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 <128 g/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.Bacterial protein secretion is an attractive target for antimicrobial chemotherapy because the secretion machinery is highly conserved among bacterial species but is distinct from its eukaryotic counterparts (10,11,14,15,27,42,43). In Escherichia coli, approximately 20% of the total cellular protein is secreted across the cytoplasmic membrane (30). The Sec-dependent pathway of secretion is a multicomponent system consisting of at least seven proteins, five of which are essential for cell viability (for a review, see reference 8). Homologs for several of the components have been identified in both gramnegative and gram-positive bacteria, and therefore it is considered likely that an inhibitor of this pathway would have broadspectrum activity.Secreted proteins are produced as precursors containing signal sequences. Those which are secreted via the Sec pathway are accompanied to the membrane by SecB, a chaperone molecule (Fig. 1). SecA, an ATPase required for translocation at the multisubunit translocase SecY-SecE-SecG, is found associated with both the signal sequence of secretable proteins, and also with the membrane at SecE-SecY-SecG. During translocation ATP is hydrolyzed, the protein is inserted across the membrane, the signal sequence is cleaved, and the protein is released.Expression of SecA appears to be a focal point of regulation for this process (20,24,33) (Fig. 2). SecA translation is autogenously regulated in response to changes in secretion levels. secA is transcribed as the second gene in an operon after geneX, an open reading frame of unknown function. Under normal secretion conditions, SecA binds to its own mRNA, blocking the ribosomal binding site, thus inhibiting initiation of translation. If secretion is blocked, SecA releases its mRNA and translation levels increase (25).An E. coli cell-bas...

Antibiotic Resistance in Bacteria and Its Future for Novel Antibiotic Development

“…The genomics provide many molecular targets to be identified and effectively screened: 5,96) peptide deformylase (PDF), 109) aminoacyl-tRNA synthetase, 110) fatty acid biosynthesis, 111) isoprenoid biosynthesis, 112) lipid A biosynthesis, 113) efflux pump, 114) aromatic amino acid biosynthesis, 96) DNA replication (GyrB subunit inhibitor), 115) protein secretion, 116) cell wall biosynthesis (sortase), 117) proteasome, 118) glyoxylate cycle, 119) and signal networks (two-component systems and quorum sensing pathways). 120,121) 1.…”

A Mechanism-Based Whole-Cell Screening Assay to Identify Inhibitors of Protein Export in Escherichia coli by the Sec Pathway

“…15 In strain EC626, β-gal is exported to the periplasm via the Sec pathway, but in strain EC627, β-gal lacks an export motif and therefore stays in the Our previous assay for inhibition of the Sec pathway 15 was suboptimal for screening in that it used three microtiter plates: one for inducing β-gal expression, a second for permeabilizing the cells, and a third for assaying β-gal activity. Following the precedent of Alksne et al, 5 we now perform these steps in a single plate as follows. Strain EC626 is grown overnight at 37 °C with vigorous aeration in LB media with 0.2% glucose, which suppresses lamB/lacZ expression.…”

“…The plate is incubated at 37 °C for 60 min. Then, 12.5 to 25 µL of "ZOB" buffer, 5 which contains the detergents hexadecyltrimethylammonium bromide (CTAB) and sodium deoxycholate to permeabilize cells and the β-gal substrate ortho-nitrophenyl-beta-galactoside (ONPG) to assess β-gal activity, is added. After 40 min at room temperature, 12.5 to 25 µL of 8 M urea with 1 M sodium carbonate is added to enhance color development, and absorbance is read at 405 nm to gauge the production of ortho-nitrophenol.…”

“…4 Although the druggability of the pathway is uncertain, SecA's crystal structure has been interpreted as containing druggable binding pockets. 3 The literature on experimental Sec-targeted screens [5][6][7][8][9][10] indicates that there is considerable interest in this pathway from the perspective of bacterial drug development, but previous screens have not generally led to "developable" hits. For example, compounds identified as hits by Alksne et al 5 had pleiotropic effects such as damage to the inner membrane, whereas a SecA inhibitor isolated from a natural product screen had very weak antibacterial activity.…”

“…3 The literature on experimental Sec-targeted screens [5][6][7][8][9][10] indicates that there is considerable interest in this pathway from the perspective of bacterial drug development, but previous screens have not generally led to "developable" hits. For example, compounds identified as hits by Alksne et al 5 had pleiotropic effects such as damage to the inner membrane, whereas a SecA inhibitor isolated from a natural product screen had very weak antibacterial activity. 7 A structure-based virtual screen for SecA inhibitors likewise did not identify superlative hits; potencies of the two best compounds, initially reported as K i s of 18 µM and 40 µM, 11 were later revised to IC 50 s of ~100 µM.…”