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D. Barra - Peptide effectors of innate immunity
with each other and found that the isomers A and B,
which are only weakly active on Gram-negative bacteria,
could synergize when combined separately
with temporin L, overcoming the bacterial resistance
imposed by the LPS protective layer. This effect
depends on the length of the LPS sugar chain on the
target microorganism.
To understand the underlying mechanism, we investigated
the effect of LPS from two strains of E. coli
with different LPS moiety on the structural organization
of temporins, alone and when mixed one with
each other. Our data have indicated that the synergistic
effect is related to the ability of temporin L to
prevent the oligomerization of the A and B isomers,
thus allowing their translocation across the bacterial
cell wall into the target cytoplasmic membrane.
Overall such studies have revealed two important
findings:
(i) temporins A and B are not active on Gram-negative
bacteria, because of their oligomerization when
in contact with the outer membrane: their larger size
should interfere with the peptide diffusion through
the cell wall into the target inner membrane;
(ii) the synergistic activity between temporins on
Gram-negative bacteria is related to the ability of
temporin L to assist A and B in traversing the LPS
layer by preventing their oligomerization. This effect
is highly dependent on the LPS structure.
In addition, we have shown that the same temporin
combinations suppress the endotoxin effects of LPS,
by inhibiting TNF-α release (considered to be a primary
mediator of endotoxemia) from macrophages.
Furthermore, by means of spectroscopic and thermodynamic
studies, we have demonstrated that the
synergism of temporins in the anti-endotoxin activity
relies on the peptide’s ability to dissociate LPS
aggregates to smaller sized vesicles and that this
property is inversely related to the length of the
polysaccharide region of LPS.
Another interesting family of frog skin AMPs is
given by bombinins H, isolated from amphibia of
Bombina genus and containing isomers with a single
D-amino acid, resulting from a post-translational
epimerization of the corresponding L-residue. Indepth
studies on the biological activity of these molecules
and the underlying mode/s of action have
shown that bombinin H2 (IIGPVL-
GLVGSALGGLLKKI-NH 2 ) and H4, differing by
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only the configuration of the second amino acid (an
L-isoleucine in H2 and a D-alloisoleucine in H4) display
antibacterial and anti-parasitic activities, with a
stronger potency and faster killing kinetic of the Damino
acid-containing bombinin H4. This peptide
was also found to have a higher haemolytic capacity
on human erythrocytes. In all cases, the isoforms H2
and H4 were able to perturb the membrane of the
target cells, causing leakage of large cytosolic components
(e.g. proteins) or diffusion of smaller molecules
through local membrane disruptions.
We have therefore demonstrated the importance of a
single L- to D-epimerization as a new approach
developed by nature to modulate not only the bioavailability
(e.g. higher solubility) and biostability
(i.e. protection from proteolytic degradation) of
AMPs, but also their biophysical properties (peptide
structure and organization within membranes) and
antimicrobial activities.
These studies help getting insight into the molecular
basis accounting for the quantitative difference in the
biological activities of H2 and H4 epimers and the
role of the D-amino acid in their different behaviour,
and in general to have a deeper understanding of the
mechanisms of action of these new potentially useful
weapons to fight infections.
Selected publications
Mangoni ML, Maisetta G, Di Luca M, Marcellini
HGL, Esin S, Florio W, Brancatisano FL, Barra D,
Campa M, Batoni G. Comparative analysis of bactericidal
activity of amphibian peptide analogues
against multidrug-resistant nosocomial bacterial
strains. Antimicrob Agents Chemother. 2007, 52:85-91.
Mangoni ML, Marcellini HGL, Simmaco M.
Biological characterization and modes of action of
temporins and bombinins H, multiple forms of short
and mildly cationic anti-microbial peptides from
amphibian skin. J Pept Sci. 2007, 13:603-13.
Mangoni ML, Epand RF, Rosenfeld Y, Peleg A,
Barra D, Epand RM, Shai Y. Lipopolysaccharide, a
key molecule involved in the synergism between
temporins in inhibiting bacterial growth and in
endotoxin neutralization. J Biol Chem. 2008,
283:22907-17.