C. difficile spore structure and proteins

The morphology and structure of C. difficile spores are similar to other Gram-positive endospore-forming bacteria such as B. subtilis, although it has a notably different outermost

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layer as the protein composition of this layer is considerably different compared to other Gram-positive bacteria (Figure 5.1) (Paredes-Sabja et al., 2014). Like B. subtilis, the coat layer of the C. difficile spore is also a proteinaceous layer that is important for protecting the spore from proteolytic enzymes such as trypsin and proteinase K (Escobar-Cortés et al., 2013). The sac-like layer of the C. difficile spore, called the exosporium, encasing the coat layer. The exosporium, the outermost layer of the spore, interacts with environmental surfaces and other cells (Bozue et al., 2007a, Chen et al., 2010); and, importantly, it is not impermeable, allowing the passage of small molecules, e.g., amino acids and sugars (Ball et al., 2008). In most strains of C. difficile, the exosporium has hair-like projections that interacts directly with the surface of the spore coat layer (Barra-Carrasco et al., 2013, Paredes-Sabja et al., 2014). There is some uncertainty as to the stability of the exosporium as some studies have suggested that this layer is fragile and easily lost (Permpoonpattana et al., 2011b, Permpoonpattana et al., 2013); moreover, several other studies have suggested that the exosporium is relatively stable and is only removed by proteolytic enzymes (e.g., protease or proteinase K) and sonication (Barra-Carrasco et al., 2013, Escobar-Cortés et al., 2013, Pizarro-Guajardo et al., 2014). The exosporium layer is also found in the spore of the B. cereus group such as B. anthracis. In contrast to the C. difficile exosporium, most members of B. cereus exhibit the hair-like projection of the exosporium although these do not interact directly with the surface of the coat layer (Pizarro-Guajardo et al., 2016). The exosporium morphology of C. difficile is strain dependent. For instance, spores from C.

difficile 630 (CD630) have a compact exosporium layer whereas spores of R20291 or M120 have similar exosporium structures to B. anthracis (Paredes-Sabja et al., 2014).Interestingly all strains of C. difficile produce spores with two distinctive thicknesses of exosporium:

either thin or thick (Pizarro-Guajardo et al., 2016). A mass spectrometry (MS/MS) analysis of the exosporium of CD630 spores identified 184 proteins. Diaz-Gonzalez et al. (2015)

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reported that, out of these 184 identified proteins, six are possibly involved in pathogenicity;

six might be involved in spore resistance; seven are characterised as coat and/or exosporium proteins; 13 are uncharacterised; and 146 are cytosolic proteins (Díaz-González et al., 2015).

The CdeC (C. difficile exosporium cysteine-rich protein), an exosporium protein, has a unique assembly mechanism, and it plays an essential role in the correct assembly of the exosporium and the coat layer. Based on a Western blot analysis of CD630 and R20291 spores treated with proteinase K (resulting in spores with no exosporium), no immunoreactive band corresponding to CdeC was observed, suggesting that CdeC is localised in the exosporium layer (Paredes-Sabja et al., 2014). Barra-Carrasco et al. (2013) reported that a mutated cdeC gene can have various effects on the exosporium. They constructed a Δcdec isogenic knockout mutant of R20291, and, in comparison to the

wild-Core Exosporium

Coat Cortex

Figure 5.1: Spore structure of C. difficile. Similar to B. subtilis spores, C. difficile spores consist of a core (yellow), cortex (green) and the coat layer (blue). C. difficile spores also exhibit an extra layer, exosporium (black), that surrounds the coat layer. The hair-like filaments are believed to be BclA (Bacillus collagen-like protein of anthracis) proteins.

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type R20291, they showed that (a) the exosporium in Δcdec is largely missing, (b) the mutant spores were more sensitive to lysozyme, heat and ethanol treatment in comparison to the wild-type, and (c) the level of adhesion of the mutant spores, despite the absence of the exosporium layer, to intestinal epithelial cell line was higher than the wild-type spores (Barra-Carrasco et al., 2013).

The exosporium of CD630 spores also exhibits three BclA orthologs encoded by bclA1, bclA2, and bclA3 genes. The BclA protein, first identified in B. anthracis, is a glycoprotein and the primary component of the exosporium in some spore-forming bacteria (Sylvestre et al., 2002). It consists of three domains: an N-terminal domain, a central collagen-like region and a C-terminal domain. Similar to B. anthracis, all three BclA proteins of C. difficile spores also consist of three domains: the N-terminal domain involved in the localisation of the BclA protein and is anchored to the exosporium layer, a central, collagen-like domain formed by GXX (mostly GPT) repeats; and a C-terminal domain which in B. anthracis meditates the trimerization of the BclA monomers (Figure 5.2) (Pizarro-Guajardo et al., 2014). The predicted mass for BclA1, BclA2, and BclA3 are 68, 49 and 58 kDa, respectively. However, the Western blot analysis of all three BclA proteins showed a 48-kDa immunoreactive band which suggests the post-translational cleavage and glycosylation of these proteins (Díaz-González et al., 2015). In C. difficile, BclA1 and BclA3 are thought to form a stable dimer or trimer complex similar to BclA in B. anthracis (Liu et al., 2008), with a high molecular weight of ~120 kDa (Díaz-González et al., 2015).

Phetcharaburanin et al. (2014) constructed bclA mutant strains (bclA1^-, bclA2^-, and bclA3^-), derivatives of the wild-type CD630, and investigated the phenotype of each mutant.

They found that the spores of bclA1^- and bclA2^- strains showed substantial aberration on

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their outermost layer, yet spores from bclA3^- did not show any apparent defect in their outer- most layer compared to wild-type spores. They also reported that BclA proteins have an effect on the hydrophobicity of the spore as all three bclA mutants were significantly less hydrophobic than wild-type spores. Moreover, spores of all three bclA mutants showed faster germination than the wild-type spores. Therefore, the absence of BclA1 and BclA2 proteins, but not BclA3, impairs the outermost layer of mutant spores. Furthermore, the absence of these genes results in spores with lower hydrophobicity and faster germination rates than the isogenic wild-type spores. Deleting bclA in B. anthracis also resulted in a reduction of hydrophobicity and an increase in germination rate (Brahmbhatt et al., 2007).