Investigating the mode of transcriptional regulation controlling plantaricin 423 expression in Lactobacillus plantarum 423

Vermeulen, Ross Rayne (2019-03)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: The discovery and use of antibiotic therapies was one of the most significant achievements of the twentieth century. However, the current rate at which antibiotic resistance develops heavily outweighs the rate that novel treatments are introduced. Without a doubt, antibiotic resistance is one of the biggest challenges scientists of the twenty-first century are facing. With a global realization of finite resource availability and continuous climate change, the transition toward developing more sustainable systems is becoming a focal point for science, technology, and society as a whole. Such an approach also must be applied in the fight against antibiotic resistance and is already being observed through the emergence of novel fields such as biotherapeutics. Antibiotics have only recently been employed as a therapy, yet antimicrobial compounds and mechanisms for resistance have existed for millennia. Therefore, systems must already exist to ensure the sustainability of an antimicrobial compound’s use by a microorganism within its environment. Such systems are likely to use a diverse array of approaches within a microbiota, with the chemical diversity of antimicrobials being one. While researching the role and effect of antimicrobials within a microbiota may elucidate new approaches and schemes to manage antimicrobial resistance, a diverse group of antimicrobials, known as bacteriocins, has already been discovered. In the past, these antimicrobial peptides have received considerably less attention than antibiotics, however due to the urgent need for alternatives they warrant serious consideration. This study concerns the native transcriptional regulation of a subclass IIa bacteriocin, plantaricin 423, produced by Lactobacillus plantarum 423, one of the strains in the probiotic EntiroTM developed by our research group. The mode of transcriptional regulation for class IIa bacteriocins in the absence of local regulatory genes, as observed for plantaricin 423, is unknown. Through the development of a fluorescent promoter-reporter system, it was observed that the transcriptional regulation of plantaricin 423 responded to manganese-limiting conditions. During this research, significant progress was made for methods concerning bacteriocin classification, heterologous expression, and real-time in vivo transcriptional monitoring. Based on findings obtained using a fluorescent promoter-reporter system and the fact that L. plantarum 423 requires high intracellular Manganese concentrations for aerobic respiration, plantaricin 423 might aid in Manganese acquisition from target cells via cell wall poration. This research represents the first steps towards understanding how L. plantarum 423 and Enterococcus mundtii ST4SA, the other strain in the EntiroTM, interact with each other, the gastrointestinal microbiota and the host. Future research in this direction will be done with the hope of discovering sustainable alternatives to current problems, such as antibiotic resistance.

AFRIKAANSE OPSOMMING: Die ontdekking van antibiotika was een van die belangrikste deurbrake van die twintigste eeu. Die tempo waarteen weerstandigheid teen antibiotika huidiglik onwikkel is egter vinniger as wat nuwe behandelings geimplimenteer kan word. Dus is antibiotika-weerstandigheid 'n uitdaging wat in die een-en-twintigste eeu oorkom moet word. Met 'n wêreldwye besef van klimaatsverandering en die beperktheid van natuurlike hulpbronne, word die ontwikkeling en oorgang na meer volhoubare stelsels 'n fokuspunt vir die wetenskap en die samelewing as geheel. So 'n benadering is ook toepaslik in die stryd teen antibiotika-weerstandigheid en word reeds waargeneem deur die opkoms van nuwe velde soos bio-terapeutika. Alhoewel antibiotika eers relatief onlangs as 'n terapie ingespan is, bestaan die antimikrobiese verbindings en meganismes vir weerstandigheid alreeds vir millennia. Daarom moet daar reeds stelsels in mikroorganismes bestaan wat die volhoubaarheid van hul eie antimikrobiese middels in hulle omgewing verseker. Sulke stelsels sluit waarskynlik'n verskeidenheid meganismes in, onder andere die chemiese diversiteit van antimikrobiese middels. Terwyl verdere ondersoek tot die rol en effek van antimikrobiese middels binne die gastro-intestinale mikrobiota nuwe meganismes kan ontbloot wat antimikrobiese weerstandigheid mag help bestuur, is daar reeds 'n diverse groep antimikrobiese peptiede, bekend as bakteriosiene, beskryf. Hierdie antimikrobiese peptiede het in die verlede aansienlik minder aandag ontvang as antibiotika, maar in die lig van die noodsaaklikheid vir alternatiewe, verdien hulle verdere ondersoek. Hierdie proefskrif het betrekking op die inheemse transkripsie regulasie van 'n subklas IIa bakteriosien, plantarisien 423, wat vervaardig word deur Lactobacillus plantarum 423, een van die stamme in die probiotikum EntiroTM wat deur ons navorsingsgroep ontwikkel is. Die modus van transkripsionele regulering vir klas IIa bakterieosiene, in die afwesigheid van plaaslike regulatoriese gene soos waargeneem vir plantarisien 423, is onbekend. Met die ontwikkeling van 'n fluoresserende promotorverslaggewerstelsel is daar waargeneem dat plantarisien 423 regulasie reageer op Mangaanbeperkende toestande. Tydens hierdie navorsing is beduidende vordering gemaak tot metodologiee rakende bakteriosien klassifikasie, heterologiese uitdrukking en monitering van in vivo translasie in “real-time”. In die lig van resultate verkry met die gebruik van 'n fluoresserende promotorverslaggewerstelsel en die bevinding dat Lactobacillus plantarum 423 hoë intra-sellulêre mangaan konsentrasies vir aërobiese respirasie benodig, kan plantarisien 423 moontlik help met die verkryging van mangaan vanuit teiken selle deur middel van selwand porie formasie. Hierdie navorsing verteenwoordig die eerste stappe om die interaksie te verstaan tussen L. plantarum 423 en Enterococcus mundtii ST4SA (die ander stam in die probiotika EntiroTM), asook interaksies met die gastro-intestinale mikrobiota en die gasheer. Verdere navorsing in die rugting sal gemik wees om volhoubare alternatiewe oplossings vir hedendaagse proebleme te vind, soos vir antibiotikaweerstandigheid.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105662
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