Development of Host-Cleavable Antibody-Bactericide Conjugates against Extracellular Pathogens.

Novel antimicrobial agents with potent bactericidal activity are needed to treat infections caused by multidrug-resistant (MDR) extracellular pathogens, such as Pseudomonas aeruginosa. Antimicrobial peptides (AMPs) and peptidomimetics are promising alternatives to traditional antibiotics, but their therapeutic use is limited due to the lack of specificity and resulting off-target effects. The incorporation of an antibody into the drug design would alleviate these challenges by localizing the AMP to the target bacterial cells. Antibody-drug conjugates (ADCs) have already achieved clinical success as anticancer therapeutics, due to the ability of the antibody to deliver the payload directly to the cancer cells. This strategy involves the selective delivery of highly cytotoxic drugs to the target cells, which enables a broad therapeutic window. This platform can be translated to the treatment of infections, whereby an antibody is used to deliver an antimicrobial agent to the bacterial antigen. Herein, we propose the development of an antibody-bactericide conjugate (ABC) in which the antibacterial oligothioetheramide (oligoTEA), BDT-4G, is coupled to an anti-P. aeruginosa antibody via a cleavable linker. The drug BDT-4G was chosen based on its efficacy against a range of P. aeruginosa isolates and its ability to evade mechanisms conferring resistance to the last-resort agent polymyxin B. We demonstrate that the ABC binds to the bacterial cell surface, and following cleavage of the peptide linker, the oligoTEA payload is released and exhibits antipseudomonal activity.

Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products.

The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover - or autolysis - of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditional LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and, contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.