Yes / Engrailed-2 (EN2) is a homeodomain-containing transcription factor that has roles in boundary formation and neural guidance in early development, but which is also expressed in a range of cancers. In addition to transcriptional regulation, it is secreted by cells and taken up by others through a mechanism that is yet to be fully elucidated. In this study, the distribution of EN2 protein in cells was evaluated using immunofluorescence with a set of antibodies raised against overlapping epitopes across the protein, and through the use of an EN2-GFP construct. MX2 expression in primary prostate tumors was evaluated using immunohistochemistry. We showed that EN2 protein is present in the cell membrane and within microvesicles that can be secreted from the cell and taken up by others. When taken up by normal cells from the stroma EN2 induces the expression of MX2 (MxB), a protein that has a key role in the innate immune response to viruses. Our findings indicate that EN2 secretion by tumors may be a means of preventing viral-mediated immune invasion of tissue immediately adjacent to the tumor. / The Ringrose Family Trust supported this study through a studentship awarded to N.P.

Komagataella pastoris, formerly known as Pichia pastoris, and hereafter referred to as Pichia, is a methylotrophic yeast widely employed as a recombinant protein factory for biotechnical and industrial purposes. P. pastoris boasts the ability to thrive at high cell densities, executes numerous post-translational modifications, and exhibits minimal secretion of endogenous proteins, thus greatly facilitating the expression and purification of recombinant proteins. Despite these advantages, Pichia still presents certain challenges as an expression system. Occasionally, recombinant proteins are retained within the cell and subject to degradation. Furthermore, Pichia falls short in matching the production capabilities of more widely used systems like Escherichia coli in terms of sheer numbers of recombinant proteins generated. When incubating a strain of Pichia expressing reporter protein enhanced green fluorescent protein (eGFP) (yJC100:pDT300) with fetal bovine serum (FBS), western blot analysis revealed a novel, higher molecular band in addition to the expected band, suggesting that FBS was altering recombinant protein expression. The alterations to this higher molecular weight variant were confirmed to happen intracellularly, but the molecular mechanisms behind it remain unclear. To elucidate the intracellular molecular mechanisms behind the production of the novel recombinant protein variant, our lab utilized site-directed mutagenesis and mass spectrometry. Through site-directed mutagenesis, we were able to localize the alteration facilitated by FBS to the C-terminus of eGFP, demonstrating that post-translational modifications incurred by FBS incubation occur at the C-terminus. Analysis of intracellular lysate of yJC100:pDT300 revealed proteomic alteration caused by treating P. pastoris with fetal bovine serum, presenting possible key players in FBS’s interaction with yJC100:pDT300. LC-MS was also used to analyze the extracellular media of FBS-treated yJC100:pDT300, revealing peptide discrepancies in the C-terminus of eGFP between FBS-treated and FBS-untreated samples. These results confirm that FBS’s interaction with yJC100:pDT300 significantly affects secretion through interaction with eGFP’s C-terminus. Our research serves to characterize the FBS’s interaction with yJC100:pDT300 and set precedence for future work in further characterization and optimization of the novel mechanism occurring due to FBS’s presence in growth media.