Answer Key to Problem Set #1, Question #2
Question 2:
A) By the inhibition of intracellular functions.
Example 1:
Short, proline-rich antibacterial peptides from insects, pyrrhocoricin, drosocin, and
apidaecin interact with the bacterial heat shock protein DnaK and inhibit two major
functions of DnaK, ATPase activity and refolding of misfolded proteins.
Kragol et al. (see Kragol et al, Biochemistry, 2001, 40: 3016-3026) showed that:
1) Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase
activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the
membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit
the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP).
Pyrrhocoricin binding was not observed to the homologous DnaK fragment of
Staphylococcus aureus, a pyrrhocoricin nonresponsive strain.
2) pyrrhocoricin also inhibited the ability of Dnak to refold misfolded proteins.
They showed that incubation with L-pyrrhocoricin or drosocin reduced the
enzymatic activity of alkaline phosphatase and beta–galactosidase in live bacteria.
In contrast, D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide
involved in binding to bacterial nucleic acids (see below), had only negligible
effect.
Example 2:
Binding to DNA and inhibit DNA synthesis.
1) In studies by Xiong and coworkers using thrombin-induced PMP-1 (tPMPs), S. aureus
cells remained viable long after rapid membrane permeabilization, indicating that
disruption of membrane is not the major contributor to tPMP antimicrobial activity.
tPMP-mediated inhibition of DNA and/or RNA synthesis corresponded temporally with
cell death but was not observed until 30 or more minutes after membrane
permeabilization (see Xiong et al, J Infect Diseases 2002, 186: 668-677). Interestingly,
staphylocidal effects did not appear to result from global cellular dysfunctions, since
protein synthesis was inhibited to an equivalent extent in strains susceptible or resistant to
tPMP-1. Moreover, pre-exposure to agents that selectively inhibit protein synthesis (30 S
or 50 S subunit inhibitors) or DNA metabolism (DNA gyrase) mitigated subsequent
tPMP-1 induced killing of an otherwise susceptible S. aureus strain in vitro. These
findings implicate a direct inhibition of nucleic acid synthesis by tPMPs. The relatively
strong negative charge of nucleic acids is consistent with the hypothesis that cationic
peptides bind to and inhibit these molecules, not unlike histone proteins.
2) Buforin II. Using FITC-labelled buforin II and a gel retardation experiment, Park et al
(Biochemical and Biophysical Research Communications 1998, 244: 253-257) showed
that buforin II killed E. coli without lysing the cell membrane. The gel-retardation
experiment showed that buforin II bound to DNA and RNA after penetrating the cell
membranes, resulting in the rapid cell death.
B) Disruption of physicochemical properties of target membrane.
Examples, Magainin (Xenopus skin), tachyplesins (horseshoe crab hemolymph) and
cecropin (Drosophila). For a fine review on how the mode of interaction between AMP
is strongly dependent on the physicochemical properties of both the peptide and the target
membrane, see Matsuzaki, Biochemica et Biophysica Acta 1999, 1462:1-10. Basically,
cationic antimicrobial peptides, such as magainin electrostatically recognizes anionic
lipids that are abundant in bacterial membranes, forming a peptide-lipid supramolecular
complex pore, whereas the peptide does not effectively bind to zwitterionic phospholipids
constituting the outer leaflets of mammalian cell membranes because of the low
hydrophobicity of the peptide.
C) Pore Formation:
Pores formed by magainin and protegrin in membranes were directly demonstrated by
crystallization, see Yang et al, Biophysical J 2000, 79: 2002-2009.