Identification and analysis of bacterial protein secretion inhibitors utilizing a SecA-LacZ reporter fusion system

Abstract

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 < or =128 microg/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.

FIG. 1

Sec-dependent secretory pathway.

FIG. 2

Autogenous regulation of SecA expression. (A) Under conditions of normal secretion, SecA binds its own mRNA and blocks its ribosomal binding site, inhibiting translation. (B) Under conditions in which secretion is blocked, SecA releases its mRNA and translation is increased.

FIG. 3

Structures of potential secretion inhibitors.

FIG. 4

ELISA and Western blot analyses of secretion of TSST-1 in S. aureus MN8. ELISA and Western blot analysis were performed to examine the extracellular and intracellular presence of TSST-1 in the presence of various potential secretion inhibitors. Cells were grown in the presence of compounds at the concentrations designated. The mobility of TSST-1 in the Western blot is noted with an arrow, while the mobility of a non-TSST-1 protein nonspecifically recognized by the antibody is noted with an asterisk. In panel D, nonspecific recognition of a marker protein is apparent in the empty middle lane. Percent inhibition is defined as for growth of TSST-1 secretion over 6 h in comparison with an untreated control. Western blots were scanned using a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Hercules, Calif.), and graphics were added with Adobe Photoshop 5.0. (A) cerulenin; (B) compound 3; (C) compound 4; (D) compound 5; (E) compounds 1 (left, Western blot; top, ELISA) and 2 (right, Western blot; bottom, ELISA).

FIG. 5

Pulse-chase immunoprecipitation analysis of MBP secretion in E. coli Mc4100. Cells were grown in minimal maltose medium as described in Materials and Methods and were left untreated or were treated at 0.25 times the MIC of each compound (0.5 times the MIC for compound 3) for 15 or 30 min prior to pulse labeling. A chase period with cold methionine was applied for 15 s to 5 min. Mature MBP mobility is designated by an arrow. Nonprocessed MBP is designated by an asterisk. Autoradiograms were scanned using a GS-700 Imaging Densitometer (Bio-Rad Laboratories), and graphics were added using Adobe Photoshop 5.0. (A) Cerulenin; (B) chloramphenicol; (C) polymyxin B; (D) compound 5; (E) compound 6; (F) compound 3.