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Globally, fungal infections affect more than one billion people annually, with an estimated mortality of approximately two million people. The genus Candida is a highly heterogeneous group containing several opportunistic pathogens responsible for the increasing number of life-threatening mycoses, especially in immunocompromised subjects, including cancer patients, human immunodeficiency virus (HIV) positive patients, and organ transplant recipients. Compared to antibacterial counterparts, the number of available antifungals is limited, and the increase in antifungal resistance further complicates this. Resistance or tolerance towards all the available classes of antifungals has been reported. Strikingly, Candida krusei exhibits innate resistance to fluconazole (FLC) and rapid adaptive resistance to other antifungal drugs. Moreover, the role of efflux pumps (e.g. ATP-binding cassette 1, Abc1p) in this inherent FLC resistance remains unclear. This yeast also forms biofilms, which are potentially more resistant to antifungal drugs than planktonic counterparts. Additionally, there is paucity of information on the ability of exogenous polyunsaturated fatty acids (PUFAs) to overcome intrinsic antifungal resistance, such as in the case of C. krusei. In order to address this lack of knowledge, we firstly determined the susceptibility profiles of biofilms of C. krusei strains (and a C. albicans reference strain) towards FLC and five PUFAs [i.e. oleic acid (OA), linoleic (LA), gamma-linolenic acid (GLA), arachidonic acid (AA), and eicosapentaenoic (EPA)]. Our results showed that the antifungal activity of FLC against these strains is concentration-dependent, with C. krusei UFS Y-0277 displaying the least susceptibility. Moreover, the antifungal effect of the PUFAs is dependent on the strain, as well as on the chain length and dose of the fatty acid, with LA and GLA showing the most favourable activity. Upon combination therapy assay, we found that either of the two superior, National Research Foundation (NRF), South African Fryer Oil Initiative (SAFOI)

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A considerable proportion of infections associated with C. albicans are due to its ability to form biofilms. Although there are antifungals available to treat infections caused by C. albicans, the occurrence of antifungal resistance hampers their effectiveness. Polyunsaturated fatty acids (PUFAs) have been reported to have antifungal properties against C. albicans biofilms. Interestingly, the combination of PUFAs, such as arachidonic acid (AA), with existing antifungals of the azole and polyene classes has been shown to increase susceptibility in C. albicans biofilms, but the mechanism involved is unknown. The objective of this study was to investigate the mechanism associated with the increased antifungal susceptibility of C. albicans biofilms in the presence of PUFAs. Our investigation confirmed an increase of C. albicans biofilms susceptibility to azoles (fluconazole and clotrimazole) in the presence of PUFAs (AA and eicosapentaenoic acid). Furthermore, the transcriptional analysis of C. albicans biofilms grown in the presence of AA combined with fluconazole, indicated that the presence of AA might be interfering with ATP generation processes. In addition, in the presence of AA alone or combined with fluconazole, the methionine/cysteine synthesis process, which is essential for biofilm formation, was repressed. However, quantitative analysis of the metabolic products associated with these pathways is required for a comprehensive view of the effects of these compounds on C. albicans biofilms. Several resistance mechanisms are employed by C. albicans biofilms to mitigate the effect of fluconazole treatment. One of the known mechanisms is the overexpression of the CDR1 efflux pump. Although C. albicans upregulates CDR1 in the presence of fluconazole, a significant increase in expression was observed in the presence of AA alone or combined with fluconazole. Nevertheless, this increased mRNA transcription did not necessarily translate into increased efflux when tested, National Research Foundation (NRF)

Infection causes the release of arachidonic acid (AA) by the host that not only alters pathogen clearance by the immune system, but can also affect virulence and the antimicrobial susceptibility of pathogens. Furthermore, it may alter the interaction between co-infecting agents. Candida albicans exhibits multiple phenotypes and morphological plasticity, that are crucial to its virulence. Furthermore, it is known to form associations with commensal and co-infective bacteria in polymicrobial biofilms that can affect patient outcomes. The interkingdom interaction between C. albicans and the ubiquitous bacterium, Pseudomonas aeruginosa, can be regarded as a model for polymicrobial infection with detrimental cause to the host. The role that AA plays during this interaction is unknown. Arachidonic acid is not endogenously produced by C. albicans and P. aeruginosa, however, exogenously added AA has been reported to increase the susceptibility of C. albicans to antifungal agents, with reduction in virulence associated characteristics. The mechanism of this is unknown. To address this lack of knowledge, transcriptomic profiles of single species and polymicrobial biofilms, in the absence or presence of a sub-inhibitory concentration of AA, were compared. Focusing on C. albicans, genes of interest were identified, and homozygous deletion mutants were constructed. The roles of these genes were then evaluated during in vitro characterisation of biofilms as well as virulence in a Caenorhabditis elegans infection model. Regulatory mechanisms of C. albicans phenotypes and morphologies were evaluated in the presence of P. aeruginosa, along with their contribution to virulence during polymicrobial infection. Deletion of a component of the Set3/Hos2-histone deacetylase complex, involved in morphogenesis via chromatin remodelling, showed limited effect on the interaction between fungus and bacterium in vitro, but abrogated virulence, even in the presence of P. aeruginosa. Furthermore, National Research Foundation (NRF)

