Download Multiple Activities in Natural Antimicrobials

Multiple Activities in Natural
Antimicrobials
Multiple activities of natural agents such as defensins and bacteriocins
suggest a change in strategy when developing new antimicrobials
Hans-Georg Sahl and Gabriele Bierbaum
any antibiotics, such as ␤-lactams, tetracyclines, and chloramphenicol, target specific molecules and act in a single, welldefined manner. However, other
larger antibiotic molecules typically have more
complex inhibitory patterns and multiple activities. This rule of thumb applies to host-defense
peptides such as the defensins from multicellular
organisms and also bacteriocins from bacteria,
and may also hold true for lipopeptide and
glycopeptide antibiotics. This multiplicity of activities provides an attractive strategy when designing antiinfective drugs.
M
Summary
• Nature’s most abundant antibiotics, cationic
amphiphilic host defense peptides, cause pleiotropic effects on membrane processes suggesting that they act like what medicinal chemists
call “dirty” or “promiscuous” drugs; unlike
with targeted, small molecule antibiotics there is
less in the way of resistance development
• Bacterial defense peptides, called bacteriocins,
combine pleiotropic membrane activities with
targeting of specific molecules such as the cell
wall precursor Lipid II, strongly increasing their
potency
• Clinically used and newly developed large natural antimicrobials such as lipopeptide and
glycopeptide antibiotics may also have multiple activities
• Designing truly effective multiply-active antibiotics appears an attractive strategy, but is likely
to require a good deal of basic research
Host-Defense Peptides Act
Like Promiscuous Drugs
Host-defense peptides (HDPs) are amphiphilic
cationic peptides of 20 –50 amino acids and
include the ␣- and ␤-defensins, cathelicidins,
cecropins, magainins, bactenecins, and protegrins. HDPs are part of the innate immune systems of almost every life form, with highly conserved and very broad activities against bacteria,
viruses, and fungi in the micromolar range of
concentration. The designation “host-defense
peptide” was coined when researchers realized
that these antimicrobial peptides have not only
direct antibiotic activities but also immune modulatory functions. For example, some of
them recruit and activate immune cells, enhance bacterial clearance, alter gene expression, and neutralize lipopolysacharides
(LPS).
A crucial physicochemical feature for the
antibiotic activity of HDPs is their amphiphilic character, which enables them to
adopt conformations in which polar and
charged amino acid side chains orient to one
side and apolar residues to the other (Fig. 1).
Thus, these peptides can bind to negatively
charged microbial surfaces, and then integrate into and disrupt the underlying cytoplasmic membranes. Despite substantial evidence that charge-mediated binding of
HDPs is critical for their antibiotic activity,
most knowledge about their lipid bilayerperturbing activities is based on analysis of
model membranes, leaving many questions
as to how they kill microbes.
Further, the disturbance of bilayer functions may be only one of several ways that
Hans-Georg Sahl is
Professor of Medical and Pharmaceutical Microbiology
and Gabriele Bierbaum is Professor
of Medical Microbiology at the Institute of Medical Microbiology,
Immunology and
Parasitology, University of Bonn,
Germany. This article is based on an
invited presentation
given at the 47th
ICAAC in Chicago,
September 2007.
Volume 3, Number 10, 2008 / Microbe Y 467
Sahl Believes Science Requires “Quite a Bit of ‘Creative Playing’”
Hans-Georg Sahl says that doing
science satisfies him more than do
the results. “Too many people are
pushing science toward applications,” he says. “It would have
been a nightmare for me to put a
scientifically interesting project
aside just because business people
had a different opinion. We need
curiosity-driven basic research in
the first place, and then we will
always have something to apply—
the other way around simply will
not work.”
Nevertheless, Sahl hopes that
his research will benefit public
health. He studies antibiotic
mechanisms—what happens at
the molecular level when an antibiotic kills bacteria—a focus that
could lead to new therapeutics
when resistance to available drugs
is increasing. “There is an urgent
need for new antibiotics, particularly compounds with unprecedented mechanisms,” he says.