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that is a substantial threat, particularly in hospital settings, causing severe infections in immunocompromised patients that may lead to death. Pseudomonas aeruginosa harbors a multitude of virulence factors that enable this pathogen to establish both acute and chronic infections in humans. A key determinant of acute infections is a hollow molecular needle structure used for injecting toxins into a host cell, called the type three secretion system (T3SS). The secretion machinery itself is highly complex and, together with the specific secreted factors, requires expression of more than 30 genes. Due to the high energy cost of its synthesis to the organism this system is highly regulated to finely time gene expression to coincide with host contact. ExsA, a member of the AraC-type transcription factor family, is the main transcriptional activator of all the genes necessary for expression of the T3SS. Members of the AraC family are characterized by the presence of two helix-turn-helix (HTH) motifs, which bind to the promoter DNA and activate transcription. ExsA uses its HTH containing C-terminal domain (CTD) to regulate gene expression from 10 different promoters. The N-terminal domain (NTD) of ExsA mediates dimerization and regulation of ExsA-activity. While most AraC-type activators are regulated by a small molecule ligands, ExsA is regulated by another protein, ExsD. As part of a four-protein signaling cascade, ExsD interacts directly with ExsA to prevent transcription of T3SS-associated genes under non-inducing conditions prior to host cell contact. The entire regulatory cascade includes of two additional proteins, ExsC and ExsE. ExsA, ExsC, ExsD, and ExsE follow a partner-switching mechanism to link expression of the secretion system with host cell contact. Our laboratory is working to understand this unique signaling mechanism by determining the molecular basis for the regulation of this important virulence factor. Previous studies in the laboratory have solved the structures of ExsE, ExsC and ExsD, and shed light on how these proteins interact and compete for overlapping binding sites. However, it is still unclear as to how the ExsA and ExsD interact and thus how regulation is mediated at the molecular level. In the presented study, we sought to map the molecular interface between ExsA and ExsD. First, the crystal structure of ExsA-NTD is presented wherein the dimerization interface of the protein was identified. Two of the well-studied AraC-type proteins, AraC and ToxT crystal structures have been solved by others in the presence of their respective ligands. Residues that were involved in ligand binding in AraC and ToxT were aligned with the residues in ExsA and analyzed for interaction with ExsD. However, this canonical binding pocket appeared to be not involved in the interaction between ExsA and ExsD. Structure directed site-specific mutagenesis was carried out to construct many different variants of ExsD and ExsA. Thus constructed variants were purified and analyzed in a functional assay. Using this approach, we were able to identify regions on ExsD and ExsA that are crucial for the interaction and for the regulation of ExsA-dependent transcription. It turns out that backbone interactions between the amino-terminal residues of ExsD and the beta-barrel region of the ExsA-NTD are pivotal. This result explains how ExsA and ExsC compete for ExsD binding, since both target the same regions on ExsD. / PHD / Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for causing severe infections in immunocompromised individuals. Acute Pseudomonas aeruginosa infections are characterized by immediate adverse effects. An initial acute infection may become chronic, leading to long-term morbidity and mortality in affected individuals. During the initial stages of infection P. aeruginosa uses the type three secretion system, a syringe-like structure, to puncture the host cell and inject potent toxins. The activation of the genes required for forming this structure is tightly controlled by an activator protein, ExsA. When P.aeruginosa is not invading a host, ExsA is inhibited by another protein called ExsD, to prevent the needless production of the secretion apparatus. The presented work explores the mechanism of how ExsD achieves this inhibition of ExsA. This information is of potential biomedical interest because a clear understanding of the molecular basis for the interaction could inform the development of a small-molecule mimic of ExsD to be used in therapy. In Chapter 2 we report the structure of the domain of ExsA that is known to bind ExsD. Also, in this chapter and more so in Chapter 3, we performed a detailed analysis of potential interacting regions and ultimately succeeded in identifying key interacting regions in both ExsA and ExsD.