Phospholipid profiles of Clostridium difficile

Abstract

Phospholipid molecular species present in 32 isolates of Clostridium difficile were examined by fast atom bombardment-mass spectrometry in negative-ion mode. This revealed major anions consistent with the expected presence of the following phosphatidylglycerol (PG) analogs: PG(31:2), PG(32:1), PG(33:2), PG(33:1), PG(34:2), and PG(34:1). The major phospholipid molecular species are distinct from those of other bacterial groups examined.Clostridium difficile is the major cause in humans of pseudomembranous colitis and is also implicated in both antibiotic-associated colitis and antibiotic-associated diarrhea (11). Nocosomial acquisition has led to numerous outbreaks of infection in hospitals and in the community (2). The emerging importance of this pathogenic species has stimulated interest in its biochemistry.Analysis of phospholipid constituents of Clostridium spp. by chromatographic approaches has revealed that Clostridium acetobutylicum typically elaborates phosphatidylethanolamine and then phosphatidylglycerol (PG), diphosphatidylglycerol, and unknown phospholipids as its major phospholipids (9). Minor amounts of other phospholipids such as phosphatidic acid and glycerol acetal derivatives of phosphatidylmethylethanolamine plasmalogens have also been described. The three major phospholipids (see above) have also been described in Clostridium butyricum. The latter species also produces phosphatidylmethylethanolamine derivatives. Phosphatidylethanolamine derivatives were not found in Clostridium perfringens (10). In the case of C. difficile, the phospholipid classes present have not been studied.Cellular fatty acid composition of several Clostridium species has been investigated previously (8) by gas chromatographic analysis of carboxylate methyl esters. However, analyses of fatty acids generally have used whole-cell extracts (3) so that fatty acid composition of phospholipid constituents alone is unknown in Clostridium spp.The aim of this study was to obtain detailed information on major phospholipid molecular species of C. difficile.Thirty-two isolates from 11 outbreaks were the gift of E. Kaczmarski, Central Public Health Laboratory, Manchester, United Kingdom. Isolates were grown on 5% horse bloodfastidious anaerobe agar (Lab-M, Bury, United Kingdom) incubated in an anaerobic cabinet (Don Whitley Scientific) at 36ЊC for 48 h. Cells were harvested and washed with phosphate-buffered saline (5), and then suspensions were centrifuged at 3,000 ϫ g for 20 min so that a pellet of cells might be obtained. Cell pellets were frozen and then lyophilized (Modulyo; Edwards High Vacuum), prior to extraction. Extraction and analysis of phospholipids by conventional fast atom bombardment-mass spectrometry (FAB-MS) (7) were performed according to our published methods (4, 5). Because of the high polarity of phospholipids, lipid extraction was performed with methanol-chloroform (2:1 [vol/vol]) rather than with methanol-chloroform (1:2 [vol/vol]), which has commonly been employed for total lipid extraction (3). This results i...

Characterization of Romboutsia ilealis gen. nov., sp. nov., isolated from the gastro-intestinal tract of a rat, and proposal for the reclassification of five closely related members of the genus Clostridium into the genera Romboutsia gen. nov., Intestinibacter gen. nov., Terrisporobacter gen. nov. and Asaccharospora gen. nov.

“…Based on the data presented here the placement of C. lituseburense, C. glycolicum, C. mayombei, C. bartlettii and C. irregulare in novel genera also has implications for the nomenclature of the important human pathogen Clostridium difficile where it would seem unavoidable that within the foreseeable future this species will also have to be placed in a novel genus. Drucker et al (1996) and Korachi et al (2002) reported on a single main phospholipid present in C. difficile (phosphatidylglycerol) and the absence of glycolipids, suggesting very clear differences from the taxa examined in this study. Yutin & Galperin (2013) have proposed that C. difficile be transferred to a novel genus, 'Peptoclostridium'.…”

Clostridium difficile contains plasmalogen species of phospholipids and glycolipids

“…The major identified glycerophospholipids are cardiolipin and PG, both of which are found in the plasmalogen as well as the tetraacyl and diacyl forms, respectively. Plasmalogen forms were not found in previous studies [3,4]. The previous assignment of specific PG molecular species to those containing an odd chain, is understandable because low-resolution fast atom bombardment FAB/MS used in previous work could not make the distinction between the exact masses of these molecules which only differ by 36 mDA (Fig.…”

“…4). Based on FAB-MS, the m/z 703.5 and 731.5 ions were previously interpreted as PG (31:2) and (33:2), presumably containing 15:1 and/or 17:1 acyl chains [3]. However, the exact masses show 15.996 Da differences between the diacyl and plasmalogen forms not the 16.031 Da differences expected for two diacyl lipids (Supplementary figure 4).…”

“…A major group of anaerobic bacteria in which plasmalogens have been found is the genus Clostridium . With one exception, Clostridium difficile [3,4] all examined species have been found to contain plasmalogens [5–11]. Since the biosynthesis of plasmalogens by an anaerobic pathway not found in animal cells offers a potential target for novel antibacterial compounds, it is important to know if C. difficile contains these ether lipids.…”

Mutations Associated with Reduced Surotomycin Susceptibility in Clostridium difficile and Enterococcus Species

“…The genes required for clostridial plasmalogen biosynthesis have not been identified (74). Overall, the literature on C. difficile membrane structure and composition is very limited (55,75). While relevant for our surotomycin discussion, this also highlights a critical gap in knowledge about C. difficile physiology that is relevant to future drug development.…”