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

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Abstract

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 of Salmonella enterica ATCC 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 of S. enterica cells; 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 of S. enterica are 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.

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About the authors

A. E. Grigor’eva

Institute of Chemical Biology and Biochemistry of the Siberian Branch of the Russian Academy of Sciences

Email: lenryab@yandex.ru
Russian Federation, Novosibirsk, 630090

A. V. Tupitsyna

Institute of Chemical Biology and Biochemistry of the Siberian Branch of the Russian Academy of Sciences

Email: lenryab@yandex.ru
Russian Federation, Novosibirsk, 630090

E. S. Ryabova

Institute of Chemical Biology and Biochemistry of the Siberian Branch of the Russian Academy of Sciences

Email: lenryab@yandex.ru
Russian Federation, Novosibirsk, 630090

A. V. Bardasheva

Institute of Chemical Biology and Biochemistry of the Siberian Branch of the Russian Academy of Sciences

Email: lenryab@yandex.ru
Russian Federation, Novosibirsk, 630090

D. A. Zadvornykh

Institute of Chemical Biology and Biochemistry of the Siberian Branch of the Russian Academy of Sciences

Email: lenryab@yandex.ru
Russian Federation, Novosibirsk, 630090

L. S. Koroleva

Institute of Chemical Biology and Biochemistry of the Siberian Branch of the Russian Academy of Sciences

Email: lenryab@yandex.ru
Russian Federation, Novosibirsk, 630090

V. N. Silnikov

Institute of Chemical Biology and Biochemistry of the Siberian Branch of the Russian Academy of Sciences

Email: lenryab@yandex.ru
Russian Federation, Novosibirsk, 630090

E. I. Ryabchikova

Institute of Chemical Biology and Biochemistry of the Siberian Branch of the Russian Academy of Sciences

Author for correspondence.
Email: lenryab@yandex.ru
Russian Federation, Novosibirsk, 630090

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2. Fig. 1. Chemical structure of the cationic amphiphile DL412 (a, D — DABCO (1,4-diazabicyclo[2.2.2]octane), L4 — tetramethylene linker, 12 — dodecyl residue) and ciprofloxacin (b).

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3. Fig. 2. Changes in the ultrastructure of S. enterica cells in ultrathin sections in TEM after exposure to DL412: a, b — homogeneous substance of medium electron density in the periplasmic space; c–d — penetration of homogeneous substance into the cytoplasm and formation of inclusions; f–j — morphological variants of inclusions; l — membrane structure of high electron density, an enlarged portion of the structure is shown in the frame. Black arrows show the outer membrane of the cell wall; white thin arrows — the inner membrane; white thick arrows — homogeneous substance in the periplasmic space; dotted arrows show the connection between the homogeneous substance in the periplasmic space and in the cytoplasm; # — cytoplasm; asterisk — homogeneous substance as part of inclusions. Incubation time with DL412 compound is 15 min; a–f, i, l — fixation according to the Reiter-Kellenberger method; g–h, j — fixation with formaldehyde. Scale: 100 nm.

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4. Fig. 3. Cytoplasm of S. enterica cells in ultrathin sections in TEM: a – intact cells; b – 15 min of incubation with DL412; c – 15 min of incubation with ciprofloxacin. Ribosomes are shown by arrows in images a and c, in b – are not visualized. Fixation with formaldehyde. Scale: 100 nm.

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5. Fig. 4. TEM images of S. enterica cells on ultrathin sections after 15 min of incubation with DL412 (a–c) and ciprofloxacin (d–f); g–i — intact cells. a–c — changes in the nucleoid (shown with white arrows), black arrows indicate DNA strands; asterisk — cells with eccentric location of the nucleus. Inset — bundles of thickened DNA strands (b), “filling” substance under them; d, f — disruption of the nucleoid structure, DNA strands are not visible; g, h — intact cells with a clear nucleoid of irregular shape. Fixation with formaldehyde. Scale: 2 µm (a, d, g), 500 nm (b, e, h) 200 nm (c, f, i), 100 nm (inset).

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6. Fig. 5. Images of S. enterica cells. (a, g) — 15 min. of incubation with DL412; (b, d) — ciprofloxacin; (c, e) — intact cells: a–e — changes in the nucleoid (shown by thick white arrows). Black arrows show indentations of the bacterial membrane at the points of contact between the nucleoid and the inner membrane. Asterisk — cells with the nucleoid adjacent to the inner membrane. g — clear disorganized bundles of DNA strands. Thin white arrows show individual DNA strands; black arrow — outer membrane of the cell membrane; arrowhead — inner membrane. d — short fragments of DNA strands after incubation with ciprofloxacin. e — in intact cells, thin DNA strands are shown by a white arrow; black arrow — outer membrane; arrowhead — inner membrane. Fixation according to Reiter-Kellenberger. Scale: a–c — 200 nm, g, d, e — 100 nm.

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