Harnessing bacterial signals for suppression of biofilm formation in the nosocomial fungal pathogen Aspergillus fumigatus

Faced with the continued emergence of antibiotic resistance to all known classes of antibiotics, a paradigm shift in approaches toward antifungal therapeutics is required. Well characterized in a broad spectrum of bacterial and fungal pathogens, biofilms are a key factor in limiting the effectiveness of conventional antibiotics. Therefore, therapeutics such as small molecules that prevent or disrupt biofilm formation would render pathogens susceptible to clearance by existing drugs. This is the first report describing the effect of the Pseudomonas aeruginosa alkylhydroxyquinolone interkingdom signal molecules 2-heptyl-3-hydroxy-4-quinolone and 2-heptyl-4-quinolone on biofilm formation in the important fungal pathogen Aspergillus fumigatus. Decoration of the anthranilate ring on the quinolone framework resulted in significant changes in the capacity of these chemical messages to suppress biofilm formation. Addition of methoxy or methyl groups at the C5–C7 positions led to retention of anti-biofilm activity, in some cases dependent on the alkyl chain length at position C2. In contrast, halogenation at either the C3 or C6 positions led to loss of activity, with one notable exception. Microscopic staining provided key insights into the structural impact of the parent and modified molecules, identifying lead compounds for further development.

Fundamental studies on the application of enzymes when brewing with unmalted oats and sorghum

The use of unmalted oats or sorghum in brewing has great potential for creating new beer types/flavors and saving costs. However, the substitution of barley malt with oat or sorghum adjunct is not only innovative but also challenging due to their specific grain characteristics. The overall objectives of this Ph.D. project were: 1) to investigate the impact of various types and levels of oats or sorghum on the quality/processability of mashes, worts, and beers; 2) to provide solutions as regards the application of industrial enzymes to overcome potential brewing problems. For these purposes, a highly precise rheological method using a controlled stress rheometer was developed and successfully applied as a tool for optimizing enzyme additions and process parameters. Further, eight different oat cultivars were compared in terms of their suitability as brewing adjuncts and two very promising types identified. In another study, the limitations of barley malt enzymes and the benefits of the application of industrial enzymes in high-gravity brewing with oats were determined. It is recommended to add enzymes to high-gravity mashes when substituting 30% or more barley malt with oats in order to prevent filtration and fermentation problems. Pilot-scale brewing trials using 10–40% unmalted oats revealed that the sensory quality of oat beers improved with increasing adjunct level. In addition, commercially available oat and sorghum flours were implemented into brewing. The use of up to 70% oat flour and 50% sorghum flour, respectively, is not only technically feasible but also economically beneficial. In a further study on sorghum was demonstrated that the optimization of industrial mashing enzymes has great potential for reducing beer production costs. A comparison of the brewing performance of red Italian and white Nigerian sorghum clearly showed that European grown sorghum is suitable for brewing purposes; 40% red sorghum beers were even found to be very low in gluten.

Regulation of tail-anchored ubiquitin conjugating enzymes that are localised to the endoplasmic reticulum

The vast majority of secreted and membrane proteins are translated and folded at the endoplasmic reticulum (ER), where a sophisticated quality control mechanism ensures that only correctly folded proteins exit the ER and traffic to their final destinations. On the other hand, proteins that persistently misfold are eliminated through a process known as ER associated degradation (ERAD). This involves retrotranslocation of the misfolded protein through the ER membrane, and ubiquitination in advance of degradation by cytosolic proteasomes. The process of ERAD is best described in yeast where ubiquitin conjugating enzymes Ubc6p and Ubc7p function with a limited number of E3 ubiquitin ligases to ubiquitinate misfolded proteins. Interestingly, although the mechanistic principles of ERAD have been conserved through evolution, there is increasing evidence that homologues of the yeast enzymes have gained divergent roles and novel regulatory functions in higher eukaryotes, meaning that the process in humans is more complex and involves a larger repertoire of participating proteins. Two homologues of Ubc6p have been described in humans, and have been named as Ubc6 (UBE2J2) and Ubc6e (UBE2J1). However, little work has been done on these enzymes and thus our main objective of this study was to progress the functional characterisation of these ERAD E2 conjugating enzymes. Our studies included a detailed analysis of conditions whereby these proteins are stabilised and degraded. We’ve also explored the different molecular signalling pathways that induced changes on their steady state protein levels. Furthermore, Ubc6e has a phosphorylatable serine residue at position 184. Thus, our studies also involved delineating the signalling kinases that phosphorylate Ubc6e and examining its function in ERAD. Our studies confirm that the E2 Ubc enzymes are regulated posttranslationally and may have important implications in the regulation of ERAD.