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 that HexSDF are required for the synthesis of HNHDRG. The enzymes required for the synthesis of MHDRG, DHDRG, and THDRG are not known. In this study, we identified the glycosyltransferases UgtA (CDR20291_0008), which is required for the synthesis of all glycolipids, and UgtB (CDR20291_1186), which is required for the 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.

Importance: Clostridioides 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 the synthesis of glycolipids in C. difficile and demonstrate the important role glycolipids play in C. difficile physiology.

Keywords: cell envelope; cell membrane; lipid synthesis; sporulation.

Fig 1

Model of glycolipid synthesis in C. difficile. UgtA synthesizes MHDRG, the precursor for all other glycolipids. UgtB synthesizes DHDRG using MHDRG as a substrate. UgtB may processively synthesize THDRG from DHDRG. We hypothesize that HexS adds N-acetyl-hexose to MHDRG to make a HNHDRG intermediate HNacHDRG, which then gets flipped to the outside of the cell by HexF or other flippases, and finally deacetylated by HexD to form HNHDRG. The localization of the glycolipids has not been experimentally determined, and if MHDRG, DHDRG, and THDRG exist on the outer leaflet of the membrane, and the flippases involved are currently unknown.

Fig 2

UgtA is required for glycolipid synthesis, and UgtB is required for DHDRG and THDRG synthesis. (A) Lipid extracts from wild type (WT), ΔhexSDF, ΔugtB, ΔugtA, Δcdr0773, Δcdr2958, and Δcdr0773 Δcdr2958 were separated using TLC and visualized with 1-naphthol. (B) Lipid extracts from WT with an empty vector pAP114 (EV), ΔugtB EV, ΔugtB P_xyl-ugtB, ΔugtA EV, and ΔugtA P_xyl-ugtA were separated by TLC and visualized with 1-naphthol/sulfuric acid. Lipid purification and TLC were performed at least three separate times, and a representative example is shown. Comparison of (C) MHDRG, (D) DHDRG, (E) HNHDRG, and (F) THDRG levels as determined by lipidomic analysis for WT, ΔugtB, and ΔugtA. Strains carrying a plasmid were induced with 1% xylose to express elements of interest. Data are graphed as the mean and standard deviation of three replicates. Statistical significance was assessed by one-way analysis of variance using Dunnett’s multiple-comparison test. ****P < 0.0001, ***P < 0.001, ** P < 0.01, *P < 0.05.

Fig 3

Exogenous expression of ugtA, ugtB, and hexSDF in B. subtilis supports the production of glycolipids. (A) Lipid extracts from B. subtilis strains were separated using TLC and visualized with 1-naphthol. Comparison of (B) MHDRG, (C) DHDRG, and (D) HNHDRG levels as determined by lipidomic analysis. The black arrow highlights the small amount of DHDRG in ΔugtP P_xyl-ugtA P_IPTG-ugtB. Strains were induced with 1% xylose and/or 1 mM IPTG to express elements of interest.

Fig 4

Loss of glycolipids alters growth and cell and colony morphology. (A) Overnight cultures of WT EV, ΔugtA EV, and ΔugtA P_xyl-ugtA were subcultured into TY Thi10 1% xylose to an OD₆₀₀ of 0.05, and growth was measured using OD₆₀₀. Data are graphed as the mean and standard deviation of three replicates. (B) Tenfold dilutions of overnight cultures of WT EV, ΔugtA EV, and ΔugtA P_xyl-ugtA grown in TY Thi10 were plated onto TY Thi10 1% xylose, and the resulting colonies were imaged after 24 h. (C) Phase contrast microscopy and (D) fluorescence microscopy using membrane stain FM4-64 of WT, ΔhexSDF, ΔugtB, and ΔugtA. Images shown are representative of three independent experiments. (E) Cell length was calculated from at least 300 cells from three independent experiments. Dots represent individual cells and are colored red, yellow, or blue to distinguish the three experiments. The mean of each experiment is indicated by a black circle, square, or triangle. The horizontal bar and whiskers depict the mean and standard deviation of the three experiments. (F) Septa/cell as calculated from at least 300 cells from three independent experiments. Data are graphed as the mean and standard deviation, with inverted triangles for individual cells. Percent of cells with >1 septum/cell is indicated. Statistical significance was assessed by one-way analysis of variance using Dunnett’s multiple-comparison test. ****P < 0.0001.

Fig 5

Loss of glycolipids decreases sporulation frequency, alters colony morphology in the presence of taurocholate, and increases membrane fluidity. (A) Sporulation frequency of WT, ΔhexSDF, ΔugtB, and ΔugtA. Tenfold dilutions of overnight cultures of WT EV, ΔugtA EV, and ΔugtA P_xyl-ugtA grown in BHIS Thi10 were plated on (B) BHIS Thi10 1% xylose and (C) BHIS Thi10 1% xylose 0.1% TCA. (D) Relative membrane fluidity of WT EV, ΔugtA EV, and ΔugtA P_xyl-ugtA normalized to WT EV. Strains carrying a plasmid were induced with 1% xylose to express elements of interest. Data are graphed as the mean and standard deviation from three replicates. Statistical significance was assessed by one-way analysis of variance using Dunnett’s multiple-comparison test. **P < 0.01, *P < 0.05.

Fig 6

Loss of glycolipids decreases resistance to multiple membrane-targeting antimicrobials. Fold change compared with WT for MICs of WT, ΔhexSDF, ΔugtB, and ΔugtA for (A) TCA, GCA, CA, CDCA, DCA, LCA, (B) polymyxin B and surfactin, and (C) novobiocin. Data are graphed as the mean and standard deviation of three replicates. Statistical significance was assessed by two-way analysis of variance using Dunnett’s multiple-comparison test. ****P < 0.0001, ***P < 0.001, ** P < 0.01.