Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities

Abstract

SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF•YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the Translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg2+, mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes.

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

“…We determined the functional activities of SecA purified from Gram-negative E. coli (EcSecA), P. aeruginosa (PaSecA) and S. typhymurium (StSecA) in the presence of these Class C inhibitors in comparison with published RB [26] or SCA-50. [13] Both the channel activity and protein translocation in the SecA-only liposomes systems were strongly inhibited by these compounds with IC 50 values in the range of 1.0–3.5 μM for SCA-107 and SCA112 (Table 5A, B).…”

Inhibitors of protein translocation across membranes of the secretory pathway: novel antimicrobial and anticancer agents

“…These compounds are predicted to occupy the ATP binding site in SecA [ 102 ]. Inhibition of ATPase activity was indeed competitive at low ATP concentrations, however, more recent data shows that rose bengal non-competitively inhibits the translocation activity of SecA at high ATP concentrations [ 103 ]. They have both bacteriostatic and bactericidal effects.…”

Dark Antibacterial Activity of Rose Bengal

“…However, at high concentrations they are known to possess a certain dark activity and to inactivate cells in the dark. The dark activity of Rose Bengal on several bacteria was described by Bond et al [31], Hsieh et al [32], Shrestha et al [33], Coulson et al [34], Nakoniecna et al [35], and Goulart et al [36], although the reason for this phenomenom is not clear [37]. In the present work we examined the dark activity of Rose Bengal alone and in the presence of several antibiotics against S. aureus by detection of minimum inhibitory concentrations (MIC).…”