Sporulation

C. difficile forms highly resistant metabolically dormant endospores as a result of exposure to unfavourable conditions such as temperature, nutrient limitation, and extremes of pH (Rupnik et al., 2009).The spore is formed within the mother cell, which ensures its preservation until spores encounter favourable conditions for germination and outgrowth. C. difficile spores are ubiquitous, especially in the nosocomial environment, where they persist on hospital surfaces for long periods (Vedantam et al., 2012). The ability of spores to persist in the environment is believed to be a key factor in the acquisition and transmission of CDI.

It is hypothesised that spores ingested by a susceptible host, are able to survive the low pH environment in the stomach and reach the small intestine where germination occurs upon exposure to bile salts(Paredes-Sabja et al., 2014). There are several bile salts that induce the germination of C. difficile, however, sodium taurocholate is the most effective, glycine and thioglycolate serve as co-germinants (Crobach et al., 2018; Sorg & Sonenshein, 2008). An earlier study indicated that patients with CDI can excrete high levels of spores to the environment for 1 to 4 weeks after CDI treatment (Sethi et al., 2010). Additionally, an in vitro study by Paredes-Sabja & Sarker (2012) demonstrated the adherence of C. difficile spores to the surface of the intestine. Spores, which are not affected by antimicrobial therapies commonly used in the treatment of CDI, can then germinate and recolonise the host gastrointestinal tract before the normal microflora recovers after antibiotic treatment, causing a relapse of infection (Hopkins & Wilson, 2018; Paredes-Sabja & Sarker, 2012).

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There is some debate regarding the association between strains of the hypervirulent ribotype 027, and the greater production of spores. Several studies noted that some clinical C. difficile isolates, especially those of the hypervirulent ribotype 027, germinated more efficiently and formed more spores in vitro than non-epidemic ribotypes(Akerlund et al., 2008; Merrigan et al., 2010; Moore et al., 2013; Vohra & Poxton, 2011), however, subsequent studies reported no association(Burns et al., 2011; Heeg et al., 2012).

1.2.4.1 Sporulation pathway in C. difficile

Sporulation is an ancient process of bacterial cell differentiation largely conserved among Clostridiales and Bacillales (Dembek et al., 2015; Higgins & Dworkin, 2012). Sporulation in C. difficile, like other spore-forming bacterial organisms, is a complex process that results in the formation of a resistant spore from a vegetative cell. This process is largely regulated by external signals which trigger a signalling cascade of sigma factors to initiate sporulation (Fimlaid et al., 2013). Little is known about the signals that trigger C. difficile sporulation, however, studies suggest that it could be related to environmental stimuli such as nutrient starvation, quorum sensing, and other unidentified stress factors (Higgins & Dworkin, 2012). Although the sporulation pathway in C. difficile has been extensively studied, the molecular mechanisms that govern their composition and formation remain poorly understood. Sporulation studies in Bacillus subtilis (B. subtilis) provides insight into the sporulation pathway in C. difficile (Fimlaid et al., 2013).

The entry into sporulation of B. subtilis is controlled by Spo0A, a master regulatory protein which becomes activated by a phosphorelay cascade (Higgins & Dworkin, 2012). Accordingly, Spo0A activates downstream regulators which initiate the sporulation process and repress vegetative cell functions. Activated Spo0A triggers an asymmetric division, yielding two distinct cells, a pre-spore (also known as forespore), and the mother cell (Figure1-7) (Pereira

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et al., 2013). Soon after division, sporulation-specific RNA sigma factors required for gene expression in the forespore and mother cell are sequentially activated. The four-cell type- specific sigma factors (σF, _σE_{, σ}G_{,and σ}K_{) are alternated between the two cells. Sigma F and E}

control the early stages of development in the two cells and are replaced by sigma G and K in the later stages of development (Fimlaid et al., 2013; Pereira et al., 2013). Sigma F is activated in the forespore, while sigma E is activated in the mother cell. Sigma E activates production of spore coat proteins and communicates with the forespore for the activation of sigma G. Sigma G initiates the signalling cascade that results in the activation of sigma K, this leads to the assembly of the spore outer coat (Pereira et al., 2013).

During the asymmetric division, a septum is formed which divides the prespore from the mother cell. Whilst the septum is being formed, the chromosome replicates to form an axial filament (Dembek et al., 2015; Fimlaid et al., 2013). The remaining section of the chromosome is actively transported into the forespore and segregated from the mother cell with the completion of the septum formation. This results into two distinct cells, both with a complete chromosome. The mother cell surrounds the forespore and compresses the membrane off in the way to completely engulf the forespore. Following this, the chromosome in the forespore is reconstructed into a circular structure, a thick cell wall and the protective protein coat is formed, this completes the full synthesis of the spore. Subsequently, the mother cell is lysed (programmed cell death), liberating the mature spore (Pereira et al., 2013).

Although the sporulation activities in B. subtilis are quite similar to those in other Clostridium species, recent studies have highlighted some differences (Pereira et al., 2013). The main periods of activity for the four cell-type sigma factors are highly conserved in C. difficile

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relative to B. subtilis (Pereira et al., 2013). The main observable difference with B. subtilis was that sigma E activity was partially independent of sigma F and sigma G or K did not require sigma E or sigma G. Pereira et al suggested that the connection between the forespore and mother cell lines of gene expression in C. difficile were weaker compared to those observed in B. subtilis (Pereira et al., 2013).

Pre-divisional cell _Asymmetric division Forespore engulfment Engulfment completion Spore coat assembly Mature spore MC FS MC MC FS FS

Figure 1-7 Schematic of the main morphogenetic stages in the process of sporulation. Mother cell

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