Funkcijska transkriptomika in kvantifikacija biofilma bakterij Campylobacter jejuni

Volk, Manca (author), Klančnik, Anja (mentor)

Biofilmi bakterij Campylobacter jejuni pomembno prispevajo k njihovemu preživetju v okoljih zunaj gostitelja ter hkrati otežujejo zanesljivo kvantifikacijo živih celic, molekularni mehanizmi, vključeni v nastanek in razvoj biofilma, pa še niso v celoti pojasnjeni. Namen doktorske disertacije je bil vpeljati metodo PMA v kombinaciji z digitalnim PCR (ddPCR) za kvantifikacijo bakterij C. jejuni v enovrstnih in večvrstnih biofilmih ter preučiti molekularne in fiziološke mehanizme, vključene v razvoj biofilma. Metodo PMA-ddPCR smo vpeljali in ovrednotili za kvantifikacijo bakterij C. jejuni v enovrstnih in večvrstnih biofilmih. Ugotovili smo, da je dvakratno tretiranje z reagentom PMA učinkoviteje zmanjšalo signal DNA iz mrtvih celic kot enkratno tretiranje, vendar popolnega utišanja signala ni omogočilo. Z uporabo sekvenciranja RNA smo analizirali izražanje genov bakterij C. jejuni med razvojem enovrstnih in večvrstnih biofilmov ter ugotovili, da biofilmski način rasti spremljajo usklajene metabolne in strukturne prilagoditve. Razvoj enovrstnega biofilma je bil povezan s povišanim izražanjem genov, vključenih v biosintezo aminokislin, odziv na oksidativni stres in nastanek bičkov, hkrati pa z znižanim izražanjem genov, povezanih z energijskem metabolizmom in transportom. V zrelih fazah biofilma se je dodatno znižalo izražanje genov, vključenih v biosintezo kapsularnih polisaharidov in lipooligosaharidov. V večvrstnih biofilmih ob prisotnosti bakterij Pseudomonas fragi so bakterije C. jejuni izkazale izrazitejši in širši transkripcijski odziv, ki je odražal kompetitivne interakcije v biofilmu. Odziv je vključeval obsežno preoblikovanje površinskih struktur, časovno odvisne spremembe v odzivih na stres in energijskem metabolizmu ter aktivacijo obrambnih mehanizmov. Na podlagi rezultatov sekvenciranja smo izbrali gene Cj0735, Cj0736 in Cj1465 ter pripravili ustrezne mutante. Fenotipska analiza mutantov je pokazala spremembe v agregaciji in gibljivosti, lastnostih, ki sta povezani z nastankom in razvojem biofilma bakterij C. jejuni.

• Language: Slovenian
• Keywords: Campylobacter jejuni, biofilmi, enovrstni biofilmi, večvrstni biofilmi, kvantifikacija, PMA-ddPCR, sekvenciranje RNA, diferenčno izraženi geni
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Volk]
• Year: 2026
• PID: 20.500.12556/RUL-190192
• UDC: 579.22/.26
• COBISS.SI-ID: 302400259
• Publication date in RUL: 24.10.2026

Secondary language

• Language: English
• Title: Functional transcriptomics and quantification of Campylobacter jejuni biofilm

Abstract

Biofilms of Campylobacter jejuni contribute to bacterial survival in environments outside the host while simultaneously complicating reliable quantification of viable cells, and the molecular mechanisms involved in biofilm formation and development remain incompletely understood. The aim of this doctoral dissertation was to establish a PMA-based method in combination with digital PCR (ddPCR) for the quantification of C. jejuni in monospecies and multispecies biofilms and to investigate the molecular and physiological mechanisms involved in biofilm formation. The PMA-ddPCR method was established and evaluated for the quantification of C. jejuni in monospecies and multispecies biofilms. We found that double PMA treatment reduced the DNA signal from dead cells more effectively than single treatment, although complete signal suppression was not achieved. Using RNA sequencing, we analysed gene expression of C. jejuni during the development of monospecies and multispecies biofilms and showed that biofilm growth is accompanied by coordinated metabolic and structural adaptations. Development of monospecies biofilms was associated with increased expression of genes involved in amino acid biosynthesis, oxidative stress response, and flagellar formation, together with reduced expression of genes related to energy metabolism and transport. In mature biofilms expression of genes involved in capsular polysaccharide and lipooligosaccharide biosynthesis was further reduced. In multispecies biofilms in the presence of Pseudomonas fragi, C. jejuni exhibited a broader and more pronounced transcriptional response reflecting competitive interactions within the biofilm. This response included extensive remodelling of surface-associated structures, temporally dependent changes in stress responses and energy metabolism, and activation of defence mechanisms. Based on the sequencing results, genes Cj0735, Cj0736, and Cj1465 were selected and corresponding mutants were constructed. Phenotypic analysis of the mutants revealed changes in aggregation and motility, traits associated with the formation and development of C. jejuni biofilms.

• Keywords: Campylobacter jejuni, biofilms, monospecies biofilms, multispecies biofilms, quantification, PMA-ddPCR, RNA sequencing, differentially expressed genes