Microcin E492 antibacterial activity: evidence for a TonB‐dependent inner membrane permeabilization on Escherichia coli

Abstract

SummaryThe mechanism of action of microcin E492 (MccE492) was investigated for the first time in live bacteria. MccE492 was expressed and purified to homogeneity through an optimized large-scale procedure. Highly purified MccE492 showed potent antibacterial activity at minimal inhibitory concentrations in the range of 0.02-1.2 m m m m M. The microcin bactericidal spectrum of activity was found to be restricted to Enterobacteriaceae and specifically directed against Escherichia and Salmonella species. Isogenic bacteria that possessed mutations in membrane proteins, particularly of the TonB-ExbB-ExbD complex, were assayed. The microcin bactericidal activity was shown to be TonBand energy-dependent, supporting the hypothesis that the mechanism of action is receptor mediated. In addition, MccE492 depolarized and permeabilized the E. coli cytoplasmic membrane. The membrane depolarization was TonB dependent. From this study, we propose that MccE492 is recognized by ironsiderophore receptors, including FepA, which promote its import across the outer membrane via a TonB-and energy-dependent pathway. MccE492 then inserts into the inner membrane, whereupon the potential becomes destabilized by pore formation. Because cytoplasmic membrane permeabilization of MccE492 occurs beneath the threshold of the bactericidal concentration and does not result in cell lysis, the cytoplasmic membrane is not hypothesized to be the sole target of MccE492.

Microcin Amyloid Fibrils A Are Reservoir of Toxic Oligomeric Species

“…This data suggest that the process of amyloid formation is highly dynamic and that biologically inert Mcc fibrils can be a reservoir of toxic soluble oligomeric structures. Although the specific Mcc conformation that is responsible for cytotoxicity is still unclear, several lines of evidence indicate that Mcc exert its toxicity by forming pores on the bacterial cell membrane (6,8), which requires the oligomerization of Mcc monomers. Strikingly, the current view in protein misfolding diseases is that the toxic species might be oligomeric pore-like structures that may cause cell damage by permeabilizing membranes and altering cellular homeostasis (15,35,36).…”

“…Antibacterial activity of Mcc appeared to be restricted to the Enterobacteriaceae species, closely related to the producer strain that directly competes with Klebsiella to occupy a spatial niche in the ecosystem (5,7). Mcc has been shown to exert its toxic effect by forming ion channels on the cell membrane of target cells in a receptor-mediated fashion, which leads to a rapid depolarization and thus permeabilization of the cell membrane (6,8,9). Unlike other bacteriocins, toxic forms of Mcc are produced mainly in the exponential phase and comparatively less toxic in the stationary phase (5,10).…”

The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

“…Bioassays were performed with the bacterial strains described previously [21,22], as well as with S. enterica Paratyphi SL369 [23], S. enterica Typhimurium LT2 [23], P. agglomerans K4 [24] (generously provided by Professor Volkmar Braun, University of Tübingen, Germany) and Pseudomonas aeruginosa A.T.C.C. 27853 (a gift from Professor Alain Reynaud, Hospital of Nantes, France).…”

“…MccJ25 and FhuA were purified as described previously [2,28] and quantified by amino acid composition analysis as in [21] , and peptide quantification was performed by amino acid composition analysis as described previously [29]. Peptide antibacterial activity was finally controlled according to the protocol described below.…”

“…Peptide antibacterial activity was assayed using the liquid growth inhibition assay described previously [21]. Briefly, serial dilutions of MccJ25 or t-MccJ25 (10 µl) were incubated in a 96-well microtitre plate with 90 µl of a mid-exponential phase culture of bacteria at a starting attenuance of 0.001 at 620 nm in Poor-Broth nutrient medium (1 % bactotryptone and 0.5 % NaCl).…”