Endospore Cuticle Interactions

The infection of second-stage root-knot juveniles by P. penetrans endospores is initiated when viable endospores adhere to the cuticle surface of migrating nema-todes as they move through the soil. Attachment can therefore be seen as the key to the commencement of infection. Studies (Stirling 1985; Davies et al. 1988, 1990; Channer and Gowen 1992; Sharma and Davies 1996; Espanol et al. 1997; Mendoza de Gives et al. 1999; Wishart et al. 2004) have shown that different populations of endospores do not adhere to all cuticles of all populations of nematodes and inter-and intra-attachment specificity is usual. Indeed, it has been shown that cuticle heterogeneity as exhibited by endospore attachment is not linked in any simple way to the phylogeny of the nematode (Davies et al. 2001) and, in addition, in standard attachment assays differences can also be found between different stages of the same nematode population (Davies and Williamson 2006). Interestingly, inter- and intra-specific functional variation as measured by Pasteuria spore attachment assays has shown an equal amount of variation even between amphimictic and parthenogeneti-cally reproducing species of root-knot nematodes (Davies et al. 2008). If biological control is to work in a predictive manner and the correct spore populations applied to control susceptible nematode populations it will be important to understand the nature and mechanism of spore/cuticle attachment compatibility.

Henriques and Moran (2007) recently reviewed the structure and function of bacterial endospores. The endospore coat is the outermost layer of the spore; however in some bacterial species the spore is surrounded by an additional layer called the exosporium. Pasteuria penetrans is a species that possesses an exosporium, which provides it with resistance to chemical and enzymic treatments and is likely to give the spore its adhesive properties (Kozuka and Tochikubo 1985; Takumi et al. 1979). Fibrils are known to be important in the attachment of many bacteria to host surfaces and their decoration with sugars has been observed to confer host specificity (Benzi and Schmidt 2002; Power and Jennings 2003; Takeuchi et al. 2003). The exposure of endospores to HCl removes its central body to reveal a structure containing fibrils (Persidis et al. 1991) and scanning electron microscope studies on intact endospores have revealed that the parasporal fibres are positioned in such a way around the central body of the endospore to produce a skirt-like structure in which the under-surface of the endospore is in intimate contact with the nematode cuticle. Electron micrographs of the endospore reveal that the surface of the skirt-like structure, made up of parasporal fibres, are covered with other fine fibres both on the upper and lower surface and that the fibres on the concave surface of the endospore are more densely distributed than on the upper surface and it has been proposed that these fibres are involved in attachment of the mature endospore to the nematode cuticle (Davies 2009).

The structure of the exosporium in other closely related bacteria, B. cereus, B. thuringiensis and B. anthracis is species and strain specific (Plomp et al. 2005a, b) and it is clear that they also have an outer surface covered with a hair-like nap (Wehrli et al. 1980; DesRosier and Lara 1981) similar to P. penetrans. In B. anthracis the hair-like nap appears to be formed by a single collagen-like protein BclA in which the length of the filaments is related to the number of G-X-Y repeats (Sylvestre et al. 2002, 2003, 2005; Boydston et al. 2005). Homologous genes to bclA have been identified in other Bacillus spp. and they reside in a rhamnose cluster operon that contains around 30 genes within which are a number of glycosyl-transferases that form an exosporium island (Charlon et al. 1999; Steichen et al. 2003; Todd et al. 2003). In the initial genomic survey using Sanger sequencing of P. penetrans (Bird et al. 2003) four genes, with e-values <e-14, were recognised using BlastP against B. anthracis, B. cereus and B. thuringiensis, within the rhamnose cluster including a collagen-like sequence that was phylogenetically more closely related to the bacterial collagens (Davies and Opperman 2006); subsequent 454 sequencing increased this to 12 genes and also included collagen-like sequences (Fig. 4.1). Several collagen-like sequences were identified each containing 28, 36 and 87, collagen-like G-X-Y repeats respectively and from which it was possible to predict that the P. penetrans hair-like nap would be made-up of filaments with lengths ranging from 56 to over 200 nm in length (Davies and Opperman 2006). Transmission electron microscope studies of endospores of P. penetrans have so far not provided evidence of fibres with a length significantly greater than 100 nm, but exosporial filaments ranging in length from 20 to over 100 nm have been identified (van de Meene, Rowe and Davies unpublished data). Conclusive evidence showing that these fibres on the surface of the endospore are collagen-like will need further investigation. However, results from a series of experiments in which endospores were either incubated in collagenase (Davies and Danks 1993) or were pretreated with fibronectin (Davies and Redden 1997; Mohan et al. 2001) suggested that the fibres are collagen-like, as these treatments reduced the ability of endospores to adhere to the nematode cuticle.

Rhamnose cluster operon cloP BclA rfbA rfbC rfbB rfbD fab1 yjbX cotZ-1 cot Y

cloP BclA rfbA rfbC rfbB rfbD fab1 yjbX cotZ-1 cot Y

cotX cotW cotV yjcA yjcB yjcC cotZ-2

yjcC

yjcD yjcE yjcF yjcG yjcH

cotX cotW cotV yjcA yjcB yjcC cotZ-2

yjcC

yjcD yjcE yjcF yjcG yjcH

Fig. 4.1 Rhamnose cluster operon with alignments of genes for Bacillus anthracis (Ba), B. thuringiensis (Bt) and B. subtilis (Bs) BlastP hits with E-values < e-14 from Sanger sequencing (black arrows) and 454 sequencing (grey arrows); X gene missing (Adapted from Todd et al. 2003)

Ba Bt

Fig. 4.1 Rhamnose cluster operon with alignments of genes for Bacillus anthracis (Ba), B. thuringiensis (Bt) and B. subtilis (Bs) BlastP hits with E-values < e-14 from Sanger sequencing (black arrows) and 454 sequencing (grey arrows); X gene missing (Adapted from Todd et al. 2003)

The nature of the receptor on the nematode cuticle has not as yet been determined, however it is thought to involve some form of carbohydrate - lectin interaction (Davies and Danks 1993). Mucins are a family of polypeptides associated with both the innate and adapted immune systems and can be secreted or membrane bound to form a protective barrier that covers epithelial surfaces (Strous and Dekker 1992; Magalhaes et al. 2010). The surface coat of C. elegans is a thin layer that is secreted onto the cuticle surface known to contain both mucin-like proteins amongst other glycosylated protein secretions (Hemmer et al. 1991; Gems and Maizels 1996). Mucin-like proteins are rich in serine and threonine and are highly glycosalated and it has been suggested that they play a role in immune defence (Hall and Altun 2008). Mucin-like proteins identified in C. elegans appear to have orthologues in Meloidogyne spp. (Davies 2009). RNAi experiments knocking down these mucin-like proteins have been shown to lead to changes in the recognition of the adult cuticle surface of C. elegans (Davies et al. 2009). It can therefore be hypothesised that similar proteins may be involved, directly or indirectly, in the endospore attachment process.

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