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis disease, contains five copies of the ESAT-6 gene cluster, each encoding a dedicated ESX protein secretion system which has been defined as a novel Type-VII secretion system. The ESX have been implicated in virulence and survival of M. tuberculosis, and as such present a promising target for novel treatment interventions. This study has investigated the evolution, regulation, functions and substrates of the ESX secretion systems. The evolutionary history of the ESX secretion systems was established using in silico and phylogenetic analyses of the sequenced mycobacteria, closely related actinomycetes and WXG-FtsK clusters from other bacteria. The ESX-4 gene cluster appears to have evolved with the start of the evolution of the mycomembrane, followed by the duplication of ESX-3, which marks the evolution of the genus Mycobacterium. The ESX-1 duplication occurred next, followed by ESX-2 and ESX-5 which occur only in the slow growing mycobacteria. Five additional ESX gene clusters were newly identified and named ESX-P1 to - P5. These additional ESX clusters occur, or are predicted to occur, on plasmid DNA, and appear to be progenitors of the genomic ESX-1 to -5 gene clusters, possibly indicating a plasmid-mediated mechanism of ESX duplication and evolution. The promoters expressing the M. tuberculosis ESX-1 to ESX-5 secretion systems were investigated using a promoter probe assay, and characterised using in silico analyses. Promoters were identified for ESX-1, -2, -3 and -5. The functions of the mycobacterial ESX secretion systems were investigated using whole proteomic, secretomic and metabolomic analyses of the fast growing, non-pathogenic M. smegmatis, which contains three of the ESX secretion systems, ESX-1, 3, and 4. ESX knockout strains of M. smegmatis were generated and used in comparative analyses with wild-type M. smegmatis. ESX-1 was highly expressed in wild-type M. smegmatis, however no specific pathways showed considerable variation when ESX-1 was deleted. Deletion of ESX-3 resulted in substantial variation to multiple cellular pathways, including amino acid, carbohydrate and fatty acid metabolism and oxidative stress. These and other differences indicate possible perturbed polyamine metabolism in the absence of ESX-3. Although no ESX-4 protein components were detected in wild type M. smegmatis, the ESX-4 knockout displayed substantial proteomic variation. Reduced levels of ESX-3 component proteins in the ESX-4 knockout suggest that ESX-4 influences ESX-3 expression. Other variation linked ESX-4 to cell division and molybdenum metabolism. Secretomic analyses of wild-type and ESX knockout M. smegmatis strains were used to search for novel substrates of the M. smegmatis ESX secretion systems. No prototype ESX substrates were identified in the culture filtrates, however 10 possible substrates of the ESX-1, -3 and -4 secretion systems, containing the general ESX secretion signal, YxxxD/E, were identified. The functions of some of these proteins correlate with the ESX functions identified in the proteomic and metabolomic analyses. This study sets the groundwork for future work in understanding the functional roles and expression patterns of these ESX secretion systems and in using global proteomic and metabolomic analyses to understand cellular changes in response to specific signals or genomic changes. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die veroorsakende agent van tuberkulose, bevat vyf kopieë van die ESAT-6 geengroep, wat elk 'n toegewyde ESX proteïen sekresiesisteem, omskryf as 'n nuwe Tipe-VII sekresiestelsel, kodeer. Die ESX sekresiesisteme is betrokke by virulensie en oorlewing van M. tuberculosis, en is dus belowende teikens vir nuwe behandelings. Hierdie studie het die evolusie, regulasie, funksies en substrate van die ESX sekresiesisteme ondersoek. Die evolusionêre geskiedenis van die ESX sekresiesisteme is bepaal met behulp van in silico en filogenetiese analises van die volgordebepaalde mikobakterieë, nouverwante actinomisete en WXG-FtsK groepe van ander bakterieë. Die ESX-4 geengroep het