(813.7) Lacritin Bactericidal Peptide N-104 Targeting of Inner Membrane Transporters FeoB and PotH (SpuG) Individually Absent in N-104 Resistant Strains of Human Opportunistic Pathogen, P. aeruginosa PA14

Poster Board Number: A370

Mohammad Sharifian Gh. (University of Virginia), Mihaela Gadjeva (Harvard University), Henrietta Venter (University of South Australia), Gordon Laurie (University of Virginia)

Through an unbiased screen of the E. coli Keio collection of 3,985 nonessential gene knockouts, we have discovered that inner membrane putrescine and ferrous iron transporters PotH and FeoB each play roles in the bactericidal mechanism of tear lacritin peptide ‘N-104’ - possibly involving outer-membrane translocation by lipoprotein YaiW with which it binds and when absent confers N-104 resistance. The results were confirmed in the human opportunistic pathogen, P. aeruginosa PA14, and in a robust C. elegans-based liquid infection assay. Does periplasmic N-104 directly or indirectly target PotH and/or FeoB? His-tagged FeoB (P. aeruginosa) and PotH (E. coli) extracted in lysis buffer containing 2% Dodecyl-b-D-maltopyranoside were purified on Ni-Sepharose columns, concentrated on 50 kDa (FeoB) or 30 kDa (PotH) Amicon cutoff membranes, and then added in equal amounts in 150 mM NaCl column buffer to SulfoLink® immobilized Cys-N-104 or to negative control Cys-C-95 columns. After overnight incubation at 4°C and collection of the flow through, columns were washed with 25 column volumes of 150 mM KCl before elution via a gradient of column buffer containing 300, 500, or 1000 mM KCl. Whereas FeoB and PotH were detected only in the flow through off of C-95, 500 mM KCl was required for elution off of N-104. PotH inwardly transports putrescine, the predominant polyamine in bacterial cells (10 - 30 mM) that is essential for optimal bacterial cell growth and proliferation. FeoB is the major mediator of ferrous iron uptake in P. aeruginosa, E. coli, H. pylori, and some other species, and is a well-recognized virulence factor. Given that none of those proteins are essential for the bacterial survival, we hypothesize that N-104 might either disturb putrescine or ferrous iron regulation, or employ each to trigger programmed cell death via uncharacterized YbdM predicted to have transcription regulatory region activity also discovered to bind N-104, and when absent confers N-104 resistance. This study uncovers new virulence factors, and a novel method of bacterial cell death by a natural peptide component of human tears and apparently of plasma.

NIH R01 EY026171;
NIH R01 EY022054;
Unrestricted gift from TearSolutions Inc.