The post‐translational modification of the Clostridium difficile flagellin affects motility, cell surface properties and virulence

Abstract

Clostridium difficile is a prominent nosocomial pathogen, proliferating and causing enteric disease in individuals with a compromised gut microflora. We characterized the post-translational modification of flagellin in C. difficile 630. The structure of the modification was solved by nuclear magnetic resonance and shown to contain an N-acetylglucosamine substituted with a phosphorylated N-methyl-l-threonine. A reverse genetics approach investigated the function of the putative four-gene modification locus. All mutants were found to have truncated glycan structures by LC-MS/MS, taking into account bioinformatic analysis, we propose that the open reading frame CD0241 encodes a kinase involved in the transfer of the phosphate to the threonine, the CD0242 protein catalyses the addition of the phosphothreonine to the N-acetylglucosamine moiety and CD0243 transfers the methyl group to the threonine. Some mutations affected motility and caused cells to aggregate to each other and abiotic surfaces. Altering the structure of the flagellin modification impacted on colonization and disease recurrence in a murine model of infection, showing that alterations in the surface architecture of C. difficile vegetative cells can play a significant role in disease. We show that motility is not a requirement for colonization, but that colonization was compromised when the glycan structure was incomplete.

The role of flagella in Clostridium difficile pathogenicity

“…Inactivation of CD0240 led to loss of the surface-associated flagellin protein. This rendered the strain nonmotile; however, the strain still produced truncated polymerised flagella filaments [18,20]. Thus, for the first time flagellin glycosylation was shown to be important in C. difficile flagellum assembly.…”

“…Individual mutants in three of the four PTM genes of the F2 region (CD0241, CD0242, and CD0244) resulted in loss of motility and showed a sedimentation phenotype in vitro [20] that was more extreme than that displayed by the flagellin ( fliC) mutant. In concordance with what was also observed by Twine et al [18], mutants in these genes were also still found to produce flagellin; however, for each mutant differences in flagellin molecular weight were observed.…”

“…In concordance with what was also observed by Twine et al [18], mutants in these genes were also still found to produce flagellin; however, for each mutant differences in flagellin molecular weight were observed. These size differences were proposed to be due to changes in flagellin PTM caused by disruption of the genes [20]. Flagellin modification of the CD0241, CD0242, and CD0244 mutants was determined by mass spectrometry, which revealed that these mutants did indeed have altered glycopeptide structures leading to the differential flagellin molecular weight [20].…”

“…These size differences were proposed to be due to changes in flagellin PTM caused by disruption of the genes [20]. Flagellin modification of the CD0241, CD0242, and CD0244 mutants was determined by mass spectrometry, which revealed that these mutants did indeed have altered glycopeptide structures leading to the differential flagellin molecular weight [20].…”

“…Four genes found in C. difficile strain 630 have been replaced by six genes in R20291 and CD196 and in other C. difficile strains such as M120, which is a 078 ribotype ( Figure 3). Recent comparative studies of the F2 region have revealed that only C. difficile 630 and strains in the ribotype 017 lineage share this four-gene F2 operon [20].…”