The overall structure and morphology of C. difficile spores
is similar to what has been seen in other endospore-forming
bacteria (Box 2), but the outermost layers are particularly different. Transmission electron microscopy (TEM) of
C. difficile spores [19,21–23,25,34,35] revealed that the
spore coat has distinctive laminations (i.e., lamellae),
which are similar to those observed in B. subtilis but
different from those in members of the B. cereus group
(i.e., B. anthracis) (Figure 1A,B) [36,37]. It is remarkable
that despite these structural similarities, only 25% of the
genes encoding spore coat proteins have homologs in C.
difficile, suggesting that the components that govern spore
coat assembly are divergent to those defined for B. subtilis
and/or the B. cereus group [11].
In members of the B. cereus group, the outermost layer is the exosporium, defined for these species as a distinguishable spore layer with hair-like projections formed in the mother cell compartment, and it does not have direct interactions with the spore coat layers that surround the cortex [38–40]. In C. difficile, the exosporium is the outermost layer. Although the C. difficile exosporium has hair-like projections, it lacks many of the B. cereus group exosporium orthologs and the frequently observed gap that separates the spore coat from the exosporium. Nevertheless, the C. difficile exosporium exhibits a conserved genetic signature to the B. cereus group, because the exosporium glycoproteins encoded by bclA1, bclA2, and bclA3 are also expressed in the mother cell (see below). The stability of the exosporium, remains a matter of controversy because several reports suggest that this layer is fragile and easily lost [21,22], or absent in spores developed during biofilm formation [39],
whereas other reports indicate that it is a reasonably stable layer that is only removed by proteases and/or sonication treatments [19,23,25,34,35]. The stability ofthe exosporium could be related to strain type or the use of proteases in purification procedures. Indeed, recent evidence indicates that proteinase K can remove the exosporium layer while leaving the spore coat intact [34]. Interestingly, the morphology of the exosporium seems to be strain-dependent (Figure 1A,B) [34,35]. C. difficile 630 spores have an electron-dense, compact exosporium layer (Figure 1A), whereas spores derived from strains R20291, M120, TL176, and TL178 have a hair-like exosporium layer reminiscent of the B. anthracis exosporium (Figure 1A,B). Interestingly, the ultrastructural organization of the exosporium surface of R20291, M120, and TL176 is identical to that of TL178 spores (Figure 1C), suggesting that the exosporium layer of C. difficile strain 630 may be an outlier among C. difficile strains.
Although the roles ofthe outermostlayers (i.e., spore coat and exosporium) on CDI and pathogenesis are unclear, recent reports have shed light on their potential functions. The C. difficile spore surface was shown to interact with unidentified surface receptor(s) of intestinal epithelium cells [25]. In vitro infection of macrophages with C. difficile spores revealed that spores become cytotoxic to macrophages possibly by disrupting the phagosomal membrane through spore surface-membrane interactions [41]. The exosporium layer was also shown to confer hydrophobicityto the spore surface affecting its adherence to inert surfaces [35]. C. difficile R20291 spores with a defective exosporium layer adhere better to Caco-2 cells than those with an intact exosporium layer suggesting a potential role in adherence to epithelial surfaces and in the transmission of CDI [23]. Removal of the exosporium layer also increases the ability of C. difficile spores to outgrow into colonies in vitro [34], raising the possibility that C. difficile spores lacking an exosporium are not only able to adhere better to colonic surfaces but also germinate more efficiently. The fraction of spores that have a firmly attached exosporium layer and the half-life of this layer in the environment remain to be determined.
Adapted from:Clostridium difficile spore biology: sporulation, germination, and spore structural proteins,Daniel Paredes-Sabja et al.Trends in Microbiology July 2014, Vol. 22, No. 7
Macherki M E
In members of the B. cereus group, the outermost layer is the exosporium, defined for these species as a distinguishable spore layer with hair-like projections formed in the mother cell compartment, and it does not have direct interactions with the spore coat layers that surround the cortex [38–40]. In C. difficile, the exosporium is the outermost layer. Although the C. difficile exosporium has hair-like projections, it lacks many of the B. cereus group exosporium orthologs and the frequently observed gap that separates the spore coat from the exosporium. Nevertheless, the C. difficile exosporium exhibits a conserved genetic signature to the B. cereus group, because the exosporium glycoproteins encoded by bclA1, bclA2, and bclA3 are also expressed in the mother cell (see below). The stability of the exosporium, remains a matter of controversy because several reports suggest that this layer is fragile and easily lost [21,22], or absent in spores developed during biofilm formation [39],
whereas other reports indicate that it is a reasonably stable layer that is only removed by proteases and/or sonication treatments [19,23,25,34,35]. The stability ofthe exosporium could be related to strain type or the use of proteases in purification procedures. Indeed, recent evidence indicates that proteinase K can remove the exosporium layer while leaving the spore coat intact [34]. Interestingly, the morphology of the exosporium seems to be strain-dependent (Figure 1A,B) [34,35]. C. difficile 630 spores have an electron-dense, compact exosporium layer (Figure 1A), whereas spores derived from strains R20291, M120, TL176, and TL178 have a hair-like exosporium layer reminiscent of the B. anthracis exosporium (Figure 1A,B). Interestingly, the ultrastructural organization of the exosporium surface of R20291, M120, and TL176 is identical to that of TL178 spores (Figure 1C), suggesting that the exosporium layer of C. difficile strain 630 may be an outlier among C. difficile strains.
Although the roles ofthe outermostlayers (i.e., spore coat and exosporium) on CDI and pathogenesis are unclear, recent reports have shed light on their potential functions. The C. difficile spore surface was shown to interact with unidentified surface receptor(s) of intestinal epithelium cells [25]. In vitro infection of macrophages with C. difficile spores revealed that spores become cytotoxic to macrophages possibly by disrupting the phagosomal membrane through spore surface-membrane interactions [41]. The exosporium layer was also shown to confer hydrophobicityto the spore surface affecting its adherence to inert surfaces [35]. C. difficile R20291 spores with a defective exosporium layer adhere better to Caco-2 cells than those with an intact exosporium layer suggesting a potential role in adherence to epithelial surfaces and in the transmission of CDI [23]. Removal of the exosporium layer also increases the ability of C. difficile spores to outgrow into colonies in vitro [34], raising the possibility that C. difficile spores lacking an exosporium are not only able to adhere better to colonic surfaces but also germinate more efficiently. The fraction of spores that have a firmly attached exosporium layer and the half-life of this layer in the environment remain to be determined.
Adapted from:Clostridium difficile spore biology: sporulation, germination, and spore structural proteins,Daniel Paredes-Sabja et al.Trends in Microbiology July 2014, Vol. 22, No. 7
Macherki M E
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