Fluorescein analogues inhibit SecA ATPase: the first sub-micromolar inhibitor of bacterial protein translocation

Abstract

SecA is a central component of the general secretion system that is essential for bacterial growth and thus an ideal target for antimicrobial agents. A series of fluorescein analogues were first screened against the ATPase activity using the truncated unregulated SecA catalytic domain. Rose bengal (RB) and erythrosin B (EB) were found to be potent inhibitors SecA with IC(50) values of 0.5 μM and 2 μM, respectively. RB and EB inhibit the catalytic SecA ATPase more effectively than the F(1) F(0) -proton ATPase. We used three assays to test the effect of these compounds on full-length SecA ATPase: in solution (intrinsic ATPase), in membrane preparation, and translocation ATPase. RB and EB show the following trend in terms of IC(50) values: translocation ATPase<membrane ATPase<intrinsic ATPase. Very importantly, the potency of these fluorescein analogues in inhibiting the truncated SecA ATPase correlates with their ability to inhibit the biologically relevant protein translocation activity of SecA. The in vitro translocation of proOmpA precursors into membrane vesicles is strongly inhibited by RB with IC(50) values of approximately 0.25 μM, making RB the most potent inhibitor of SecA ATPase and SecA-dependent protein translocation reported thus far. The ability of these compounds to inhibit SecA also directly translates into antibacterial effects. Our findings show the value of fluorescein analogues as probes for mechanistic studies of SecA functions and for the potential development of new antimicrobial agents with SecA as the target.

Figure 1

The chemical structures of DI, RB, EB, and CJ-21058.

Figure 2. The inhibitory effect of RB and EB against different ATPases

ATPase activities of the catalytic domain of SecA (EcN68) and the F₁F₀-proton ATPase were assayed with different concentrations of RB and EB. The inhibitory effects were illustrated by the percentage (%) of remaining ATPase activity as compared to the controls in the absence of inhibitors.

Figure 3. The inhibitory effects of RB and EB against the SecA-dependent in vitro translocation of proOmpA

The translocation of proOmpAprecursors into membrane vesicles was assayed in the presence of RB and EB. The insert is the expanded presentation for RB. The inhibitory effects were illustrated by the percentage (%) of translocated proteins as compared to the controls in the absence of inhibitors. The results were presented as line graphs with standard error of the mean.

Figure 4. The bactericidal effect of RB on Gram-positive and Gram-negative bacteria

The bactericidal activities are illustrated by the number of surviving cells as CFU after one hour treatment with various concentrations of inhibitors (gray bar) as compared to the controls (white bar) in the absence of inhibitors.(A)Permeability leaky mutant E. coli NR698; (B) B. subtilis 168.

Figure 5. The docking conformations of DI, EB, RB and CJ-21058 at the EcSecA ATP-site