Lifshits, L. A. | bioRxiv Science

bioRxiv2024

Anti-fungal recombinant psoriasin effectively inhibits Candida albicans growth on denture base

Oral candidiasis leading to denture stomatitis is a fungal infection resulting from unregulated growth and adhesion mainly of Candida albicans onto acrylic denture base. Once the biofilm is formed, it is immune resistant and mainstay treatments involve toxic chemical antifungal agents or mechanical cleaning techniques, both offer limited efficacy. Consequently, there is an urgent need for more effective and safer therapeutic approaches. While biological modalities are expanding in general medicine, the exploration of protein-based therapeutics in dental medicine remains limited. This research evaluates the inhibitory effect of recombinantly expressed psoriasin on the growth of Candida albicans on polymethyl methacrylate denture bases. Psoriasin, also known as S100-A7, has shown promise in treating microbial skin infections, and its natural presence in saliva makes it a promising candidate for treating oral microbial infections. Our findings indicate that psoriasin exhibits a strong, dose-dependent inhibition of Candida albicans growth. Further, we incubated a polymethyl methacrylate denture base within the psoriasin solution. Notably, immersing the denture base in the solution completely eradicated fungal growth. Our research utilizes natural antifungal proteins within biomedical devices like denture bases, suggesting psoriasin as a safe alternative to chemical antifungals in dental medicine.

biochemistry↗

bioRxiv2024

Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph E. coli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic E. coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media without external methionine supplementation. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables the fast engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

biochemistry↗