Lacritin bactericidal peptide N‐104 targets FeoB and PotH through interaction with the surface‐exposed lipoprotein YaiW

Putrescine and iron transporters PotH and FeoB play roles in bactericidal mechanism of tear lacritin peptide ‘N‐104’. Given that N‐104 doesn't appear to disrupt bacterial lipid membranes, we asked how N‐104 bactericidal activity involves the inner‐membrane proteins in Gram‐negative bacteria, for example, ocular pathogenic P. aeruginosa PA14. We synthesized N‐104, and negative control lacritin peptide C‐95 with cysteine added to the N‐terminus, and coupled them to SulfoLink® Coupling Resin. Overnight cultures of PA14 were washed in PBS, lysed and incubated overnight (4ºC) in lysis buffer containing 200 mM octyl β‐D‐glucopyranoside (‘OG’) to allow membrane proteins getting into OG vesicles. The total lysate supernatant was passed through Pierce TM Agarose Resin precolumns prior to overnight incubation (4ºC) with N‐104 or C‐95 columns. After washing nonspecifically bound proteins, interacting proteins were eluted by gradient addition of 50, 75, 100, 125, 150, 300, 500 or 1000 mM KCl. The 500 mM KCl eluted proteins from the two columns were subjected to mass spectrometry for protein identification. The surface‐exposed lipoprotein ‘YaiW’ was the sole identified outer‐membrane protein hit with 7.4x higher enrichment from N‐104 vs negative control C‐95 columns, while no hits were obtained from biotinylated surface proteins of PA14 (possibly due to amine‐reactive interference). We know that YaiW contributes to the transport of proline‐rich Bac7 peptides across the outer membrane, thus YaiW appears to be a receptor for N‐104 and potentially capable of transporting the N‐104 into the periplasm. We also studied bactericidal efficacy of N‐104 against PA14 in the free living nematode C. elegans , as a model mammalian system. Adult worms grown on a lawn of E. coli strain OP50 were harvested in M9 buffer. Approximately, 10 7 cfu/ml of bacteria strains, including PA14, PA14 feoB mutant, PA14 potH mutant (from PA14NR Set; Harvard), and OP50 as a negative control were untreated or treated with 10 and 20 μM N‐104 in 0.1xPBS for 5 hours (35°C). 75‐100 worms were incubated with the cells in sterile black 96‐well plate with the final composition of 90% M9 buffer and 10% LB broth (in a humidified chamber at room temperature). After 3 days, bacteria and debris were aspirated from the wells and worms were stained with 2.5 μM SYTOX Orange in M9 and imaged next day by capturing both transmitted (all worms) and epi‐fluorescence (dead worms) images. While all PA14 strains (wild‐type, feoB mutant, and potH mutant), but not OP50, were able to kill >75% of the worm population, N‐104 treated samples of wild‐type PA14 couldn't damage any worms. In contrast, N‐104 treated samples of feoB mutant, and potH mutant strains could kill about 35% and 25% of the worm populations confirming the roles for PotH and FeoB in N‐104's bactericidal mechanism.