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Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria

Institute of Infectious Diseases and Public Health, University of Ancona, Ancona, Italy,¹ Department of General Surgery, I.N.R.C.A. I.R.R.C.S., University of Ancona, Ancona, Italy,² Biotechnology Centre, Research Department, I.N.R.C.A. I.R.R.C.S., Ancona, Italy,³ Department of Biomedical Sciences and Technologies, University of L'Aquila, L'Aquila, Italy,⁴ Department of Biochemical Sciences "A. Rossi Fanelli," S. Andrea Hospital, and CNR Molecular Biology Centre, University of Rome "La Sapienza," Rome, Italy,⁵ Department of Biomedical Sciences and Technologies, University of Cagliari, Monserrato (CA), Italy⁶

Received 6 December 2005/ Returned for modification 12 February 2006/ Accepted 8 April 2006

Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used ß-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and ß-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF- levels, resulting in the highest survival rates.

We dedicate this work to the memory of Prof. Augusto Rinaldi, colleague, friend, and guide in life, who prematurely passed away on 7 October 2005.

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