saam met die evolusie van die mikomembraan ontwikkel, gevolg deur die duplisering van ESX-3, wat die evolusie van die genus Mycobacterium merk. Die ESX-1 duplisering het volgende plaasgevind, gevolg deur ESX-2 en ESX-5, wat slegs in die stadiggroeiende mikobakterieë voorkom. Vyf addisionele ESX geengroepe is nuut geïdentifiseer in hierdie studie en is ESX-P1 tot -P5 genoem. Hierdie addisionale ESX groepe is op, of word voorspel om op, plasmied DNS voor te kom, en mag voorlopers van die genomiese ESX-1 tot -5 geengroepe wees, wat moontlik dui op 'n plasmied-gemedieërde meganisme van ESX duplisering en evolusie. Die promoters wat verantwoordelik is vir die uitdrukking van die M. tuberculosis ESX-1 tot ESX-5 sekresiesisteme is ondersoek deur middel van 'n promoter aktiwiteitstoets, en gekarakteriseer deur in silico analises. Promoters is geidentifiseer vir ESX-1, -2, -3 en -5. Die funksies van die mikobakteriële ESX sekresiesisteme is ondersoek deur proteomiese, sekretomiese en metabolomiese analises van die vinnig-groeiende, nie-patogeniese mikobakterium M. smegmatis, wat ESX- 1, -3 en -4 sekresiesisteme besit. ESX uitslaanmutante van M. smegmatis is gegenereer en gebruik in die vergelykende analises met die wilde-tipe M. smegmatis. ESX-1 is hoogs uitgedruk in wilde-tipe M. smegmatis, maar geen spesifieke metabolise weë het aansienlike variasie getoon wanneer ESX-1 verwyder is. Delesie van ESX-3 het gelei tot aansienlike variasie in verskeie sellulêre weë, insluitend aminosuur-, koolhidraat- en vetsuur-metabolisme en oksidatiewe stres. Hierdie en ander verskille dui op moontlike versteurde poli-amien metabolisme in die afwesigheid van ESX-3. Hoewel geen ESX-4 proteïenkomponente opgespoor is in wilde-tipe M. smegmatis nie, vertoon die ESX-4 uitslaanmutant aansienlike proteomiese variasie. Laer vlakke van ESX-3 proteïne dui daarop dat ESX-4 die uitdrukking van ESX-3 beinvloed. Baie van die proteomiese variasie kan geassosieer word met verlaagde ESX-3 uitdrukking, maar ander variasie mag ESX-4 koppel met seldeling en molibdeen metabolisme. Sekretomiese analises van wilde-tipe en ESX uitslaanmutant M. smegmatis stamme is gebruik om nuwe substrate van die M. smegmatis ESX sekresiesisteme te identifiseer. Geen prototipe ESX substrate is geïdentifiseer in die kultuurfiltraat, maar 10 moontlike substrate van die ESX-1, -3 en -4 sekresiesisteme, met die algemene ESX sekresiesein, YxxxD/E, is geïdentifiseer. Die funksies van sommige van hierdie proteïene korreleer met die funksies geïdentifiseer in die proteomiese en metabolomiese analises. Hierdie studie stel die grondslag vir toekomstige werk in die begrip van die funksionele rol en uitdrukkingspatrone van die ESX sekresiesisteme en in die gebruik van globale proteomiese en metabolomiese analises om sellulêre veranderinge in reaksie op spesifieke seine of genomiese veranderinge te verstaan. / The National Research Foundation / German Academic Exchange Service (DAAD), / The Harry Crossley Foundation / The Ernst and Ethel Erikson Trust / Stellenbosch University

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein comprising an amino-terminal adenylate cyclase (AC) domain and a carboxy-terminal Repeat-in-Toxin (RTX) domain. The RTX domain is a hallmark of the family of RTX proteins, which are secreted from the cytosol of Gram-negative bacteria to the cell environment through the Type I Secretion System (T1SS). The RTX domain of CyaA consists of five blocks of RTX nonapetide repeats with a consensus sequence X-(L/I/V)-X-G-G-X-G- X-D. The aim of this work was to determine the role of the RTX domain in biological activities of CyaA and its role in the secretion of the toxin molecule from Bordetella pertussis. Systematic deletion analysis revealed that none of the prepared CyaA constructs was able to translocate its AC domain across the cytoplasmic membrane of host cells and make pores in target membranes. Moreover, deletion of individual RTX repeat blocks resulted in a very low efficacy of secretion of CyaA mutants into cell exterior. These data suggested that structural integrity of the RTX domain of CyaA is essential not only for cytotoxic activities of the toxin molecule but also for its secretion through the T1SS.

