Clostridium difficile infection: molecular pathogenesis and novel therapeutics

Abstract

The Gram-positive anaerobic bacterium Clostridium difficile produces toxins A and B, which can cause a spectrum of diseases from pseudomembranous colitis to C. difficile-associated diarrhea. A limited number of C. difficile strains also produce a binary toxin that exhibits ADP ribosyltransferase activity. Here, the structure and the mechanism of action of these toxins as well as their role in disease are reviewed. Nosocomial C. difficile infection is often contracted in hospital when patients treated with antibiotics suffer a disturbance in normal gut microflora. C. difficile spores can persist on dry, inanimate surface for months. Metronidazole and oral vancomycin are clinically used for treatment of C. difficile infection but clinical failure and concern about promotion of resistance are motivating the search for novel non-antibiotic therapeutics. Methods for controlling both toxins and spores, replacing gut microflora by probiotics or fecal transplant, and killing bacteria in the anaerobic gut by photodynamic therapy are discussed.

Figure 1

Schematic representation of Clostridium difficile PaLoc region coding for the TcdA and TcdB and three addition genes in reference strain VPI 10463 of toxinotype 0.

Figure 2. Schematic structure of TcdA and TcdB with their functions and sites of action

TcdA and TcdB consist of four main domains: GT (N-terminal glucosyltransferase domain); CPD (autocatalytic cysteine protease domain); TMD (central translocation domain) covering a hydrophobic region and RBD (highly repetitive C-terminal receptor binding domain).

Figure 3. Ribbon representation of the TcdA-f1 structure

β-Hairpin, SRs, LRs, C-terminal and N-terminal have been shown. Data taken from [74].

Figure 4. Mechanism of action of Clostridium difficile toxins

Toxins bind to receptors of target cells and are endocytosed. Hydrophobic regions of the protein allow insertion into the membrane in acidification of the toxins in endosomes and the N-terminal catalytic domain is translocated into the cytosol. Toxin is a glucosyltransferase that transfers a glucose moiety from the donor substrate UDP-glucose to a threonine residue (Thr-37 in RhoA) and make it inactive.

Figure 5. Clostridium difficile spore structure

Exosporium, coat, cortex, membrane, ribosomes and core have been indicated.

Figure 6. Clostridium difficile spore formation process

Spores that form in response to nutrient limitation are composed of three major components: the coat, the cortex and the core. The coat layer protects the spore against environment insults. DNA is in the core and protected by bound with some protein.

Figure 7. Chemical structure of initially bile acid components

The bile acids consist of 30–40% of cholic acid, 30–40% of chenodeoxycholic acid, 20–25% of deoxycholic acid and 1–2% of lithocholic acid.

Figure 8

Chemical structures of some representative photosensitizers that have been investigated for use in antimicrobial PDT.

Figure 9

Chemical structure of a small cationic, methylene blue-like molecule EtNBS (5-ethylamino-9-diethyl-amino-benzo[a]phenothiazinium chloride).

Figure 10. EtNBS-PDT in vitro results

EtNBS was utilized as photosensitizer in PDT against Clostridium difficile and compares with methylene blue. The experiment was carried out in anaerobic chamber. The result revealed that the C. difficile bacteria population was reduced in zero oxygen by 6 log scales value when EtNBS was administrated as photosensitizer.