after translation the CDO protein is rapidly degraded . Translation may be blocked until the L-cystine is not supplemented. In T. mentagrophytes both transcriptional and post-translational regulations of CDO takes place.

Keywords

cystein dioxygenáza, dermatofyty, keratinolýza, vakcinácia

Language used: Czech

Date on which the thesis was submitted / produced: 11. 9. 2014

Accessible from:: 11. 9. 2014

Thesis defence

• Date of defence: 15. 1. 2015

PALACKÝ UNIVERSITY IN OLOMOUC

Faculty of Medicine and Dentistry

Doctoral programme / field:
Medical Immunology / Medical Immunology