Endolysins: insights into their mode of action and regulation for enhanced applications

Gram-positive (G+) bacteria are normally considered more susceptible to the exolytic action of endolysins due to the absence of an outer membrane. However, they can still exhibit some tolerance to the lytic enzymes, especially under isotonic conditions that support growth. Previous studies have shown that this tolerance requires both an intact proton motive force (PMF) and the presence of wall teichoic acids (WTA). We investigated how the interconnected PMF and WTA factors modulate the activity of the anti-S. aureus endolysin Lys11. Our findings showed that the chemical (ΔpH) and electrical (Δψ) gradients of the PMF affect Lys11's functional domains differently. Dissipation of ΔpH primarily enhanced the lytic action via the enzyme’s peptidase domain, while Δψ dissipation unexpectedly boosted binding through the amidase domain. WTA acted as major tolerance determinants by inhibiting peptidoglycan (PG) cleavage and by severely impairing binding mediated by the canonical cell binding domain. The enhanced lytic activity under PMF-collapsed conditions mirrors the natural mechanism of phage infection, where endolysins act only after holins dissipate the PMF. Supporting this, we showed that antimicrobial peptides mimicking holins eliminate PMF/WTA-mediated tolerance, greatly increasing S. aureus susceptibility to endolysins. Moreover, endolysins targeting G+ bacteria are often termed single-use agents due to strong binding to CW debris post-lysis. We found that removing the amidase domain can reduce binding, while improving bacteriolysis kinetics. Finally, PG-cleaving domains from virion-associated lysins (VALs), which naturally evolved to target energized cells externally, may offer alternatives to overcome tolerance.