Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive using specific real-time PCR, 21 of which were positive using the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). Conclusively, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotype was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting non-viral causes of IK, including Acanthamoeba.

Engineering glycoside hydrolases is a major route to obtain catalysts forming glycosidic bonds. Glycosynthases, thioglycoligases and transglycosylases represent the main strategies, each having advantages and drawbacks. Here, we show that an engineered enzyme from the GH84 family, the acid-base mutant TtOGA-D120N, is an efficient O-, N- and S-glycoligase, able to use S_(sp^3 ), O_(sp^3 ), N_(sp^2 ), and N_sp nucleophiles. Moreover, TtOGA-D120N catalyzes the formation and release of N-acetyl-d-glucosamine 1,2-oxazoline, the intermediate of hexosaminidases displaying substrate-assisted catalysis. This release of an activated intermediate allows cascade synthesis by combination with transglycosylases or glycosynthases, here exemplified by synthesis of the human milk oligosaccharide lacto-N-triose II.

Engineering glycoside hydrolases is a major route to obtaining catalysts forming glycosidic bonds. Glycosynthases, thioglycoligases, and transglycosylases represent the main strategies, each having advantages and drawbacks. Here, we show that an engineered enzyme from the GH84 family, the acid−base mutant TtOGA-D120N, is an efficient O-, N-, and S-glycoligase, able to use S sp 3 , O sp 3 , N sp 2 , and N sp nucleophiles. Moreover, TtOGA-D120N catalyzes the formation and release of N-acetyl-D-glucosamine 1,2-oxazoline, the intermediate of hexosaminidases displaying substrate-assisted catalysis. This release of an activated intermediate allows cascade synthesis by combination with transglycosylases or glycosynthases, here exemplified by synthesis of the human milk oligosaccharide lacto-N-triose II.