“Multiresistant bacteria are really
a major health threat, and the
medical need for new antibiotics
is not adequately met by the pharmaceutical industries. Identifying
new antibiotic paradigms is a very
rewarding business that can certainly contribute to overcoming
resistance problems.”
Sahl is professor of pharmaceutical and medical microbiology at
the University of Bonn in Bonn,
Germany. “How are complex
biosynthetic pathways organized
in space?” he asks. “How do multiple enzymes function in a coordinated fashion so that a complex
macromolecule such as the bacterial cell wall keeps growing from
one cell to the next generation of
cells without losing functionality?
Antibiotics are wonderful tools to
468 Y Microbe / Volume 3, Number 10, 2008
interfere with such processes, and,
from disturbing a process, you
can learn much about the process
itself.”
Sahl, 58, was not always
pointed toward a career in biology. He grew up in Engers, a small
town near Koblenz in the central
Rhine valley, where the river
Mosel enters the Rhine. As a
child, he had an aquarium and a
terrarium, and received his first
microscope when he was 15. But
he was also interested in geography, listening to rock and pop
music and playing the guitar,
sports, and studying German, English, and American literature.
Tom Sawyer and Huckleberry
Finn are among his favorite
books. “I definitely cannot say
that I wanted to become an academic researcher from childhood
on,” he says.
Nevertheless, growing up, Sahl
nurtured a love for nature and the
outdoors, a fondness inspired by
his childhood environment. “It
was not too far from a city, but far
enough to have fields, a small forest, small lakes and the river
Rhine,” he says. “What I remember most is that we had endless
time for playing outside and never
got tired; that certainly implanted
my love for outdoor activities.
“I definitely kept that love for
nature and activities, and playing
outdoors,” he adds. “Building a
small dam in a creek is still a fascinating thing to do. I guess I have
kept a lot of the homo ludens
[man at play] of my childhood.
Science actually allows you to do
that—it requires quite a bit of
‘creative playing’—it is just that
the toys are getting so much more
expensive.”
During his final year of high
school, his biology and chemistry
teacher led him to think about a
career in biology. “He taught us
exciting new things which just
had appeared in textbooks,” Sahl
says. “I remember very well when
we learned about the chemistry
behind converting sugars into energy. This was really hot stuff in
those days for high school teaching. It gave me, for the first time, a
concrete molecular idea for an essential process of life, energy generation. Those experiences actually made me enroll in biology
and chemistry—rather than geography, languages, or other things.”
Sahl received his Diploma in Biology from the University of Bonn
in 1975, and his Ph.D. in microbiology, also from the University of
Bonn, in 1978. Between 1979 and
1983, he held a postdoctoral fellowship in medical microbiology,
which included a year at Purdue
University. “I spent almost all of
my scientific life at the University
of Bonn, which is a bit unusual,
but it was perfect for me and for
the University, so why not?” he
says. “Bonn is a beautiful place to
live.”
After deciding to study biology,
“I just did what I found most interesting, and I was lucky enough
to find support, positions, and the
right mentors,” Sahl says. “After
undergraduate studies, I specialized in microbiology because I
thought that a bacterium would
be biggest organism that I could
possibly understand on the molecular level. I had tried virology before, because a virus was much
simpler and seemed to be more
easy to understand, but I returned
to bacteria when I found out that
you cannot study a virus without
studying eukaryotic cell biology.”
Sahl, who is unmarried, enjoys
travel and takes long trips whenever he can spare the time. “I love
to be in other places of the world,
although getting there is more and
more of a nuisance,” he says. “My
love for nature and outdoor activities is one of the reasons why I
have to get away every once in a
while— on a three-week canoe
trip into the wilderness, or to the
outback and the desert in Australia, or to go hiking in the mountains.”
When he is not traveling or engaged in his research, he works on
his house. “I have quite a bit of
land around it, which I turned
into a little park,” he says. “It
always keeps me busy over the
weekend, in winter even more
than in summer, trimming bushes
or cutting a tree, planting new
trees, chopping wood, cutting the
grass. It is the right exercise, physically and mentally, for somebody
who sits at a desk most of the
week.”