Das Typ III Sekretionssystem (T3SS) ist ein Proteinkomplex den Gramnegative Bakterien nutzen um in einem Schritt Effektorproteine (Effektoren) aus dem Zytosol über die Doppelmembran zu sekretieren. Für viele Bakterien ist das T3SS ein essenzieller Virulenzfaktor, der es ihnen erlaubt mit ihrem Wirt zu interagieren und diesen zu manipulieren. Charakteristisch für das T3SS ist die strukturelle Komponente, der Nadelkomplex. Dieser ähnelt strukturell einer Spritze, deren Basalkörper die bakteriellen Membranen und das Periplasma durchspannt und einer Nadel, die vom Basalkörper aus dem Bakterium ragt. Basierend auf dem Modell einer Spritze wird angenommen, dass Effektoren entfaltet und anschließend durch Basalkörper und Nadelkanal sekretiert werden. Trotz der kontinuierlichen Forschung an T3SS entbehrt dieses Modell einer experimentellen Grundlage und der Mechanismus ist nicht vollständig erklärt. Ziel der Arbeit war es, eine experimentelle Basis für den Sekretionsmechanismus des T3SS zu schaffen. Um zu verstehen, wie das T3SS Effektoren sekretiert, wurden zunächst Fusionsproteine konstruiert, welche aus einem Effektor und einem stabil gefalteten Knotenprotein bestehen. Aufgrund des Knotens in der Fusion ist davon auszugehen, dass dieser während der Sekretion nicht entfalten kann. Die Effektordomäne wird sekretiert während der Knoten im Kanal verbleibt und diesen verstopft. Nach unseremWissen ist diese Arbeit die erste Visualisierung von Effektorfusionen an isolierten Nadelkomplexen. Die Effektorfusion wird N-terminal voran durch den Kanal sekretiert, wobei der Kanal das Substrat umschließt und gegen Proteasen und chemische Modifikationen abschirmt. Die Ergebnisse dieser Arbeit untermauern eine Grundidee der Funktionsweise des T3SS und liefern eine vielversprechende Strategie für in situ-Strukturanalysen. Dieser Ansatz lässt sich auch auf andere Proteinsekretionssysteme übertragen, bei welchen Substrate vor dem Transport entfaltet werden müssen. / The Type III Secretion System (T3SS) is a complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. This work aimed to provide an experimental basis for the model of the T3SS mechanism. In order to elucidate details of the effector secretion mechanism, fusion proteins consisting of an effector and a bulky protein containing a knotted motif were generated. It is assumed that the knot cannot be unfolded during secretion of the chimera. Consequently, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. This is, to our best knowledge, the first time effector fusions have been visualized together with isolated NCs and it demonstrates that effector proteins are secreted directly through the channel with their N-terminus first. The channel encloses the substrate and shields it from a protease and chemical modifications. These results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.

No description available.

Species of the Bordetella genus cause the highly contagious whooping cough disease in humans (B. pertussis, B. parapertussis) and related respiratory diseases in other mammals (B. bronchiseptica, B. parapertussis). One of the virulence systems of Bordetellae is the type III secretion system (T3SS) employed for translocation of effector proteins directly from bacterial cytosol into the cytosol of host cells. The T3SS protein BopN protein has been categorized as a Bordetella effector protein. Nevertheless, the homologous proteins in other gram-negative bacteria function in establishing the secretion hierarchy through T3SS and some of them block T3SS secretion in high calcium environments before bacteria-host cell contact has been established. In this thesis I examined the function of the BopN protein and the role of calcium ions in T3SS activity of B. bronchiseptica. Two independent methods have been used for determination of T3SS secretion activity. Addition of 2 mM calcium ions into bacterial media decreased secretion of the T3SS reporter, while no such effect was observed in a B. bronchiseptica strain lacking the bopN gene. Mass spectrometry data confirmed the inhibition of T3SS activity in the presence of calcium ions. Enhanced calcium levels resulted in decreased mobilization and secretion of...

