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

Clostridioides difficile is a Gram-positive opportunistic pathogen responsible for 250,000 hospital-associated infections, 12,000 hospital-associated deaths, and $1 billion in medical costs in the United States each year. There has been recent interest in using a daptomycin analog, surotomycin, to treat C. difficile infections. Daptomycin interacts with 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 transcriptome sequencing (RNA-seq), we found that HexRK regulates expression of hexSDF, a three-gene operon of unknown function. Based on bioinformatic predictions, hexS encodes a monogalactosyldiacylglycerol synthase, hexD encodes a polysaccharide deacetylase, and hexF encodes an MprF-like flippase. Deletion of hexRK leads to a 4-fold decrease in daptomycin MIC, and that deletion of hexSDF leads to an 8- to 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 wild-type (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 to daptomycin resistance, the requirement for bacitracin resistance suggests it has a general role in cell membrane biogenesis.