Transmembrane delivery of antimicrobial peptides by engineered eCIS into macrophages for intracellular mycobacteria elimination.

Mycobacterium tuberculosis (M. tuberculosis, Mtb) is an intracellular bacterium persisting in macrophages, which can cause chronic infection and drug resistance. To address transmembrane limitation and improve treatment accuracy, we employed the Photorhabdus virulence cassette (PVC), a typical bacterial extracellular Contractile Injection System (eCIS), to deliver antimicrobial peptides (AMPs) into macrophages for intracellular mycobacteria clearance. We demonstrated that the survival of various intracellular mycobacteria, including Mycobacterium smegmatis (M. smegmatis), Mycobacterium bovis (M. bovis), M. tuberculosis and its isoniazid-resistant strain, can be significantly inhibited by PVC-mediated AMP transmembrane delivery. The engineered PVC, loaded with mycobacteriophage-derived AMPs and targeting alveolar macrophages (AMs) through binding to surface receptor CD11b, eliminated AM-dwelling mycobacteria and alleviated inflammation in murine infection models. Notably, the pulmonary mycobacterial load in M. tuberculosis H37Rv-infected mice was reduced by more than tenfold. In summary, this work represents a pioneering application of engineered PVC-based intracellular delivery of biomacromolecules into macrophages, providing a novel therapeutic strategy against mycobacterial infection.