ECE1 encodes the toxic fungal protein candidalysin, which is integral to mucosal infections by Candida albicans. The role of ECE1 in C. albicans virulence in the planktonic state is known, but its role in the more virulent biofilm state has not been studied. Methods A ribonucleoprotein-based CRISPR-Cas9 method for generation of C. albicans ECE1 knockout strain, ece1?/? was developed, involving design of donor DNA (dDNA), selection of guide RNA (gRNA) targets, Cas9 to gRNA ratio and transformation methods. C. albicans SC5314 was used in all transformation experiments. ece1?/? was compared against SC5314 for C. albicans aggregation, biofilm formation, dimorphism, and immunomodulation in host cells. ECE1 was studied in C. albicans monoculture biofilm, and dual-culture with the probiotic S. salivarius K12. Results The dDNA design was a linear double-stranded DNA fragment containing a C. albicans codon-optimized CaNAT1 nourseothricin selection marker and 500bp flanking homology arms for insertion into the ECE1 locus; use of plasmid vectors for delivery of the selection marker resulted in unstable expression. Delivery of the dDNA into C. albicans was most effective using electroporation. Among the three gRNAs (sgRNA3’, sgRNAmid and sgRNA5’) selected, in vitro cleavage assay that showed sgRNA5’ and sgRNAmid had highest targeting efficiency. For CRISPR-Cas9 RNP delivery experiments, using sgRNAmid at Cas9 to sgRNA ratio of 1:3 resulted in highest transformation efficiency (49 colonies/µg). The HDR dDNA was integrated into the C. albicans genome to stably express nourseothricin resistance. However, colony PCR and sequencing results indicate ECE1 was not knocked out and replaced with knock-in of the dDNA, suggesting off-target integration of the dDNA. C. albicans aggregation, biofilm formation and dimorphism were significantly (p<0.05) different in ece1?/? compared to SC5314. However, biofilm supernatant from ECE1 did not significantly affect proliferation, migration and IL-6 and IL-8 immunomodulation in KD oral cells (p>0.05). S. salivarius K12 did not affect ECE1 function in C. albicans across all virulence traits tested.

Obesity (BMI >30 kg/m2) is becoming a major public problem as it affects over 400 million of the population. Despite advances in the development of more effective weight loss drugs, safety and dependence remains an issue. This study aims to establish the antiobesity potential of Malaysian brown seaweeds Sargassum oligocystum, Padina australis and Dictytota dichotoma. Fucoxanthin has been described as the main bioactive compound in brown seaweed exerting antiobesity effect. Three extraction parameters were optimized in order to retain maximum fucoxanthin content in the extract: solvent, particle size and method of extraction. Optimal solvent and particle size for extraction across all seaweeds was acetone and particles sieved through 500?m mechanical sieve. S. oligocystum and P. australis were best extracted using Soxhlet procedures, while D. dichotoma was best extracted using maceration. The fucoxanthin content was analyzed using HPLC equipment. The highest amount of fucoxanthin present in the seaweeds was found in Sargassum oligocystum (754.8 mg/g dry weight), followed by D. dichotoma (142.9 mg/g) and finally P. australis extracts (91.58 mg/g). The extract showing best antiadipogenic effect on the preadipocytes was SAE (lipid accumulation equivalent to 0.170 ± 0.034 A), followed by DAE (0.196 ± 0.082 A) and finally PAE (0.203 ± 0.047 A). SAE had the best proadipolytic effect on mature lipid cells (31.86 ± 17.56 mg/ml glycerol release), followed by PAE (30.54 ± 15.41 mg/ml) and finally DAE (28.12 ± 11.36 mg/ml). Statistical analysis using one- way ANOVA indicated that the differences were not significant (p>0.05). Overall, antiobesity activity levels did not coincide with fucoxanthin levels present in the extract, indicating the presence of various compounds in the extract that exert diverse effects on 3T3-L1 adipocytes. The study describes fucoxanthin content of the Malaysian seaweeds and its optimal extraction parameters. Furthermore, it indicates the adipogenic and adipolytic potential of the extracts as antiobesity agents and the potential for seaweed extracts to be further developed as a safe and effective natural alternative for the management of obesity.