Lacritin cleavage-potentiated targeting of iron - respiratory reciprocity promotes bacterial death

Discovering new bacterial signaling pathways offers unique antibiotic strategies. With current antibiotic classes targeting cell wall synthesis, depolarizing the inner membrane, altering the bacterial metabolome or inhibiting replication or transcription pathways, manipulation of transporters to limit bacterial respiration and thereby pathogenesis has been a decades-long quest. Here we report an inhibitor of multiple bacterial transporters. The inhibitor is the bactericidal N-104 endogenous cleavage fragment of the prosecretory mitogen lacritin. Lacritin is now known to be widely distributed in plasma, cerebral spinal fluid, tears, and saliva. With the bactericidal mechanism determined to be nonlytic by surface plasmon resonance as confirmed by lack of SYTOX Orange entry, we performed an unbiased resistance screen of 3884 Escherichia coli gene knockout strains revealing a complex N-104 polypharmacology. Validation in the virulent Pseudomonas aeruginosa strain PA14-one of three WHO Priority 1: Critical list species-focused on an approach that sequentially couples three transporters and downstream transcription to lethally suppress respiration. By targeting the outer membrane YaiW, cationic N-104 translocates into the periplasm where it ligates inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. With FeoB favoring an anaerobic environment, N-104 promotes the expression of genes regulating anaerobic respiration while largely suppressing those involved in aerobic respiration-a strategy counterproductive under aerobic conditions. This mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with tear thrombin fragment GKY20 as tested on antibiotic-resistant clinical isolates.