INVESTIGADORES
DUPUY Fernando Gabriel
congresos y reuniones científicas
Título:
Fluorescent labeled microcin incorporation in living cells
Autor/es:
DUPUY, FERNANDO G.; NIKLISON CHIROU, MARÍA V.; FERNÁNDEZ DE ARCURI, BEATRIZ; BELLOMIO, AUGUSTO AND MORERO, ROBERTO D.
Lugar:
Montevideo, Uruguay
Reunión:
Congreso; XXXVI Reunión Anual de la Sociedad Argentina de Biofisica; 2007
Institución organizadora:
Sociedad Argentina de Biofisica
Resumen:
Microcin J25 is a
plasmid encoded 21 aminoacid antibiotic peptide with a distinctive lasso-structure
produced by Escherichia coli and
active against Escherichia coli, Salmonella enteritidis serovars and Shigela strains. It has been shown that the target in E. coli
is the b subunit
of the RNA polymerase. However, its also known that in Salmonella newport cells MccJ25 inhibits
the plasmatic membranes respiratory enzymes, suggesting a dual mechanism of
action.
To study with more detail the antibiotic activity of the peptide, a fluorescent
labeled microcin was sinthetizated. To achieve this, a mutant peptide
containing lysine instead of isoleucine(13) was purificated, the MccJ25 I13K.
This peptide was chosed because this amino acid substitution has no effect on
the biological activity of the peptide.
For the labelling reaction, the mutant peptide was incubated with the
amine-reactive probe fluorescein isothiocyanate, which form a thiourea bond
with free amine groups. A mixture of peptide and fluorescent probe 1:3 ratio
(w/w) was incubated in alcaline medium at room temperature during two hours in
the dark.
The purification of the labeled peptide was carried out in two steps.
First, the non reacted fluorescent probe was eliminated by a C-8 hydrophobic
chromatography. Then, the methanolic fraction was purified by HPLC on a C18 column
in order to separate the labeled peptide from the non labeled one. The purified
fluorescein-labeled microcin showed antibiotic activity against MccJ25
sensible-strains similar to the native peptide.
Uptake assays of the microcin derivative with several strains were performed
following the maximum emission fluorescence at 520nm. A bacterial suspension
was incubated with the peptide at sublethal concentrations at 37°C in a TRIS buffer medium supplemented with glucose. Aliquots were taken
at different times and the residual peptide in the supernadant was determined.
The sensible strains Salmonella newport and E. coli AB1133 (pGC01)
and the resistant strains E. coli SBG
231 y E. coli AB 259 (pTUC 200)
were used. The independent effect of 100 mM 2,4 dinitrophenol, 200 mM vanadate
or heat plus azide on the derivative peptide uptake were studied.
The results
indicates that MccJ25 incorporation is an active mechanism ATP driven in the
different strains tested, and the incorporation is seen both in sensitive and
resistant strains.