Tesis doctoral de Iosu Razquin Olazarán
Pseudomonas aeruginosa is a gram-negative pathogen that frequently causes life-threatening infections in immunocompromised patients. The increasing isolation of p. Aeruginosa strains highly resistant to most of the available antibiotics greatly limits the therapeutic options and underscores the need to develop antimicrobial agents with novel mechanisms of action. We previously showed that subinhibitory concentrations of certain cationic peptides derived from human lactoferricin permeabilize the cell envelope of p. Aeruginosa and greatly enhance the lethal action of co-administered antibiotics. This phenomenon depends on the peptide binding to p. Aeruginosa lipopolysaccharide (lps; endotoxin), a component of the bacterial envelope whose release during infection triggers an often fatal pathology called sepsis. We hypothesized that a structure-activity analysis of our current peptide library could provide peptides with higher antibiotic enhancing activity and the ability to neutralize lps and consequently to inhibit sepsis. Ideally, peptides could also cause postantibiotic effect (pae; i.E. An inhibition of bacterial growth caused by peptide treatment that persists after peptide removal). our structure- activity analysis used a new class of constitutional and sequence-dependent descriptors called pedes (peptide descriptors from sequence) and was based on partial least-squares (pls) regression. Although this rational optimization process could not predict accurately the permeabilizing activity of the new peptides (n=55), most of these compounds had improved antibiotic potentiating activity against a multiresistant p. Aeruginosa clinical isolate. Moreover, most of the new peptides showed an enhanced affinity to p. Aeruginosa lps in vitro, high ability to inhibit tnf-alpha release from human mononuclear cells in vitro and low cytotoxic and hemolytic activity. Finally, the combined administration of peptide p4-1 (2 doses of 17,5 mg/kg) and azithromycin (1 dose of 25 mg/kg) conferred long lasting protection to 25% of mice against lethal sepsis caused by intraperitoneal inoculation of p. Aeruginosa. in general, antimicrobial activity correlated with molecular weight, positive net charge and number of cationic residues. In contrast, a good permeabilizing activity was associated with shorter peptides, with fewer tryptophan residues, a moderate hydrophobic moment and an angle of the hydrophobic domain no greater than 200 °. we demonstrated that treatment of a p. Aeruginosa culture with p4-9 peptide at twice its minimum inhibitory concentration (mic) causes a growth delay (pae) of 1,4 hours after peptide removal. Interestingly, when the treated culture was allowed to grow in the absence of the peptide, cells remained sensitive to subinhibitory concentrations of antibiotics such as novobiocin, fosfomycin and ceftazidime during at least two hours. This long-term sensitization to antibiotics has not been described before and we have designated it as postantibiotic effect associated permeabilization (paep). Using atomic force microscopy, we demonstrated that p4-9 causes profound alterations in the bacterial cell surface and the cells need at least two hours of growth to repair those lesions. We showed that p4-9 renders p. Aeruginosa mutants overexpressing either the efflux pump mexab-oprm system or ampc β-lactamase sensitive to antibiotics that are known substrates of these resistance systems during the paep period. This phenomenon has important therapeutical implications for a combined peptide-antibiotic treatment because one of the components (the peptide) would not need to be present to exert its antibiotic enhancing activity.
Datos académicos de la tesis doctoral «Diseño racional de péptidos derivados de la lactoferricina humana con actividad permeabilizante mejorada y capacidad de producir permeabilidad asociada al efecto postantibiótico (paep)«
- Título de la tesis: Diseño racional de péptidos derivados de la lactoferricina humana con actividad permeabilizante mejorada y capacidad de producir permeabilidad asociada al efecto postantibiótico (paep)
- Autor: Iosu Razquin Olazarán
- Universidad: Navarra
- Fecha de lectura de la tesis: 29/05/2012
Dirección y tribunal
- Director de la tesis
- Guillermo Martínez De Tejada De Garaizábal
- Tribunal
- Presidente del tribunal: ignacio Moriyon uria
- carmen Ezpeleta baquedano (vocal)
- Antonio Oliver palomo (vocal)
- José Antonio Bengoechea alonso (vocal)