Objectives (i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes.

Methods We inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal α-amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant.

Results Our biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase and polysorbate 80.

Conclusions Synergistically acting cell wall degrading enzymes are potently inhibiting Mtb – which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.