Abstract
Bacteriophage endolysins (lysins) bind to a cell wall substrate and cleave peptidoglycan, resulting in hypotonic lysis of the phage-infected bacteria. When purified lysins are added externally to Gram-positive bacteria they mediate rapid death by the same mechanism. For this reason, novel therapeutic strategies have been developed using such enzybiotics. However, like other proteins introduced into mammalian organisms, they are quickly cleared from systemic circulation. PEGylation has been used successfully to increase the in vivo half-life of many biological molecules and was therefore applied to Cpl-1, a lysin specific for S. pneumoniae. Cysteine-specific PEGylation with either PEG 10K or 40K was achieved on Cpl-1 mutants, each containing an additional cysteine residue at different locations To the best of our knowledge, this is the first report of the PEGylation of bacteriophage lysin. Compared to the native enzyme, none of the PEGylated conjugates retained significant in vitro anti-pneumococcal lytic activity that would have justified further in vivo studies. Since the anti-microbial activity of the mutant enzymes used in this study was not affected by the introduction of the cysteine residue, our results implied that the presence of the PEG molecule was responsible for the inhibition. As most endolysins exhibit a similar modular structure, we believe that our work emphasizes the inability to improve the in vivo half-life of this class of enzybiotics using a cysteine-specific PEGylation strategy.
Highlights
Streptococcus pneumoniae is the first cause of otitis media and a common cause of sinusitis, communityacquired pneumonia, bacteremia, and meningitis (Jacobs, 2004,)
We recently showed that pre-dimerization of Cpl-1, which doubles the molecular weight of the enzyme, decreased its plasma clearance by a factor of ten (Resch et al, 2011,)
While introducing cysteines at several sites on Cpl-1 did not alter its bactericidal activity, PEGylation on these residues totally abrogated it. This might be related to the complex structure and mode of action of the enzyme, which makes it susceptible to bulky adducts
Summary
Streptococcus pneumoniae is the first cause of otitis media and a common cause of sinusitis, communityacquired pneumonia, bacteremia, and meningitis (Jacobs, 2004,). Antibiotic misuse and overuse has progressively selected for resistance against major drug classes, and treatment failures are widely reported (Fuller and Low, 2005,; Klugman, 2002,). This justifies the search for new drugs with different mechanisms of action. The bacteriolytic action of bacteriophage lysins enables the release of phage progeny from the bacterial sacculus. Purified pneumococcal phage lysin Cpl-1 has been used to successfully treat pneumococcal sepsis, endocarditis, meningitis, and pneumonia in rodent models (Entenza et al, 2005,; Grandgirard et al, 2008,; Loeffler et al, 2003,). Due to its short circulating half-life (~20.5 minutes) (Loeffler et al, 2003,), optimal efficacy requires
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