Endolysins isolated from C difficile phages

All C. difficile specific phages that have been isolated to date and characterised in order

to develop therapeutic agents have proven to be lysogenic phages, and no lytic phages

have been isolated (Sell et al.,1983 ; Mahony et al.,1985 ; Dei, 1989; Nagy et al., 1991;

Goh et al., 2005; Govind et al., 2006; Fortier and Moineau, 2007; Mayer et al., 2008;

Horgan et al., 2010; Sekulovic, et al., 2011; Meessen-Pinard et al., 2012; Nale et al.,

2012; Shan et al., 2012) and in this study. However, these types of phages are of limited

value as therapeutic agents (Gill and Hyman, 2010; Meader et al., 2010).

Although these phages have limited therapeutic potential in their current form, they each

carry a gene encoding an endolysin which has the ability to degrade the bacterial cell wall

when applied externally. Thus, these proteins could be developed and used as effective

therapeutic agents against C. difficile. In theory, endolysins would be well suited as

treatment of C. difficile, since they target only C. difficile cells without harming the

normal body microbiota. In this work we have characterised five endolysins from C.

difficile: φCD27, φC2, φCD119 (Myoviridae family), φCD6356 and φCD38-2

(Siphoviridae family).

Bioinformatic analysis of these endolysins revealed that they all contained N-

acetylmuramoyl-L-alanine amidase (amidase3) N-terminal catalytic domains. This class

of endolysin cut the critical amide bond between N-acetylmuramic acid and L-alanine

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This type of endolysin represents one of the most widespread classes of endolysin

(Fischetti, 2008). In contrast, the C-terminal domains of these endolysins lacked any

homology, which was somewhat surprising given that these phages are specific for C.

difficile.

All endolysin sequences were codon optimised for expression in E. coli, to maximise the

production of recombinant protein. Comparison of the level of expression of codon

optimised LysCD27 to that of the non-codon optimised version found a two-fold increase

in expression (Mayer et al., 2008). During protein induction at 37°C, the recombinant

proteins formed insoluble inclusion bodies located in inclusion bodies; these results

correlate with those generated by a protein solubility prediction program that all of our

proteins have a high insolubility percent (Table 4.3). However, the culture temperature

was reduced to 27°C, which is a temperature that has been reported to reduce the

formation of inclusion bodies (Zimmer et al., 2002; Dhalluin et al., 2005).

With regards to the level of protein expression of the other endolysins, they were

expressed at a roughly similar level, ranging from 1-4 mg/100ml.

Characterisation of the biological activity of the recombinant endolysins across a range of

pH values was determined. The endolysins maintained their activity over a wide range of

pHs(4 to 9) including physiological pH, suggesting that they would remain biologically

active in the gastrointestinal tract (GI) environment. A similar observation was made for

other previously characterised C.difficile phage endolysin (φCD27 endolysin) (Mayer et

al., 2008). The effect of temperature on lytic activity was also determined and was found

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activity was seen at temperatures of 60°C and above. With the exception of BHI, the

buffers tested had no significant effect on the lysins activity. Thus, based on these results,

we developed a standardised set of assay conditions with which to determine the level of

activity of each endolysin.

While the lytic profile of each endolysin for C. difficile PG was similar to that described

in the literature, this was not the case for LysCD27 (Loeffler et al., 2001; Schuch et al.,

2002; Son et al., 2012). Mayer and colleagues (2008) reported that the lytic activity of

their version of LysCD27 required a 10 minute incubation step before lytic activity was

initiated (Mayer et al., 2008). In contrast, our version of LysCD27 was able to lyse the

PG almost immediately upon addition. Mayer and colleagues (2008) suggested that this

delay may have been the result of incorrect folding of the recombinant lysin expressed

from their E. coli-based system (pET15b, E. coli BL21). Our results suggest that our

recombinant protein, which is expressed from different host E. coli strains to the Mayer

and colleagues (2008) group, is produced in the correct form and hence, is more active.

Given that one of the differences between the two systems is the codon usage of the gene,

it is tempting to speculate that this is responsible for the enhanced activity (Zimmer et al.,

2002).

While the level of activity of individual lysins varied from strain to strain, the

LysCD6356 endolysin was the most active against all strains of C. difficile tested

(ribotypes: 001, 027, 106, 014, 002, 005, 078, 020, 010 and 023). The second most active

endolysin was LysCD38-2, which was the most active against strains of ribotype 012,

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Interestingly, LysCD6356 shares a high percent amino acid identity (88%) with

LysCD38-2, and they belong to same phage family and same phage group

These endolysins also showed activity against other Gram-positive strains, such as C.

sordellii, which is closely related to C. difficile, and less closely related species B. subtilis

and L. monocytogenes. However, they were inactive against a number of other Gram-

positive species.

The host range of each endolysin was significantly broader than that of the phages themselves, as previously described for φCD27 (Mayer et al., 2008). Moreover, all C. difficile strains tested were at least sensitive to one of our endolysins.

This phenomenon support the therapeutic potential of endolysins compared to

bacteriophages.