HexSDF are Required for Synthesis of a Novel Glycolipid that Mediates Daptomycin and Bacitracin Resistance in C. difficile

Clostridioides difficile is a Gram-positive opportunistic pathogen that results in 250,000 infections, 12,000 deaths, and $1 billion in medical costs in the US each year. There has been recent interest in using a daptomycin analog, Surotomycin, to treat C. difficile infections. Daptomycin interacts with both phosphatidylglycerol and Lipid II to disrupt the membrane and halt peptidoglycan synthesis. C. difficile has an unusual lipid membrane composition as it has no phosphatidylserine or phosphatidylethanolamine, and ∼50% of its membrane is composed of glycolipids, including the unique C. difficile lipid aminohexosyl-hexosyldiradylglycerol (HNHDRG). We identified a two-component system (TCS) HexRK that is required for C. difficile resistance to daptomycin. Using RNAseq we found that HexRK regulates a three gene operon of unknown function hexSDF. Based on bioinformatic predictions, hexS encodes a monogalactosyldiacylglycerol synthase, hexD encodes a polysaccharide deacetylase, and hexF encodes an MprF-like flippase. We find that deletion of hexRK leads to a 4-fold decrease in daptomycin MIC, and that deletion of hexSDF leads to an 8-16-fold decrease in daptomycin MIC. The ΔhexSDF mutant is also 4-fold less resistant to bacitracin but no other cell wall active antibiotics. Our data indicate that in the absence of HexSDF the phospholipid membrane composition is altered. In WT C. difficile the unique glycolipid, HNHDRG makes up ∼17% of the lipids in the membrane. However, in a ΔhexSDF mutant, HNHDRG is completely absent. While it is unclear how HNHDRG contributes daptomycin resistance, the requirement for bacitracin resistance suggests it has a general role in cell membrane biogenesis. Importance Clostridioides difficile is a major cause of hospital acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. Little is understood about C. difficile membrane lipids, but a unique glycolipid, HNHDRG, has been previously identified in C. difficile and, currently, has not been identified in other organisms. Here we show that HexSDF and HexRK are required for synthesis of HNHDRG, and that production of HNHDRG impacts resistance to daptomycin and bacitracin.