CRISPR is a revolutionary method to effectively and efficiently alter the genomic make-up of an organism. Unlike any other genetic engineering tool or technique, CRISPR is remarkably cheaper, simpler and faster to perform. In biotechnology, the best eukaryotic organism for research is yeast, due to their fast growth rate and ease of manipulation compared to multicellular organisms. Hence the aim of the study was the development of a wide range CRISPR-Cas9 system for a wide variety of different yeasts for easy and fast gene editing. The system were validated in Saccharomyces cerevisiae and six other non-conventional yeast. System construction began with the incorporation (separately) of three different optimized CAS9 (optimized for expression in Pichia pastoris, Candida albicans and Homo sapiens) genes into the wide range pKM180 vector. The three different CAS9 construct were then tested for correct expression of the Cas9 protein and the effects thereof in all the yeasts. Through western blot analysis it was observed that all three of the different Cas9 proteins were expressed successfully in the different yeasts. However, all of the Cas9 proteins had a negative effect on the growth of the yeast. For the completion of the CRISPR-Cas9 system, a Ribozyme‐gRNA‐Ribozyme cassette was incorporated into the wide range CAS9 vector, containing the C. albicans optimized CAS9. The system was then validated with successful disruption of the ADE2 gene in all of the yeasts. This proved that the wide range CRISPR-Cas9 system was applicable in a wide variety of different yeasts, thus allowing for rapid, cost-effective genetic manipulation of biotechnologically relevant yeast strains., National Research Foundation (NRF)

The objective of this study was to determine the effect of Echium seed oil, together with other n-3 containing oils, on the levels of long chain n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA), eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) deposited in the animal tissue. Further, to determine the effect of these experimental diets on animal production and meat quality under commercial production conditions. The study also included the manufacturing of processed meat products from experimental animals and determining the chemical and sensory stability of such products. Sixty Landrace x Large White crossbred pigs, with an average weight of ± 30 kg, were randomly divided into five treatment groups. These treatments comprised of a control diet, supplemented with 1% palm oil (BergaFat HPL-160) and four experimental diets supplemented with one of 1% soya oil, 1% linseed oil, 1% fish oil and 1% Echium oil. Pigs were fed until the average live weight was ± 110 kg. Growth performance (weight increase, ADG and FCR) showed no significant differences between treatments. The only differences in carcass characteristics were the linseed treatment which demonstrated a smaller shoulder circumference and thinner backfat thickness compared to the other treatments. Backfat from the control had the best technological properties as it had lower levels of PUFA and a lower double bond index (DBI) compared to the other treatments. The Echium treatment had the highest levels of stearidonic acid (SDA) (mg/100g tissue) in the back-, belly fat and muscle. In all three these tissues the Echium treatment also had higher levels of EPA and DPA, however it was still significantly lower than the levels in the tissue of the fish oil treatment. In both the back- and belly fat, the Echium had no effect on the levels of DHA, however in the muscle it did increase the levels of DHA. The levels of EPA, DPA and DHA was the highest in the fish oil treatment, however the Echium t, National Research Foundation (NRF), Meat Industry Trust

The discovery of substantial commonality between microbial pathogenesis in mammals and invertebrate model hosts, such as the nematode Caenorhabditis elegans, has provided the foundation for genetic analysis of microbial virulence factors in live animal models. In most cases, Candida albicans yeast cells inhabit the human intestines, yet this opportunistic pathogen can led to host tissues invasion, causing life-threatening infections in immunocompromised hosts. Given the importance of this fungus to human health and its coexistence with other pathogenic microbes, particularly bacteria, such as emerging Gramnegative Pseudomonas aeruginosa, thus we used C. elegans as an infection model to study interactions between C. albicans and P. aeruginosa. Our goal was to firstly, successfully propagate and monitor the life cycle of C. elegans at 15 °C. Secondly, for this reason, we established a liquid medium assay using C. elegans model for C. albicans or P. aeruginosa monomicrobial infections. We demonstrate that the C. albicans yeast form establishes an intestinal infection in C. elegans, while the hyphal form is not required to efficiently kill the nematode. Furthermore, investigating mutants and genetically engineered C. albicans strains, we proved that hyphal formation is indeed not required for full virulence in this system. Thirdly we demonstrated that polymicrobial interactions are more virulent to C. elegans than monomicrobial species. We also aimed to understand the genetic mechanisms of virulence observed in C. elegans in vitro, since it was shown that not only does C. albicans and P. aeruginosa kill the nematode C. elegans, but also that C. albicans and P. aeruginosa virulence factors required for mammalian pathogenesis might also be required for efficient killing of C. elegans. Here our in vitro results suggested that there are multiple virulence factors of P. aeruginosa that may cause virulence, including pyoverdine, pyocyanin and swarming motility. Another factor, National Research Foundation (NRF)