The development of bacterial resistance to conventional antibiotics is a major
public health concern. For example, methicillin-resistant Staphylococcus
aureus
(MRSA), vancomycin-resistant enterococci (VRE) and Staphylococcus
aureus (VRSA)
have emerged as common nosocomial (hospital-originating) infections.
Circumvention of such resistance may be possi ble by emulating host defense
antimicrobial peptides (AMP's), which are found in a broad range of species and
have broad-spectrum antimicrobial properties. These AMP's have two structural
motifs in common: they are cationic and amphipathic. It is thought that electro
static interactions facilitate association of the peptide with the anionic
bacterial membrane and amphiphilic interactions act to form pores in the
bacterial membrane, leading to cell death. Thus, AMP's target generic
characteristics common to the mem branes of many pathogenic species, and
resistance to such natural defences evolves much more slowly than for
conventional antibiotics. The exact molecular mechanisms by which membrane
pores are formed are still not fully understood, although three major models
('barrel-stave', 'toroidal pore', 'carpet') have been proposed. Moreover, these
models do not ex haustively cover all possibilities, as AMP activity is not
always correlated with the loss of a permeability barrier. Understanding the
structural tenden cies generated in antimicrobial-membrane interactions is an
essential step to elucidating such molecular mechanisms and therefore to the
pre dictive design of synthetic AMP analogs.
Figure. (A) Electron density profile r(x,y) of the 2-D unit cell for
complexes formed by the specifically active AMO-2 and DOPG:DOPE = 20:80 lipid
membranes confirm the inverted hexagonal structure. The regions of lowest
electron density correspond to lipid chains. Circular 'rims' of high electron
density surrounding 'holes' of intermediate density correspond to lipid head
groups surrounding water channels which have a diameter of ~3.4nm. (B) A
proposed model of the unit cell is shown. The white and green spheres represent
headgroups of zero intrinsic curvature (ex. DOPG, DOPC) and negative intrinsic
curvature lipids (ex. DOPE), respectively. AMOs are represented by blue
spherocylinders embedded in the membrane.
In this work, we use synchrotron small-angle x-ray scattering (SAXS) to examine
the structures made by self-assembly of model membrane vesicles and members of
a prototypical class of synthetic antimicrobial oligomers (AMO) based on the
meta-phenylene ethynylene (mPE) family. These molecules are simple divalent
oligomeric amphiphiles. As the hydrophobic volume of these AMO's increases, 3
homologues in this family are inactive (AMO-1), specifically active against
bacteria (AMO-2), or non-specifically active against bacteria and eukaryotic
cells (AMO-3). We find that the antibacterial activity of these AMOs correlates
with their ability to induce an inverted hexagonal (HII) phase when added to
con centrated Small Unilamellar Vesicle (SUV) suspensions. This phase consists
of columnar arrays of lipid tubes threaded by water channels with diameters of
3.4 nm. When AMO is applied to dilute Giant Unilamellar Vesicle (GUV)
suspensions, encapsulated polymers with a radius of gyration less than this
diameter leak out whereas larger polymers are retained. These results suggest a
common structural tendency in membrane-AMO interactions, which is expressed as
an inverted hexagonal phase in bulk membrane systems and as permeated quasi-2D
membranes in isolated vesicles. The generation of an HII phase requires
negative curvature. This provides a potential mechanism for selective activity:
bacterial membranes are rich in PE lipids, which have a negative intrinsic
curvature, while eukaryotic cell membranes are rich in PC lipids, which have
zero intrinsic curvature. Using concentrated SUV suspensions composed of
DOPG:DOPE:DOPC , we find that the inactive AMO-1 cannot generate the inverted
hexagonal phase even when DOPE completely replaces DOPC, whereas the
specifically active AMO-2 requires a threshold ratio of DOPE:DOPC1 = 4:1. In
contrast, the nonspecifically active AMO-3, which lyses eukaryotic cells,
requires a drasti cally lower threshold ratio of DOPE:DOPC = 1.5:1. These
trends suggest an explanation for the mechanism of specific activity, since it
is well known that the PE content of eukaryotic membranes is significantly
lower than that of bacterial membranes.
Primary Citation
Over the last decade, new peptidic and nonpeptidic antimicrobial oligomers
(AMO's) with tunable structural features similar to those of natural,
membrane-spanning AMPs have been synthesized and investigated. Like AMP's,
these oligomers are also cati onic and amphiphilic. However, these synthetic
analogs have significantly lower molecular weights, and often do not span the
membrane. Synthetic AMO's have the advantage that their structures can be tuned
so that the importance of electrostatic and amphiphilic interactions to
membrane remodeling and cytotoxicity can be systematically probed.
L. Yang, V. D. Gordon, A. Mishra, A. Som, K. R. Purdy, M. A. Davis, G. N. Tew
and G. C. L. Wong, "Synthetic Antimicrobial Oligomers Induce a
Composition-dependent Topological Transition in Membranes", J. Am. Chem.
Soc. 129, 12141 (2007)
SSRL is supported by the Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences.