Engineering of fluorescent vaginal Lactobacillus species for their monitoring in nanofibers and cell models

Stojanov, Spase (author), Berlec, Aleš (mentor), Zupančič, Špela (co-mentor)

Vaginal infections are one of the main reasons why women seek medical help. They are associated with decreased number of lactobacilli and overgrowth of opportunistic pathogens. Restoring the normal vaginal microbiota with lactobacilli as probiotics can help cure the infections. However, the lack of techniques for their functional studies and the need for an appropriate delivery system hinders their use as probiotics. In the dissertation, we propose two solutions for overcoming the deficiencies. First, genetic engineering of three vaginal lactobacilli (Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii), and control Lactiplantibacillus plantarum, enabled expression of fluorescent proteins, i.e. infrared fluorescent protein (IRFP), green fluorescent protein (GFP), red fluorescent protein (mCherry) and blue fluorescent protein (mTagBFP2), thereby facilitating distinction and quantification of lactobacilli. Second, vaginal lactobacilli have been successfully incorporated into polyethylene oxide (PEO) nanofibers with addition of different excipients, thereby developing a promising delivery system. Alginate and sucrose showed the highest stimulation of growth of lactobacilli in vitro and were included in the formulations. Interaction between excipients and bacteria was assessed thermally and spectroscopically. All the species survived electrospinning, with L. gasseri demonstrating the highest survival, followed by L. jensenii and L. crispatus. The species were viable in all formulations after 56 days of storage. The lactobacilli retained their fluorescence after incorporation and after their release from nanofibers. We therefore used engineered lactobacilli expressing mCherry to study the release kinetics, where PEO nanofibers loaded with lactobacilli were completely dissolved after 30?45 min. Lactobacilli have also adhered to Caco-2 epithelial cells as observed with a confocal microscope. To conclude, this dissertation describes innovative technology for tracking vaginal lactobacilli and their successful incorporation into electrospun nanofibers. The results hold a great promise for the development of a novel medicine against vaginal infections.

• Language: English
• Keywords: Vaginal infections, lactobacilli, fluorescent proteins, electrospinning, nanofibers, delivery system.
• Work type: Doctoral dissertation
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-141668
• Publication date in RUL: 05.10.2022

Secondary language

• Language: Slovenian
• Title: Razvoj fluorescentnih vaginalnih vrst iz rodu Lactobacillus za njihovo spremljanje v nanovlaknih in celičnih modelih

Abstract

Vaginalne okužbe so eden od glavnih razlogov, zaradi katerih ženske poiščejo zdravniško pomoč. Povezane so z zmanjšanim številom laktobacilov in prekomerno rastjo oportunističnih patogenov. Obnovitev normalne vaginalne mikrobiote z laktobacili kot probiotiki lahko pomaga pri zdravljenju okužb, vendar pomanjkanje tako metod za funkcionalne študije probiotikov kot tudi ustreznega dostavnega sistema ovirata njihovo učinkovito uporabo. V doktorski disertaciji predlagamo dve rešitvi za opredeljeni pomanjkljivosti. Prvič, genski inženiring treh vaginalnih laktobacilov (L. crispatus, L. gasseri in L. jensenii) in kontrolnega Lactiplantibacillus plantarum je omogočil izražanje fluorescenčnih proteinov, tj. infrardečega fluorescenčnega proteina (IRFP), zelenega fluorescenčnega proteina (GFP), rdečega fluorescenčnega proteina (mCherry) in modrega fluorescenčnega proteina (mTagBFP2), kar je olajšalo razlikovanje in kvantifikacijo laktobacilov. Drugič, vaginalne laktobacile smo uspešno vgradili v nanovlakna iz polietilenoksida (PEO) skupaj z različnimi pomožnimi snovmi in s tem razvili obetaven dostavni sistem. Alginat in saharoza sta in vitro najmočneje spodbujala rast laktobacilov, zato sta bila vključena v nanovlakna. Interakcijo med pomožnimi snovmi in bakterijami smo ocenili termično in spektroskopsko. Vse vrste so preživele elektrostatsko sukanje, pri čemer je bilo preživetje največje pri bakteriji L. gasseri, sledili sta ji bakteriji L. jensenii in L. crispatus. Po 56 dneh shranjevanja so bile bakterije v vseh formulacijah še vedno viabilne. Laktobacili so ohranili fluorescenco po vgradnji in po sprostitvi iz nanovlaken. Zato smo za proučevanje kinetike sproščanja uporabili spremenjene laktobacile, ki izražajo mCherry, pri čemer so se PEO nanovlakna z laktobacili popolnoma raztopila po 30⠒45 min. Laktobacili so se pritrdili tudi na epitelijske celice Caco-2, kar smo opazovali s konfokalnim mikroskopom. Če povzamemo, v tej disertaciji je opisana inovativna tehnologija za sledenje vaginalnim laktobacilom in njihova uspešna vgradnja v nanovlakna. Rezultati predstavljajo velik obet za razvoj novega zdravila proti vaginalnim okužbam.

• Keywords: Vaginalne okužbe, lactobacilli, fluorescentni proteini, elektrostatsko sukanje, nanovlakna, dostavni sistem.