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Hen egg white lysozyme (HEWL; muramidase; EC 3:2:1:17) is an enzyme present in high concentrations in chicken (Gallus gallus) egg whites. It hydrolyses the link between N-acetylmuramic acid and N-acetylglucosamine in Gram positive bacterial cell walls, resulting in cell death. It is thus active against lactic acid bacteria (LAB), which may be present in grape juices and musts. These bacteria are responsible for malolactic fermentation of wines although many species, particularly of the genera Lactobacillus and Pediococcus, are considered spoilage organisms. The growth of LAB is therefore closely monitored and controlled during winemaking. The most common means of control is growth inhibition by chemical treatment (usually with SO2). Lysozyme is a commonly used wine processing aid, complementing the antimicrobial activity of SO2 . It allows for lower doses of SO2 to be used, thus improving the wholesomeness of wine. The OIV (Organisation Internationale de la Vigne et du Vin) approved its use in quantities up to 500 mg per liter of wine in 1997. This study evaluated the effect of different secretion signals on the secretion of lysozyme by the haploid auxotroph Saccharomyces cerevisiae strain FY23. Secretion by an industrial strain (VIN13) transformed with a single copy of the HEWL gene with the MF-a secretion signal under the control of the PGK1 (phosphoglycerate kinase 1) prompter and terminator was also evaluated. In the case of FY23 four secretion signals were used, namely the native lysozyme signal and the S. cerevisiae mating factor-a signal as well as mutants of these signals. These mutants incorporated two additional arginines at the N-terminus of the signals immediately downstream of the terminal methionine. The effect of these mutations was to increase the positive charge of the secretion signal N-terminals. The secretion signal-lysozyme fusions were placed under the regulation of the S. cerevisae PGK1 gene’s promoter and terminator. The resulting expression cassettes were cloned into integrating vectors YIpLac211 and pDMPOF1b and episomal vector pHVX2. These were used to transform FY23 and VIN13. FY23 as well as VIN13 transformants were evaluated in an artificial medium designed to reflect the nutrient content of grape juice, with particular attention being paid to assiminable nitrogen. Three hexose concentrations were tested in order to determine the effect thereof on lysozyme secretion titer. Lysozyme secreted under all tested growth conditions was found to be too low for detection by either enzymatic assay or HPLC-FLD. For this reason secreted lysozyme was isolated and concentrated 10x by means of cation-exchange. Subsequently, lysozyme concentrations in the concentrates was determined by means of the aforementioned techniques. SDS-PAGE analysis of lysozyme concentrates was also performed. No significant differences were found between native or MF-a secretion signals and their mutated counterparts in terms of secretion titer or proteolytic maturation. Lysozyme secreted with the MF-a signal was found to be misprocessed in all cases, with both an authentically processed and a larger form, in which the secretion signal was not completely removed, being present. Lysozyme secreted with the native signal appeared to be correctly processed in all cases. Secretion titer from high copy number episomal FY23 tranformants was similar to that of integrants containing a single copy of the gene. Sugar concentration affected lysozyme production, with higher quantities of the enzyme being secreted when higher initial sugar concentrations were used. Lysozyme titers were extremely low (< 0:25 mg/L) with all expression cassettes under all the tested conditions with both FY23 and VIN13. In the case of the VIN13’s a lower final biomass