Dual Action of Eeyarestatin 24 on Sec-Dependent Protein Secretion and Bacterial DNA

Abstract

Eeyarestatin 24 (ES24) is a promising new antibiotic with broad-spectrum activity. It shares structural similarity with nitrofurantoin (NFT), yet appears to have a distinct and novel mechanism: ES24 was found to inhibit SecYEG-mediated protein transport and membrane insertion in Gram-negative bacteria. However, possible additional targets have not yet been explored. Moreover, its activity was notably better against Gram-positive bacteria, for which its mechanism of action had not yet been investigated. We have used transcriptomic stress response profiling, phenotypic assays, and protein secretion analyses to investigate the mode of action of ES24 in comparison with NFT using the Gram-positive model bacterium Bacillus subtilis and have compared our findings to Gram-negative Escherichia coli. Here, we show the inhibition of Sec-dependent protein secretion in B. subtilis and additionally provide evidence for DNA damage, probably caused by the generation of reactive derivatives of ES24. Interestingly, ES24 caused a gradual dissipation of the membrane potential, which led to delocalization of cytokinetic proteins and subsequent cell elongation in E. coli. However, none of those effects were observed in B. subtilis, thereby suggesting that ES24 displays distinct mechanistic differences with respect to Gram-positive and Gram-negative bacteria. Despite its structural similarity to NFT, ES24 profoundly differed in our phenotypic analysis, which implies that it does not share the NFT mechanism of generalized macromolecule and structural damage. Importantly, ES24 outperformed NFT in vivo in a zebrafish embryo pneumococcal infection model. Our results suggest that ES24 not only inhibits the Sec translocon, but also targets bacterial DNA and, in Gram-negative bacteria, the cell membrane.

Dissecting antibiotic effects on the cell envelope using bacterial cytological profiling: a phenotypic analysis starter kit

“…S11). Tetracycline has been shown to have secondary effects on the cell membrane ( 42 ), and nitrofurantoin may cause lipid peroxidation ( 33 ), possibly explaining these effects. Yet, why chloramphenicol elicits Nile red foci is unclear.…”

“…This selection comprised two additional membrane-active compounds, the Na + /K + channel ionophore gramicidin (gramicidin D = a mix of gramicidin A–C) and the H + carrier ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and two additional cell wall synthesis inhibitors: D-cycloserine, inhibiting both alanine racemase and D-ala-D-ala ligase, and tunicamycin, inhibiting the lipid I synthase MraY. Additionally, we selected six compounds with targets unrelated to the cell envelope, namely, the gyrase/topoisomerase IV inhibitor ciprofloxacin, the RNA polymerase inhibitor rifampicin, the ribosome inhibitors tetracycline, kanamycin, and chloramphenicol, and the pro-drug nitrofurantoin, which generates reactive species that damage cellular macromolecules ( 33 ).…”

“…We examined the effects of these antibiotics in different phenotypic assays, focusing on techniques that are easy to implement and execute, do not require highly specialized instrumentation, and can be interpreted without excessive knowledge of bacterial cell biology. To this end, we evaluated a slightly modified BCP assay as well as methods to assess membrane potential, membrane fluidity, and cell wall synthesis based on our previous experiences with in-depth mode of action analysis of cell envelope-targeting antibiotics ( 2 , 3 , 16 – 19 , 33 , 34 ). We chose Bacillus subtilis as a model since it is already a popular model for antibiotic mode of action studies ( 19 , 35 – 39 ), can be used in biosafety level 1 labs, is easy and inexpensive to handle, and is susceptible to most antibiotics.…”

Involvement of the Bacillus SecYEG Pathway in Biosurfactant Production and Biofilm Formation

“…This is why Rose Bengal was used to drive this understanding and to demonstrate the link between SecYEG and biosurfactant secretion and biofilm formation. Rose Bengal and Eeyarestatin 24 (ES24) are known to inhibit SecYEG-mediated protein transport [ 24 , 25 , 42 ]. According to our understanding, Rose Bengal might interact with SecA or with SecDF [ 5 , 10 ].…”