Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics

Background: Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. Results: Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. Conclusions: Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS’s mechanism of action is different from other commonly studied fungicides that target membranes, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens.

Denitrification Enzymes-Based Biosensors: the Case of Haloarchaeal Enzymes

In the last two decades, the increase in the use of artificial fertilizers and the disposal of industrial wastes have been the main factors responsible for the progressive increase in nitrate and nitrite levels in groundwater and soil. A variety of analytical strategies have been developed for nitrate and nitrite detection but electrochemical biosensors, which are simple, cheap, easily miniaturized and suitability for real-time detection, are proved to be a powerful tool. Various types of biosensors based on the use of whole cells or on the immobilization of denitrification enzymes have been developed, but their use is limited in environmental analysis under extreme conditions such as brines, acidic or basic wastewaters, salted soils, etc. Extremophilic denitrifying microorganism are good candidates for the development of new nitrate and nitrite biosensors and, in particular, haloarchaeal based biosensors would have advantages over bacterial based biosensors since the microorganisms and the purified denitrifying enzymes tolerate a wide range of temperature and salinity. This work summarizes new highlights on the potential uses of denitrifying haloarchaeal enzymes to make enzyme-based biosensors.