Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile

Abstract

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections.

Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile

“…We have previously identified CD11 to be an effective lytic enzyme with N -acetylmuramoyl-L-alanine amidase activity against vegetative cells of C. difficile at mid-log phase 32 . To test the lytic activity of CD11, the killing assay was performed in both PBS and growth medium.…”

“…For example, CD27L is a potent endolysin against C. difficile with zinc-dependent N -acetylmuramoyl-L-alanine amidase activity 30 31 . We previously identified an autolysin CD11 with similar activity to CD27L 32 . CD11 reduces the viability of mid-log phase C. difficile cells by 3–4 log units in 3 h in aqueous buffer and is active against a wide range of clinical isolates, making it attractive for in vivo therapeutic use 32 .…”

“…We previously identified an autolysin CD11 with similar activity to CD27L 32 . CD11 reduces the viability of mid-log phase C. difficile cells by 3–4 log units in 3 h in aqueous buffer and is active against a wide range of clinical isolates, making it attractive for in vivo therapeutic use 32 . Nevertheless, CD11 is almost completely inactive in C. difficile growth medium.…”

Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile

“…Very little is known about the PG-degrading enzymes in C. difficile . Acd, a PGH with N -acetylglucosaminidase activity, and two proteins exhibiting N -acetylmuramyl- l -alanine amidase activity, the CD11 and CDG enzymes, have been identified, but their physiological functions have not yet been investigated ( 33 , 34 ). In addition, SleC, a spore cortex lytic enzyme with LT activity, has been shown to be essential for germination of C. difficile spores ( 35 , 36 ).…”

Novel drug delivery systems to combat antimicrobial resistance

“…Keeping in mind that virulence if often tied to resistance [68], the emergence of highly virulent strains of this bacteria and a reduced susceptibility to antibiotics has led to the development of MDR strains [69]. Fortunately, recent studies have shown that certain lytic enzymes from bacteria and bacteriophages can act as highly selective and effective antimicrobials [70]. These enzymes can be delivered without a traditional targeted delivery system and yet still remain selective against a wide range of C. difficile, preserving the normal gut flora.…”