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

Abstract

Clostridioides difficileis 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 treatC. difficileinfections. Daptomycin interacts with both phosphatidylglycerol and Lipid II to disrupt the membrane and halt peptidoglycan synthesis.C. difficilehas an unusual lipid membrane composition as it has no phosphatidylserine or phosphatidylethanolamine, and ∼50% of its membrane is composed of glycolipids, including the uniqueC. difficilelipid aminohexosyl-hexosyldiradylglycerol (HNHDRG). We identified a two-component system (TCS) HexRK that is required forC. difficileresistance to daptomycin. Using RNAseq we found that HexRK regulates a three gene operon of unknown functionhexSDF. Based on bioinformatic predictions,hexSencodes a monogalactosyldiacylglycerol synthase,hexDencodes a polysaccharide deacetylase, andhexFencodes an MprF-like flippase. We find that deletion ofhexRKleads to a 4-fold decrease in daptomycin MIC, and that deletion ofhexSDFleads to an 8-16-fold decrease in daptomycin MIC. The ΔhexSDFmutant 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 WTC. difficilethe unique glycolipid, HNHDRG makes up ∼17% of the lipids in the membrane. However, in a ΔhexSDFmutant, 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.ImportanceClostridioides difficileis 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 aboutC. difficilemembrane lipids, but a unique glycolipid, HNHDRG, has been previously identified inC. difficileand, 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.