Marlene Cimons
Marlene Cimons is a freelance writer
in Bethesda, Md.
HDPs affect target cells. For example, when we
Bacteriocins Combine Targeted and
treated staphylococcal cells with human ␤-deNontargeted Activities
fensin 3 (hBD3, Fig. 1), the cell wall stress reguBacterial peptides with antibacterial activity,
lon including the two-component regulatory
called bacteriocins—whether posttranslationsystem vraSR was upregulated at the transcripally modified (lantibiotics) or not—typically
tional level. The cell wall stress regulon consists
share features with other HDPs, including relaof a group of genes that typically is upregulated
tive size, cationicity, and amphiphilicity. Addiin response to vancomycin, oxacillin, and other
tionally, some bacteriocins contain structural
agents that inhibit cell wall biosynthesis in
motifs that also are found among eukaryotic
Staphylococcus aureus.
defensins, such as the ␥-core motif that appears
This response indicates that the peptides do
not simply disrupt cytoplasmic membranes. Instead, they also interfere with
FIGURE 1
highly coordinated, membrane-bound
processes, of which cell wall biosynthesis appears the most vulnerable. Electron transport chains may also be impaired, particularly by helical peptides
such as human cathelicidin LL37 that
have strong amphiphilic features.
In general, the antibiotic activity of
host defense peptides appears not to be
based on targeting specific molecules.
Instead, they produce pleiotropic effects at various sites—acting as if they
were what some medicinal chemists and
other experts call “dirty drugs.” Nonetheless, conventional drug-design strategies aim not at developing promiscuous drugs but at developing agents
that have high affinity for well-defined
targets. However, nature apparently
Primary sequence of the human beta defensin 3 (hBD3) and the human cathelicidin LL 37
(amino acids in the one-letter code); hBD3 has an overall positive charge of ⫹11 due to
works differently in the case of so many
a surplus of lysine (K) and arginine (R) residues over negatively charged residues (D, E);
antimicrobial peptides. Moreover, units structure is stabilized by 3 disulfide bridges. LL37 is a linear, ␣-helical peptide with a
like antibiotics which act on a single
net charge of ⫹ 6; its pronounced amphiphilicty is demonstrated in the spatial structure
with hydrophobic residues in green, negatively charged side chains in red and positively
target, there is little in the way of resischarged residues in blue.
tance developing for these antimicrobial peptides.
Volume 3, Number 10, 2008 / Microbe Y 469
onine-containing antibiotics) are
about the same molecular weight as
defensins but contain thioether amino
acids instead of disulfide bonds. The
thioether amino acids in these bacteriocins, namely lanthionine and methyllanthionine, are produced by posttranslational changes from cysteine
and serine and from cysteine and threonine residues, respectively (Fig. 2A).
Bacteriocins were thought to kill exclusively by disrupting membranes until
researchers realized that they also act
on other targets. Indeed, many of them
act like HDPs by disrupting lipid bilayer functions at micromolar concentrations. However, against a narrow
spectrum of closely related strains, lantibiotics have minimal inhibitory concentrations (MICs) in the nanomolar
range; in such cases, specific target-mediated activities typically are involved.
For about 40 of the lantibiotics described so far, the molecular target is
the membrane-bound cell wall precursor Lipid II.
Researchers assign lantibiotics that
bind Lipid II to two groups (Fig. 2B).
The first, typified by the food preservative nisin, consists of elongated molecules, while lantibiotics in the second
group, such as mersacidin, have a more
globular shape. After these lantibiotics
have formed stoichiometric complexes
with Lipid II, they sequester the cell
wall precursor, blocking its further incorporation into the cell envelope.
