Physicochemical and Proteolytic Barriers Limiting Activity of Cpl-1 and Pal Endolysins in Human Circulation

The growing prevalence of antibiotic-resistant bacterial infections poses a serious burden on healthcare systems worldwide. Endolysins are promising candidates for a new type of antibiotic due to their strong bacteriolytic activity. However, important limitations, including reduced activity and short persistence in the bloodstream, must still be addressed. We evaluated the key physicochemical and biological factors limiting the activity and stability of the endolysins Cpl-1 and Pal in blood. The analysis included ionic composition and strength, pH, bystander proteins, physiological temperature, and proteolytic activity. Our results indicate that the aforementioned factors significantly affect Cpl-1 and Pal, suggesting that physiological conditions in human circulation markedly restrict the anti-bacterial potential of endolysins. To overcome these limitations, we designed a set of Cpl-1 and Pal variants with modified amino acid compositions aimed at increasing their resistance to such physiological constraints. One variant demonstrated improved performance in an ex vivo mouse model and lacked a cleavage site for blood proteases.