Outer Membrane Protein I of Pseudomonas aeruginosa Is a Target of Cationic Antimicrobial Peptide/Protein

Yu-Min Lin,Chiu-Feng Wang,Ting-Wei Chang,Ching-Shu Suen,Ming-Jing Hwang,Shih-Jung Wu,Margaret Dah-Tsyr Chang,Yuan-Tsong Chen,You-Di Liao

doi:10.1074/jbc.m109.078725

Abstract

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and alpha-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and alpha-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric alpha-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of alpha-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.

Highlights

As a member of the RNase A superfamily, hRNase 7 is a highly positively charged protein with 128 amino acids [6, 9, 10]
We identify a polymeric lipoprotein, OprI, from the outer membrane of P. aeruginosa that is responsible for the bacterial susceptibility to ␣-helical cationic antimicrobial peptides/proteins (AMPs) [16]
P. aeruginosa was susceptible to both hRNase 7 and sheep myeloid antimicrobial peptide 29 (SMAP-29), whereas E. coli was bound P. aeruginosa with similar efficiency, but the hRNase 7 was markedly released from P. aeruginosa on treatment with MgCl2 or LPS from E. coli and P. aeruginosa (Fig. 1F, top panel)

Summary

EXPERIMENTAL PROCEDURES

Materials—The LPSs of P. aeruginosa and Escherichia coli and polymyxin B were obtained from Sigma-Aldrich; SYTOX௡ Green was from Molecular Probes (Carlsbad, CA); 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 3,3-dithiobissul fosuccinimidyl-propionate (DTSSP) were

Outer Membrane Protein Is a Target of Antimicrobial Peptide

RESULTS

Identification and Production of

The coincidence of bactericidal activity and OprI binding of hRNase

The cellular components were extruded from the outer membrane of

DISCUSSION