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

Protein secretion is an essential process for bacterial growth, yet there a re few if any antimicrobial agents which inhibit secretion. An in vivo, hig h-throughput screen to detect secretion inhibitors was developed based on t he translational autoregulation of one of the central protein components, S ecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escher ichia coli which is induced when secretion is perturbed, Several compounds, including two natural product extracts, which had the ability to induce th e reporter fusion were identified and the MICs of these compounds for Staph ylococcus aureus strain MN8 were found to be less than or equal to 128 mu g /ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipi tation techniques were used to analyze the affects of these compounds on pr otein secretion. Six representative compounds presented here appear to be b ona fide secretion inhibitors but were found to have deleterious effects on membranes, It was concluded that, while the method described here for iden tifying inhibitors of secretion is valid, screens such as this, which are d irected against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.