Chemical Genomics, Structure Elucidation, and in Vivo Studies of the Marine-Derived Anticlostridial Ecteinamycin

Abstract

A polyether antibiotic, ecteinamycin (1), was isolated from a marine Actinomadura sp., cultivated from the ascidian Ecteinascidia turbinata. ¹³C enrichment, high resolution NMR spectroscopy, and molecular modeling enabled elucidation of the structure of 1, which was validated on the basis of comparisons with its recently reported crystal structure. Importantly, ecteinamycin demonstrated potent activity against the toxigenic strain of Clostridium difficile NAP1/B1/027 (MIC = 59 ng/μL), as well as other toxigenic and nontoxigenic C. difficile isolates both in vitro and in vivo. Additionally, chemical genomics studies using Escherichia coli barcoded deletion mutants led to the identification of sensitive mutants such as trkA and kdpD involved in potassium cation transport and homeostasis supporting a mechanistic proposal that ecteinamycin acts as an ionophore antibiotic. This is the first antibacterial agent whose mechanism of action has been studied using E. coli chemical genomics. On the basis of these data, we propose ecteinamycin as an ionophore antibiotic that causes C. difficile detoxification and cell death via potassium transport dysregulation.

Figure 1

Polyether ionophores sharing the characteristic acyltetramic acid moiety. Ecteinamycin (1), tetronomycin (2), and tetronasin (3).

Figure 2

Membrane depolarization assay. Flow cytometry-based analysis of MSSA cells treated with DiOC₂(3) dye control (A), ecteinamycin (B), and CCCP (C). (D) The ratio of red to green fluorescence for MSSA cells treated with DiOC₂(3) dye and the corresponding compound. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control. CCCP is a protonophore that disrupts membrane potential.^,

Figure 3

Chemical genomic profile of ecteinamycin. The chemical genomic analysis was performed in triplicate at [1] = 62.5 μg/mL. In green, E. coli mutants deficient in sspA, rseB, cusC, and other genes proved resistant to the actions of 1. In red, E. coli sensitive to 1 are deficient in trkA, sapD, kdpD, and other indicated genes (Table 2).

Figure 4

Effect of 1 on C. difficile challenged mice. Experimental groups: healthy control (uninfected), C. difficile control, and group treated with 30 ng of ecteinamycin. Health scores from 27–48 h were averaged for each experimental group.

Figure 5

C. difficile clearance. Feces samples collected at seven different time points were diluted in PBS and plated onto selective C. difficile-Brucella agar, incubated anaerobically for 48 h, and subsequently enumerated. For healthy controls at all time points, as well as the others at time points 0 and 12 h, the CFU/g was zero, given that the limit of detection of the method is <3.00 × 10⁴ (log₁₀ = 4.4771).