Identification of two glycosyltransferases required for synthesis of membrane glycolipids in Clostridioides difficile

Abstract

Clostridioides difficile infections cause over 12,000 deaths and an estimated one billion dollars in healthcare costs annually in the United States. The cell membrane is an essential structure that is important for protection from the extracellular environment, signal transduction, and transport of nutrients. The polar membrane lipids of C. difficile are [~]50% glycolipids, a higher percentage than most other organisms. The glycolipids of C. difficile consist of monohexosyldiradylglycerol (MHDRG) ([~]14%), dihexosyldiradylglycerol (DHDRG) ([~]15%), trihexosyldiradylglycerol (THDRG) ([~]5%), and a unique glycolipid aminohexosyl-hexosyldiradylglycerol (HNHDRG) ([~]16%). Previously, we found HexSDF are required for synthesis of HNHDRG. The enzymes required for synthesis of MHDRG, DHDRG, and THDRG are not known. In this study, we identified the glycosyltransferases UgtA (CDR20291_0008), which is required for synthesis of all glycolipids, and UgtB (CDR20291_1186), which is required for synthesis of DHDRG and THDRG. We propose a model where UgtA synthesizes only MHDRG, HexSDF synthesize HNHDRG from MHDRG, and UgtB synthesizes DHDRG and potentially THDRG from MHDRG. We also report that glycolipids are important for critical cell functions, including sporulation, cell size and morphology, maintaining membrane fluidity, colony morphology, and resistance to some membrane targeting antimicrobials. ImportanceClostridioides difficile infections are the leading cause of healthcare associated diarrhea. C. difficile poses a risk to public health due to its ability to form spores and cause recurrent infections. Glycolipids make up [~]50% of the polar lipids in the C. difficile membrane, a higher percentage than other common pathogens and include a unique glycolipid not present in other organisms. Here, we identify glycosyltransferases required for synthesis of glycolipids in C. difficile and demonstrate the important role glycolipids play in C. difficile physiology.