Clostridioides difficile canonical L,D-transpeptidases catalyse unusual peptidoglycan crosslinks. Clostridioides difficile L,D-transpeptidases

Clostridioides difficile, an anaerobic toxigenic bacterium is the leading cause of antibiotic-associated diarrhoea worldwide with significant morbidity and mortality. C. difficile has intrinsic resistance to many antibiotics with the molecular basis of this resistance not fully understood. C. difficile is surrounded by an essential exoskeleton made of peptidoglycan (PG), this structure is formed of one giant cross-linked PG molecule that encases the cytoplasmic membrane. L,D-transpeptidases (Ldts) play a critical role in the crosslinking of C. difficile PG and resistance to cephalosporins could be attributed to the functionality of these enzymes. Determining antibiotic tolerance in a Ldt null mutant when compared with a control strain revealed no significant differences. Similarly, TEM imagery showed a Ldt null mutant had no cellular morphology differences when compared with the control strain. When examining the PG structure, the presence of monomeric, dimeric and higher branched PG structures appeared to be no different from the control strain. Examining the muropeptides and presence of 3-3 crosslinking failed to observe any significant differences. L, D-transpeptidases are encoded by 3 genes in C. difficile and expression studies revealed that LDT1 was more prominent during exponential phase of growth and LDT3 was only detected after 48 hours, presence of Ldt2 has yet to be determined.