Browsing by Author "Joos, Carla Grete"

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    Investigating the effects of the plaC accessory protein on disulphide bond formation in plantaricin 423 heterologously expressed in escherichia coli
    (Stellenbosch : Stellenbosch University, 2024-03) Joos, Carla Grete; Dicks, Leon Milner Theodore; Van Staden, Anton du Preez ; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH 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.