Background: SecA has been viewed as ATPase helping precursors across SecYEG channels. Results: SecA alone could promote protein translocation and ion channel activity, but loses specificity and efficiency, which can be restored by SecYEG. Conclusion: SecA plays important structural roles and can function as low affinity protein conducting channels in membranes. Significance: Establishing SecA as channels is crucial for understanding diverse mechanisms and evolution of bacterial translocation pathways.

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SecA, a key component of bacterial Sec-dependent secretion pathway, is an attractive target for exploring novel antimicrobials. Rose bengal (RB), a polyhalogenated fluorescein derivative, was found from our previous study as a potent SecA inhibitor. Here we describe the synthesis and structure-activity relationships (SAR) of 23 RB analogues that were designed by systematical dissection of RB. Evaluation of these analogues allowed us to establish an initial SAR in SecA inhibition. The antimicrobial effects of these SecA inhibitors are confirmed in experiments using E. coli and B. subtilis.

SecA, a key component of bacterial Sec-dependent secretion pathway, is an attractive target for novel antimicrobial development. Through a combination of virtual screening and experimental exploration of surrounding chemical space, we identified a hit bistriazole SecA inhibitor, SCA-21, and studied a series of analogs by systematic dissections of the core scaffold. Evaluation of these analogs allowed us to establish an initial SAR in SecA inhibition. The best compounds in this group have potent inhibition activity of SecA-dependent protein-conducting channel activity and protein translocation activity at low to sub-μM concentrations. They also have MIC values against various strains of bacteria that are correlated with the SecA and protein translocation inhibition data. These compounds are effective against methicillin-resistant Staplylococcus aureus strains with various levels of efflux pumps, indicating the ability to null the effect of multiple-drug resistance with SecA inhibitors. Results from studies of drug affinity responsive target stability and protein pull-down assays are consistent with SecA as a target for these compounds.

We have developed a sensitive method to detect the opening of SecA-dependent, protein-conducting channels in Xenopus oocytes. In this study, we determined the ionic current activities of the SecA-dependent channel from membrane vesicles depleted of SecYEG. We found that these SecYEG-depleted membranes produced SecA-dependent ionic currents in the oocytes, as did membranes containing SecYEG. However, reconstituted membranes depleted of SecYEG required higher concentrations of SecA to elicit ionic currents like those in membranes containing SecYEG. In contrast to membranes containing SecYEG, the proofreading capacity of signal peptides was lost for those membranes lacking SecYEG. These findings are consistent with loss of signal peptide specificity in channel activity from membranes of SecY suppressor or SecY plug domain mutants. The signal peptide specificity of the reconstituted membranes, like SecA-liposomes, can be restored by the addition of SecYEG proteoliposomes. On the other hand, the channel activity efficiency of reconstituted membranes was fully restored, while SecA-liposomes could only be partially enhanced by the addition of SecYEG, indicating that, in addition to SecYEG, other membrane proteins contribute to the efficiency of channel activity. The SecA-dependent channels in membranes that lacked SecYEG also lost ion selectivity to monovalent cations but retained selective permeability to large anions. Thus, the electrophysiological evidence presented here indicates that SecYEG is not obligatory for the channel activity of Escherichia coli membranes, as previously shown for protein translocation, and that SecYEG is important for maintenance of the efficiency and specificity of SecA-dependent channels.