Thus, these lantibiotics inhibit cell wall
biosynthesis in a way that resembles
(A) Primary structure of the lantibiotics nisin and mersacidin. Lantibiotics contain
numerous unusual amino acids which are produced through posttranslational modificaglycopeptide antibiotics. However, the
tion of precursor peptides; such amino acids include, e.g., didehydroalanine (Dha, from
lantibiotics bind the highly conserved
serine), didehydrobutyrine (Dhb, from threonine), lanthionine (Lan, from Dha and cyssugar-pyrophosphate moiety of Lipid II
teine), methyllanthionine (MeLan, from Dhb and cysteine). The thioether containing
residues Lan and MeLan introduce intramolecular ring structures; the two sequence
rather than the D-Ala-D-Ala peptide
motifs involved in Lipid II binding which are characteristic for each group of nisin- and
side chain of the building block for the
mersacidin-related lantibiotics are boxed. (B) Structure of the membrane-bound cell wall
cell wall (Fig. 2B).
precursor Lipid II, consisting of the disaccharide-pentapeptide cell wall subunit and the
bactoprenol-pyrophosphate membrane anchor; the interpeptide bridge attached to the
Binding to Lipid II may yet provoke
lysine residues of the stem peptide is found in most gram-positive bacteria and may
additional activities for some lantibiotconsist of five glycine residues in many staphylococci. The binding sites on Lipid II for
ics. For example, when nisin binds to
lantibiotics (light blue) and glycopeptide antibiotics (dark blue) are indicated.
Lipid II, the cell wall precursor acts like
an anchor molecule for the lantibiotic,
which subsequently inserts into the memamong class IIa bacteriocins from gram-positive
brane to form a defined and stable pore consisting
bacteria. Meanwhile, the lantibiotics (lanthiof Lipid II and nisin molecules (Fig. 3).
FIGURE 2
470 Y Microbe / Volume 3, Number 10, 2008
FIGURE 3
Dual mode of action of the lantibiotic nisin. Nisin forms a stoichiometric complex with Lipid II which triggers two fatal events (i)
the cell wall precursor is abducted and no longer available for incorporation into the growing peptidoglycan network (left); binding
of Lipid II via its N-terminal binding motif and the carbohydrate-pyrophosphate moiety on Lipid II enables the C-terminal segment
of nisin to insert into the membrane (right); several nisin: Lipid II complexes assemble to form a stable pore with an overall
diameter of 2 nanometers. Besides these Lipid II-mediated activities, nisin can induce cell wall lytic processes, particularly in
staphylococci, and, at micromolar concentrations, impair vital lipid bilayer functions.
Several other lantibiotics, including nisinrelated epidermin and gallidermin as well as
plantaricin C, which has a mersacidin-like
Lipid II binding motif, also form Lipid IIdependent pores. However, these peptides
form pores in membranes of only some bacterial strains, such as the micrococci, but not
the lactococci. The overall length of the individual lantibiotic molecule as well as the
lipid composition of the target-strain membranes determine whether pores can form.
When Lipid II-lantibiotic complexes insert
into membranes, they may dislocate from the
sites where they are needed for cell wall synthesis and may lead to septum formation at
inappropriate sites.
In addition to these Lipid II-mediated activities, lantibiotics have other effects. For instance, when treated with nisin, dividing cells
of Staphylococcus simulans prematurely split
the new cell wall between the two daughter cells,
leading to rapid lysis when the septum is in-
complete. Other cationic peptides, including
defensins, also rapidly lyse staphylococci—
possibly triggering the same cell wall lytic mechanism.
The complexity of the lantibiotics apparently
is matched by that of another prominent group
of bacteriocins—namely, the class II peptides
from gram-positive bacteria. For instance, the
Listeria-active class IIa peptides combine with
protein components of the mannose phosphotranferase sugar uptake system to form pores
through target-cell membranes.
More generally, many naturally occurring antimicrobial peptides appear to combine broad
toxicity with the targeting of specific molecules.
This property of dual or multiple activities also
helps to explain how peptide bacteriocins have
such remarkably high efficacies, with MICs typically in the nanomolar concentration range,
against some targeted bacteria; but low-level
background activity against the rest of the grampositive bacteria.
