Ribosome disorganization and other effects of artificial RNase DL412 on Salmonella enterica cells

Cationic amphiphile DL412, which has RNase activity (D — DABCO (1,4-diazabicyclo[2.2.2]octane); L4 — tetramethylene linker; 12 —dodecyl residue), was synthesized at the ICBFM SB RAS, and showed pronounced antibacterial properties. A suspension ofSalmonella entericaATCC 14028 cells was incubated with DL412 (5 µM) for 15 and 30 min, or with ciprofloxacin (5 µM, reference compound). Intact cells served as controls. Samples were fixed with formaldehyde (4%, postfixed with 1% OsO4), or by the Reiter-Kellenberger method (1% OsO4, postfixed with 0.5% uranyl acetate), dehydrated and embedded into an Epon-Araldite mixture. Ultrathin sections were examined using an electron microscope Jem 1400 (“Jeol”, Japan). Within 15 min of incubation with compound DL412, visible ribosomes disappeared throughout the cytoplasm ofS. entericacells; In the periplasmic space, a homogeneous substance of average electron density was observed, its penetration into the cytoplasm was noted, in which polymorphic inclusions appeared. The ultrastructure of the nucleoids was significantly disrupted; they became rounded, and the DNA strands “stick together” into bundles. The ultrastructure of the outer membrane remained unchanged. The observed changes in the structure ofS. entericaare due to a combination of RNase activity and amphiphilic properties of DL412 and did not differ depending on the fixation method. Such changes were not described in any publication. Our study made it possible for the first time to visualize the influence of RNase activity and the amphiphilic component of the compound DL412, which penetrated into the cell through two bacterial membranes without their visible damage.