Investigating the effects of the plaC accessory protein on disulphide bond formation in plantaricin 423 heterologously expressed in escherichia coli

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dc.contributor.advisorDicks, Leon Milner Theodoreen_ZA
dc.contributor.advisorVan Staden, Anton du Preez en_ZA
dc.contributor.authorJoos, Carla Greteen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Microbiology.en_ZA
dc.date.accessioned2024-03-05T14:57:59Z
dc.date.accessioned2024-04-26T15:36:48Z
dc.date.available2024-03-05T14:57:59Z
dc.date.available2024-04-26T15:36:48Z
dc.date.issued2024-03
dc.descriptionThesis (MSc)--Stellenbosch University, 2024. en_ZA
dc.description.abstractENGLISH ABSTRACT: The discovery of antibiotics has revolutionised society. Antibiotics have been used to treat countless diseases and improved the quality of life for many. Unfortunately, the overuse of antibiotics has led to bacteria developing mechanisms to outmanoeuvre the inhibitory effects of these pharmaceuticals. The emergence of antibiotic-resistant bacteria poses a major threat to human health, industries and the economy. Thus, the search for alternatives to antibiotics has become imperative. Lactic acid bacteria (LAB) have a long-standing association with food preservation, pharmaceutical production and use as probiotics. Amongst the myriad of metabolites produced by LAB, ribosomally synthesised peptides referred to as bacteriocins, have gained interest for their antimicrobial properties. LAB secrete bacteriocins to help outcompete other bacteria inhabiting their surrounding environment. This study focused on class IIa bacteriocins, in particular plantaricin 423 isolated from Lactobacillus plantarum 423. Class IIa bacteriocins are pediocin-like and well-known for their antilisterial properties. The production of class IIa bacteriocins has been greatly improved, particularly by their heterologous expression as fusion proteins. In this study, we further improved the heterologous expression of plantaricin 423 through the co-expression with its accessory protein, PlaC, identified in the plantaricin 423-encoding operon. A thioredoxin-fold and CXXC catalytic motif were identified in the PlaC accessory protein, which are characteristics associated with the thioredoxin superfamily. However, thioredoxin-like accessory proteins are uncommon amongst class IIa bacteriocins. An insulin reduction assay based on the dithiol-disulphide oxidoreductase activities of the thioredoxin system was performed and further confirmed that the PlaC accessory protein is a thioredoxin superfamily member. In addition, a peptidase (PEP) domain was identified in the native secretion machinery of plantaricin 423. The PEP domain shares characteristics with C39 cysteine peptidases. The PEP domain was isolated and its ability to proteolytically remove the leader peptide of precursor plantaricin 423 was evaluated. It was hypothesised that the PlaC accessory protein improved the heterologous expression of plantaricin 423 through promoting disulphide bond formation. Previous studies obtained similar results with another class IIa bacteriocin, pediocin PA-1, with its respective accessory protein, PedC. The formation of disulphide bonds in the periplasmic space of Escherichia coli involves two important enzymes, DsbA and DsbC, which function as an oxidase and isomerase, respectively. Unfortunately, disulphide bond formation is poorly understood in Gram-positive bacteria. To improve our current knowledge of disulphide bond formation in secreted proteins from Gram-positive bacteria, the PlaC accessory protein was expressed in E. coli strains with knockout mutations for DsbA or DsbC. In addition, PlaC was heterologously expressed with two plant antimicrobial peptides, VvAMP1 and VvScorpio, that both require four nonconsecutive disulphide bonds. This is the first study that provides experimental evidence confirming that the thioredoxin-like accessory protein, PlaC, identified in the plantaricin 423-encoding operon serves as a DsbC isomerase homologue during the maturation and secretion of plantaricin 423 by promoting correct disulphide bond formation. This is also the first report for a DsbC-like homologue in Firmicutes.