was found for the secretor strain tested in comparrison to the VIN13 wild-type. / AFRIKAANSE OPSOMMING: Hoendereierwitlisosiem (HEWL; muramidase, EG 3:2:1:17) is ´n ensiem teenwoordig in hoë konsentrasies in hoender (Gallus gallus) eierwitte. Dit hidroliseer die binding tussen N-asetielmuramiensuur en N-asetielglukosamien in Gram positiewe bakteriese selwande, wat tot seldood lei. Dit is dus aktief teen melksuurbakterieë (MSB), wat in druiwesap en mos teenwoordig kan wees. Hierdie bakterieë is verantwoordelik vir appelmelksuurgisting van wyne, hoewel baie spesies, veral van die genera Lactobacillus en Pediococcus, ook as bederforganismes beskou word. Die groei van MSB word dus noukeurig tydens wynbereiding gemoniteer en beheer. Die algemeenste wyse van beheer is groei-inhibisie deur chemiese behandeling (gewoonlik SO2). Lisosiem is ´n algemeen gebruikte wyntoevoegingsmiddel en vul die antimikrobiese aktiwiteit van SO2 aan. Met lisosiem kan ´n laer dosis van SO2 gebruik word, wat lei tot ´n verbetering van die heilsaamheid van die wyn. Die OIV (Organisasie Internationale de la Vigne et du Vin) het die gebruik daarvan goedgekeur tot en met 500 mg per liter wyn vanaf 1997. Hierdie studie het die effek van verskillende sekresieseine op die uitskeiding van lisosiem deur die haploïede ouksotrofe Saccharomyces cerevisiae stam, FY23, geëvalueer. Uitskeiding deur ´n industriële stam (VIN13), wat getransformeer is met ´n enkelkopie van die HEWL-gene met die MF-a sekresiesein onder die beheer van die PGK1 (Fosfogliseraat kinase 1) promotor en termineerder, is ook geëvalueer. In die geval van FY23 is vier sekresieseine gebruik, naamlik die inheemse lisosiemsein, S. cerevisiae MF- a sein, asook mutante van hierdie seine. Hierdie mutante het twee bykomende arginienresidu’s by die N-terminus van die seine direk stroom-af van die terminale metionien. Die effek van hierdie mutasies was om die positiewe lading van die uitskeidingsein N-terminale te verhoog. Die gevolglike uitdrukkingskassette is in die integrasievektor YIpLac211 en pDMPOF1b, en die episomale vektor pHVX2, gekloneer. Dit is gebruik om VIN13 en FY23 te transformeer. FY23, sowel as VIN13-transformante, is geëvalueer in ´n kunsmatige medium wat ontwerp is om die voedingsinhoud van druiwesap te weerspieël, met besondere aandag aan assimileerbare stikstof. Drie heksose konsentrasies is getoets om te bepaal wat die uitwerking daarvan op die lisosiemsekresietiter is. Onder alle groeitoestande was die isosiem wat uitgeskei is, te laag om deur ensimatiese toetse of HPLC-FLD bepaal te word. Om hierdie rede is uitgeskeide lisosiem geïsoleer en 10x gekonsentreer deur middel van katioon-uitruiling. Daarna is lisosiemkonsentrasies bepaal deur middel van bogenoemde tegnieke. SDS-PAGE-ontleding van lisosiemkonsentraat is ook uitgevoer. In terme van sekresietiter of proteolitiese maturasie, is geen beduidende verskille gevind tussen inheemse of MF-a sekresieseine en hul gemuteerde eweknieë nie. Lisosiem wat deur die MF-a sein uitgeskei is, is in alle gevalle foutief geprosesseer, met ´n teenwoordigheid van beide die regte produk en ´n groter produk, waarin die uitskeidingsein nie heeltemal verwyder word nie. Lisosiem wat met die inheemse sein uitgeskei is, blyk in alle gevalle korrek verwerk te wees. Sekresietiter van ´n aantal hoë-kopie episomale FY23-transformante was soortgelyk aan dié van integrante met ´n enkelkopie van die geen. Suikerkonsentrasie beïnvloed lisosiemproduksie, met ´n hoër hoeveelheid van die ensiem wat uitgeskei word wanneer die aanvanklike suiker in hoër konsentrasies gebruik is. Lisosiemtiters was baie laag (< 0:25 mg/L), met al die kassette onder al die getoetste toestande vir beide FY23 en VIN13. In die geval van die VIN13’s, is ´n laer finale biomassa vir die uitskeidingstam in vergelyking met die VIN13 wilde-tipe gevind.