Volume 3, Number 10, 2008 / Microbe Y 471
FIGURE 4
Structures of the glycopeptide antibiotic vamcomycin and of the lipodepsipeptide
antibiotic daptomycin; both are clinically important natural product antibiotics for treatment of multiresistant Gram-positive bacterial infections. Glycopeptide antibiotics other
than vancomycin may have increased amphiphilic properties through attached strongly
hydrophobic moieties such as chlorophenyl-benzyl rings (oritavancin) or acyl chains (e.g.,
teicoplanin) linked to the disaccharide units.
Some Lipopeptide and Glycopeptide
Antibiotics Show Multiple Activities
Some experts argue that such complex activity
patterns are found mainly among highermolecular-weight, natural compounds that are
unsuitable for commercial development. However, there are noteworthy exceptions to this
rule. For instance, established low-molecularweight drugs such as isoniazide cause pleiotropic effects.
Meanwhile, several commercially successful
antibiotics that overcome drug resistance
among some gram-positive pathogens also appear to have multiple activities. For instance,
glycopeptide antibiotics such as vancomycin
(Fig. 4) interfere with cell wall synthesis by
attaching to the Lipid II precursor at its D-AlaD-Ala peptide side chain (Fig. 2B), thereby inhibiting transglycosylation of cell wall subunits.
However, some other glycopeptide antibiotics
have pronounced amphiphilic properties, particularly those containing lipid side chains such
472 Y Microbe / Volume 3, Number 10, 2008
as teicoplanin as well as telavancin and
ramoplanin, both of which are under
development. Thus, these drugs may
impair membrane barrier functions in
addition to inhibiting cell wall biosynthesis. Another glycopeptide, oritavancin, which is also in clinical development, appears to have an additional
binding site for the interpeptide bridge,
that occurs in a modified form of Lipid
II in gram-positive cells.
The lipodepsipeptide daptomycin
(Fig. 4), which was approved for use in
the United States in 2003, supposedly
forms pores in gram-positive bacteria.
This cyclic molecule has a net negative
charge and requires calcium ions for
activity. When it forms complexes with
calcium ions, the antibiotic has an overall positive charge and is assumed to
behave like other cationic peptides with
antimicrobial activity—that is, to oligomerize within membranes, forming
pores that disrupt vital ion gradients
within target bacterial cells. However,
according to some investigators, daptomycin also may inhibit either peptidoglycan or lipoteichoic acid biosynthesis.
Daptomycin, in contrast to cationic
HDPs, is a very potent compound.
Designing Anti-Infective Drugs with
Multiple Modes of Action
During the past few decades, hybrid antibiotics
were designed with dual modes of action by combining various beta-lactam, macrolide, quinolone,
oxazolidinone, or glycopeptide antibiotics. The
goal was to have drugs with extended activity
spectrums while reducing the risk that pathogens would develop resistance to them. However, inferior physicochemical and pharmacological properties tended to counterbalance those
advantages—reducing potency and introducing
other adverse features. Whether more recently developed rifamycin-quinolone hybrids overcome
those problems and fulfill their promise remains to
be seen.
Natural peptides with multiple antimicrobial
activities, of course, differ from synthetic hybrid
antibiotics because the former combine innate,
pleiotropic antibiotic activity, which is based on
cationic amphiphilicity, with specific target-
mediated activities, mainly involving interference with cell wall biosynthesis. Cationic amphiphilicity is also an indispensable feature for the
immunomodulatory activity of HDPs. Some synthetic short-chain amphiphilic HDP derivatives
have been designed that are devoid of direct antibiotic activity, but fully retain immunoregulatory
functions and effectively control bacterial infec-
tions in mice. Thus, combining those three functions—immunomodulation, basic, and targeted
antibiotic activities—into one molecule appears a
particularly promising approach. However, succeeding with that approach is likely to require a
good deal of basic research. The increasingly dramatic buildup of antibiotic resistance among human pathogens warrants such efforts.
ACKNOWLEDGMENTS
We gratefully acknowledge the many excellent contributions by members of our research groups and collaborators. Financial
support was obtained from the German Research Foundation (DFG, various projects to both of us), the Federal Ministry of
Education and Research (BMBF, Pathogenomics network to G.B. and SkinStaph network to H.G.S.) and the BONFOR
programme of the Medical Faculty, University of Bonn.
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