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

Capparelli R, Romanelli A, Iannaccone M, Nocerino N, Ripa R, Pensato S, Pedone C, Iannelli D - PLoS ONE (2009)

Bottom Line: Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria).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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Soil, Plant, Environment and Animal Production Sciences, University of Naples Federico II, School of Biotechnological Sciences, Napoli, Italy. capparel@unina.it

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.

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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).
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pone-0007191-g002: 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).

Mentions: The study examined the antimicrobial activity of the synthetic temporins A and B in their native forms, and of the temporin B following chemical modifications (Table 1); the latter temporin was studied alone and in combination with native temporin A. The native temporin A was more active against gram positive (MIC: 10–30 µg/ml) than against gram negative (MIC: 125–400 µg/ml) bacteria (Table 2); the native temporin B and TB-Y were active against gram positive bacteria (approximately to the same extent as temporin A), but totally inactive against gram negative bacteria (data not shown). Also, tested in different ratios, the two native temporins did not display synergism. Instead TB-YK exhibited antimicrobial activity either against gram positive and gram negative bacteria (Table 2). Remarkably, when modified TB-YK (at 4–8 µg/ml) was used in combination with the native temporin A (at 5–100 µg/ml), a strong synergism was observed against gram positive as well as against gram positive bacteria (Table 2). Electron microscopy showed the ability of the combination of TA and TB-YK to perforate the bacterial membrane (Figure 2). The labelled peptides, TA-Rho and TB-Fluo, were employed in fluorescence light microscopy experiments. Several combinations of the peptides were tested; the combinations which allowed for a better visualization of the bacteria were 6 µg TA+9 µg TB-YK on S.aureus 170 and 15 µg TA+20 µg TB-YK on S.enterica Paratyphi. The end product of temporins' activity, bacterial fragmentation, is illustrated in Figure 3. An independent experimental approach, the inhibition zone test, confirmed the synergism between the two temporins (Figure 4).


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

Capparelli R, Romanelli A, Iannaccone M, Nocerino N, Ripa R, Pensato S, Pedone C, Iannelli D - PLoS ONE (2009)

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).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747021&req=5

pone-0007191-g002: 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).
Mentions: The study examined the antimicrobial activity of the synthetic temporins A and B in their native forms, and of the temporin B following chemical modifications (Table 1); the latter temporin was studied alone and in combination with native temporin A. The native temporin A was more active against gram positive (MIC: 10–30 µg/ml) than against gram negative (MIC: 125–400 µg/ml) bacteria (Table 2); the native temporin B and TB-Y were active against gram positive bacteria (approximately to the same extent as temporin A), but totally inactive against gram negative bacteria (data not shown). Also, tested in different ratios, the two native temporins did not display synergism. Instead TB-YK exhibited antimicrobial activity either against gram positive and gram negative bacteria (Table 2). Remarkably, when modified TB-YK (at 4–8 µg/ml) was used in combination with the native temporin A (at 5–100 µg/ml), a strong synergism was observed against gram positive as well as against gram positive bacteria (Table 2). Electron microscopy showed the ability of the combination of TA and TB-YK to perforate the bacterial membrane (Figure 2). The labelled peptides, TA-Rho and TB-Fluo, were employed in fluorescence light microscopy experiments. Several combinations of the peptides were tested; the combinations which allowed for a better visualization of the bacteria were 6 µg TA+9 µg TB-YK on S.aureus 170 and 15 µg TA+20 µg TB-YK on S.enterica Paratyphi. The end product of temporins' activity, bacterial fragmentation, is illustrated in Figure 3. An independent experimental approach, the inhibition zone test, confirmed the synergism between the two temporins (Figure 4).

Bottom Line: Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria).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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Soil, Plant, Environment and Animal Production Sciences, University of Naples Federico II, School of Biotechnological Sciences, Napoli, Italy. capparel@unina.it

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.

Show MeSH
Related in: MedlinePlus