Ribosome Disorganization and Other Effects of Artifitial RNAse DL412 on Salmonella enterica Cells

Abstract The cationic amphiphile DL 4 12, has been synthesized, possessing ribonuclease activity (D—DABCO (1,4-diazabicyclo[2.2.2]octane); L 4 is a tetramethylene linker; 12 is a dodecyl residue) and pronounced antibacterial properties. A suspension of Salmonella enterica ATCC 14028 cells was incubated in 5 μM DL 4 12 solution for 15 and 30 min or 5 μM ciprofloxacin solution (comparison drug); intact cells served as a control. Samples were fixed with 4% formaldehyde and 1% OsO 4 or by the Ryter-Kellenberger method with 1% OsO 4 post-fixed with 0.5% uranyl acetate, then dehydrated and embedded in an epoxy resin mixture. The obtained ultrathin sections were examined in a Jem 1400 electron microscope. After 15 min of incubation with the DL 4 12 compound, visible ribosomes disappeared from the entire cytoplasm of S. enterica cells. A homogeneous substance of medium electron density appeared in the periplasmic space, capable of penetrating into the cytoplasm, in which polymorphic inclusions were formed. Pronounced ultrastructural changes were observed in the nucleoids of the bacteria: they were rounded, and the DNA strands “glued” together into bundles. The structure of the outer membrane has not change. The observed changes in the structure of S. enterica were similar when fixed by different methods and were due to a combination of ribonuclease activity and the amphiphilic properties of DL 4 12. The ultrastructural changes we identified have not been described in the scientific literature. This study is the first to visualize the effects of ribonuclease and the amphiphilic component of the DL 4 12 compound in bacterial cells.