Synergistic antibacterial and anti-inflammatory activity of temporin A and modified temporin B in vivo

Abstract

Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria). They are 10-14 amino acid long polypeptides active prevalently against gram positive bacteria. This study shows that a synthetic temporin B analogue (TB-YK), acquires the capacity to act in synergism with temporin A and to exert antimicrobial and anti-inflammatory activity in vivo against gram positive and gram negative bacteria. Administration of 3.4 mg/Kg of temporin A (TA)+1.6 mg/Kg TB-YK, given to individual mice concurrently with a lethal dose of bacteria (gram positive or negative), rescued 100% of the animals. More importantly, the same doses of temporins, administered one week after experimental infection with a sub lethal dose of bacteria, sterilized 100% of the animals within 3-6 days. Also, it is described an animal model based on the use of sub lethal doses of bacteria, which closely mimics bacterial infection in humans. The model offers the possibility to test in a preclinical setting the true potential of TA and TB-YK in combination as antimicrobial and anti-inflammatory agents.

Figure 1. CD spectra of: TB (blue, continuous line), TB-YKβA (red continuous line) in Sodium Phosphate buffer pH 7.4 (-); TB (blue dashed line) and TB-YKβA (red dashed line) in SodiumPhosphate buffer+SDS pH 7.4.

Figure 2. Temporins initiate their antibacterial activity by drillings holes in the bacterial membrane.

A: S.aureus A170, untreated; B: S aureus A 170 treated for 10 min with TA (8 µg/ml) plus TB-YK (5 µg/ml) (B); C: S.enterica serovar Paratyphi B untreated; D: S.enterica serovar Paratyphi B treated for 10 min with TA (100 µg/ml) plusTB-YK (4 µg/ml).

Figure 3. Detection of total, viable and membrane-perturbed bacteria using temporin A or temporin B labeled with rhodamine isothiocyanate or fluorescein isothiocyanate respectively.

A- B-C: S.aureus A 170 plus TA-Rho 6 µg plus TB-Fluo 9 µg; D-E-F: S.aureus A170 plus fluorescein isothiocyanate o rhodamine isothiocyanate (control); G-H-I: S.enterica serovar Paratyphi B plus TA-Rho 15 µg plus TB-Fluo 20 µg; L-M-N: S.enterica serovar Paratyphi B plus fluorescein isothiocyanate o rhodamine isothiocyanate (control).

Figure 4. Synergism between TA and TB-YK temporins.

The diameter of the inibition was maesured after 24 h incubation.

Figure 5. In vivo antibacterial activity of temporins.

The 35/75 pool of temporins, given concurrently with bacteria, rescue 100% of mice infected with a lethal dose of S. aureus A170 10⁷ CFU/mouse (A: mice infected with S.aureus A170 and immediately treated with TA plusTB-YK (closed box) and mice infected with S.aureus A170 (open triangle) or a lethal dose of S. enterica serovar Paratyphi 10⁶ CFU/mouse (B: Mice infected with S.enterica serovar Paratyphi B and immediately treated with TA plus TB-YK (closed box) and mice infected with S.enterica serovar Paratyphi B (open triangle)

Figure 6. Temporins sterilize mice sperimentally infected with S aureus A170 or S.enterica serovar Paratyphi B.

The 35/75 pool of temporins, given concurrently with bacteria, sterilize mice infected with a lethal dose (10⁷ CFU/mouse) of S.aureus A170 (A: kidneys from mice infected with S. aureus A170 (closed rhomb) and kidneys from mice infected with S.aureus A170 and immediately after treated with TA plus TB-YK (open box), or a lethal dose (10⁶ CFU/mouse) of S.enterica serovar Paratyphi B (B: gastro instestinal tract from mice infected with S.enterica serovar Paratyphi B (closed rhomb) and gastro intestinal tract from mice infected with S.enterica serovar Paratyphi B and immediately treated with TA plus TB-YK (open box); (C: liver from mice infected with S.enterica serovar Paratyphi B (closed rhomb) and liver from mice infected with S.enterica serovar Paratyphi B and immediately treated with TA plus TB-YK (open box).

Figure 7. Temporins are effective in vivo when given 7 days after experimental infection.

Mice were infected with a sub-lethal dose (10⁵ CFU/mouse) of S.aureus A170 (A) or a sub-lethal dose(10⁵ CFU/mouse) of S.enterica serovar Paratyphi B (B–C) and 6 days later were treated with the 35/75 pool of temporins. A: kidneys from mice infected with S.aureus A170 (closed box) and kidneys from mice infected with S.aureus A170 and treated after 6 days with TA plus TB-YK (open rhomb); B: gastro instestinal tract from mice infected with S.enterica serovar Paratyphi B (closed box ) and gastro intestinal tract from mice infected with S.enterica serovar Paratyphi B and treated after 6 days with TA plus TB-YK (open rhomb); C: liver from mice infected with S.enterica serovar Paratyphi B (10⁵ CFU/mouse) (closed box) and liver from mice infected with S.enterica serovar Paratyphi B (10⁵ CFU/mouse) and treated after 6 days with the TA plus TB-YK (open rhomb).

Figure 8. Expression levels of cytokine genes in mice infected with S.enterica serovar Paratyphi B (10⁶ CFU/mouse) (In) or infected with S.enterica serovar Paratyphi B (10⁶ CFU/mouse) and immediately after treated with the 35/75 pool of temporins (Tr).

Figure 9. Expression levels of cytokine genes in mice infected with S.enterica serovar Paratyphi B (10⁵ CFU/mouse) and 6 days lose treated with temporins (35/75 pool).

Each hystogram represents the mean value measured in 2 mice, each one tested in triplicate.

Figure 10. Mice infected with GFP-labelled bacteria were analysed using the Leica macrofluo instrument (Wetzlar, Germany) equipped with the Leica application suite 3.1.0 software.

A: liver from mice infected with S. enterica serovar Paratyphi B; B: liver from mice infected with S. enterica serovar Paratyphi B and immediately treated with TA plus TB-YK; C: gastro instestinal tract from mice infected with S. enterica serovar Paratyphi B; D: gastro instestinal tract from mice infected with S. enterica serovar Paratyphi B and immediately treated with TA plus TB.