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Die ontdekking van antibiotika was revolusionêr vir die samelewing. Antibiotika word gebruik om talle siektes te behandel en het die lewenskwaliteit van baie verbeter. Ongelukkig het die oorbenutting van antibiotika daartoe gelei dat bakterieë die vermoë ontwikkel het om die inhiberende effek van hierdie middels te omseil. Die opkoms van antibiotika-weerstandige bakterieë hou 'n groot bedreiging in vir menslike gesondheid, nywerhede en die ekonomie. Die soeke na alternatiewe vir antibiotika het dus noodsaaklik geword. Melksuurbakterieë (MSB) het 'n lang verbintenis met voedselpreservering, farmaseutiese produksie en gebruik as probiotika. Onder die magdom metaboliete wat deur MSB geproduseer word, het ribsomaal-gesintetiseerde peptiede, bekend na as bakteriosiene, belangstelling ontlok weens hul antimikrobiese eienskappe. MSB skei bakteriosiene af om met ander bakterieë in hul omliggende omgewing te kompeteer. Hierdie studie het gefokus op klas IIa bakteriosiene, in die besonder plantarisien 423 wat uit Lactobacillus plantarum 423 geïsoleer is. Klas IIa bakteriosiene is pediosien-agtig en bekend vir hul antilisteriale eienskappe. Die produksie van klas IIa bakteriosiene is beduidend verbeter, veral deur hul heteroloë uitdrukking as samesmeltingsvennote. In hierdie studie het ons die heteroloë uitdrukking van plantarisien 423 verder verbeter deur die mede-uitdrukking van sy hulp-proteïen, PlaC, geïdentifiseer in die plantarisien 423-koderende operon. 'n Tioredoksien-vou en CXXC katalitiese motief is in die PlaC hulp-proteïen geïdentifiseer, wat met die tioredoksien- superfamilie geassosieer is. Tioredoksienagtige hulp-proteïene is ongewoon onder klas IIa- bakteriosiene. 'n Insulienreduksietoets gebaseer op die ditiol-disulfied oksidoreduktase aktiwiteite van die tioredoksien sisteem het bevestig dat die PlaC hulp-proteïen 'n tioredoksien superfamilielid is. 'n Peptidase (PEP) domein in die inheemse sekresie masjinerie van plantarisien 423 is ook geïdentifiseer. Die PEP domein deel kenmerke met C39 sisteïen peptidases. Die PEP-domein is geïsoleer en sy vermoë om die leierpeptied van voorloper plantarisien 423 proteolities te verwyder, is geëvalueer. Verder is die hipotese ondersoek dat die PlaC hulp-proteïen die heteroloë uitdrukking van plantarisien 423 verbeter deur disulfiedbindingsvorming te bevorder. Vorige studies het soortgelyke resultate verkry met 'n ander klas IIa-bakteriosien, pediosien PA-1, met sy betrokke hulp-proteïen, PedC. Die vorming van disulfiedbindings in die periplasmiese ruimte van Escherichia coli behels twee belangrike ensieme, DsbA en DsbC, wat onderskeidelik as 'n oksidase en isomerase funksioneer. Ongelukkig word disulfiedbindingsvorming in Gram- positiewe bakterieë swak verstaan. Om ons huidige kennis van disulfiedbindingsvorming in uitgeskeide proteïene van Gram-positiewe bakterieë te verbeter, is die PlaC bykomstige proteïen uitgedruk in E. coli stamme met uitklopmutasies vir DsbA of DsbC. Daarbenewens is PlaC heteroloog uitgedruk met twee plant antimikrobiese peptiede, VvAMP1 en VvScorpio, wat albei vier nie-opeenvolgende disulfiedbindings benodig. Dit is die eerste studie wat eksperimentele bewyse verskaf wat bevestig dat die tioredoksienagtige hulp-proteïen, PlaC, in die plantarisien 423-koderende operon as 'n DsbC isomerase homoloog dien tydens die rypwording en uitskeiding van plantarisien 423 deur korrekte disulfiedbindingsvorming te bevorder. Dit is ook die eerste verslag vir 'n DsbC-agtige homoloog in Firmicutes.af_ZA
dc.description.versionMastersen_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/130383
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshLactobacillus plantarum -- Inhibitorsen_ZA
dc.subject.lcshBacteriocins -- Genetic aspectsen_ZA
dc.subject.lcshBacteria -- Environmental aspectsen_ZA
dc.subject.lcshEscherichia coli -- Effect of stress onen_ZA
dc.subject.lcshAntimicrobial peptidesen_ZA
dc.subject.lcshLactic acid bacteriaen_ZA
dc.subject.nameUCTDen_ZA
dc.titleInvestigating the effects of the plaC accessory protein on disulphide bond formation in plantaricin 423 heterologously expressed in escherichia colien_ZA
dc.typeThesisen_ZA
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