Abstract
Purpose :
Lacritin C-terminal proteoform 'N-104' is a main source of tear bactericidal activity. By screening for N-104 resistance, we recently discovered the importance of respective putrescine and iron transporters PotH and FeoB as likely virulence factors for P. aeruginosa (PA14) ocular pathogenesis. PotH and FeoB are constituents of the inner bacterial membrane, whereas N-104 is in tears and not membrane disruptive. How then does N-104 bactericidal activity involve PotH and FeoB? Here we search for N-104 binding proteins in the outer membrane of PA14.
Methods :
N-104, and negative control lacritin peptide C-95, synthesized with cysteine added to the N-terminus, were coupled to beads of SulfoLink® Coupling Resin with an efficiency of 0.7 mmole peptide to ml resin, and then blocked with L-cysteine. Overnight cultures of PA14, either without or with prior surface biotinylation, were washed in PBS, resuspended in lysis buffer containing 200 mM octyl β-D-glucopyranoside ('OG') with rocking overnight at 4°C to allow membrane proteins to insert into OG vesicles, and centrifuged. Supernatant was passed through PierceTM Agarose Resin precolumns and then onto N-104 or C-95 columns with rocking overnight at 4°C. After washing with 20 ml of running buffer containing 20 mM KCl, proteins were eluted by progressive addition of 50, 75, 100, 125, 150, 300, 500 or 1000 mM KCl. 500 mM KCl eluted proteins from N-104 and C-95 columns were subjected to mass spectrometry.
Results :
'YaiW' was the sole outer membrane protein hit with 7.4x higher enrichment from N-104 vs negative control C-95 columns, and mutants lacking YaiW resistant to N-104 with a slope ratio threshold of 0.7. YaiW contributes to the transport of proline-rich Bac7 peptides across the outer membrane. No hits were obtained from biotinylated cells, in keeping with amine-reactive interference.
Conclusions :
YaiW is a candidate outer membrane receptor for N-104 with the potential capability to transport N-104 into the periplasm, and thus in proximity to inner membrane PotH and FeoB.
This is a 2021 ARVO Annual Meeting abstract.