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Antimicrobial Agents and Chemotherapy, June 2000, p. 1418-1427, Vol. 44, No. 6
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification and Analysis of Bacterial Protein Secretion Inhibitors Utilizing a SecA-LacZ Reporter Fusion System

L. E. Alksne,* P. Burgio, W. Hu, B. Feld, M. P. Singh, M. Tuckman, P. J. Petersen, P. Labthavikul, M. McGlynn,dagger L. Barbieri, L. McDonald, P. Bradford, R. G. Dushin, D. Rothstein,Dagger and S. J. Projan

Wyeth-Ayerst Research, Pearl River, New York

Received 10 August 1999/Returned for modification 20 December 1999/Accepted 26 February 2000

Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds for Staphylococcus aureus strain MN8 were found to be <= 128 µg/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.

* Corresponding author. Mailing address: Wyeth-Ayerst Research, 401 Middletown Rd., Pearl River, NY 10965. Phone: (914) 732-5601. Fax: (914) 732-2480. E-mail: alksnel{at}war.wyeth.com.

dagger Present address: Regeneron Pharmaceuticals, Tarrytown, N.Y.

Dagger Present address: Periodontix, Watertown, Mass.

Antimicrobial Agents and Chemotherapy, June 2000, p. 1418-1427, Vol. 44, No. 6
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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