Apomorphine Targets the Pleiotropic Bacterial Regulator Hfq

Abstract

Hfq is a bacterial regulator with key roles in gene expression. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, thanks to its binding to small regulatory noncoding RNAs. This property is of primary importance for bacterial adaptation and survival in hosts. Small RNAs and Hfq are, for instance, involved in the response to antibiotics. Previous work has shown that the E. coli Hfq C-terminal region (Hfq-CTR) self-assembles into an amyloid structure. It was also demonstrated that the green tea compound EpiGallo Catechin Gallate (EGCG) binds to Hfq-CTR amyloid fibrils and remodels them into nonamyloid structures. Thus, compounds that target the amyloid region of Hfq may be used as antibacterial agents. Here, we show that another compound that inhibits amyloid formation, apomorphine, may also serve as a new antibacterial. Our results provide an alternative in order to repurpose apomorphine, commonly used in the treatment of Parkinson's disease, as an antibiotic to block bacterial adaptation to treat infections.

Keywords: Hfq; antibacterial compound; apomorphine; bacterial adaptation; bacterial amyloid; functional amyloid; protein fibrillation inhibition; protein fibrils.

Figure 1

TEM visualization of the effect of apomorphine on CTR fibrils. (a) preassembled fibrils of CTR (control). More than 100 fibers can be observed; (b) preassembled CTR incubated with 5 mM apomorphine; Incubation time 24 h. Here, only tens of fibers are still present. (c) apomorphine was added before the fibrils’ formation, and almost no fibers can be observed in this case. Scale bars, 100 nm.

Figure 2

TEM visualization of the effect of apomorphine of Hfq-CTR bound to DNA. (a) Hfq-CTR:DNA without apomorphine (control); (b) Hfq-CTR:DNA with apomorphine. Only a few pieces of fibrils remain on the grid. Scale bars, 1 µm.

Figure 3

Disassembly of the Hfq-CTR amyloid structure by apomorphine between 0 and 15 h (blue to yellow). (a) The decrease in the signal at 215 nm, characteristic of an amyloid structure [32,35], is observed upon the addition of apomorphine. This proves that the amyloid cross-β structure is disrupted by the compound. (b) Kinetics of disassembly. Ln of the absolute value of CD at 215 nm vs. time. In our case [Apomorphin] >> [Hfq-CTR_n] (Hfq-CTR_n = Hfq-CTR fibers) and CD₂₁₅ = CD⁰_215.e^−kt. An apparent dissociation constant k = 7.8 10⁻⁵ min⁻¹ can be measured. CD⁰₂₁₅ is the absolute value of CD at 215 nm and at t = 0 min.

Figure 4

Box plots for C₅₀ apomorphine concentrations (in mM) for MG1655 WT, MG1655-∆hfq and MG1655-hfq72 strains. All three strains had different medians (All p-values for all pairwise comparisons were smaller than 2 × 10⁻⁵).