Old War, New Battle, New Fighters!
Old War, New Battle, New Fighters!
Sandra Sousa 0 1
Francisco Sarmento Mesquita 0 1
Didier Cabanes ( 0 1
0 Received and accepted 11 September 2014; electronically published 17 September 2014. ular and Cell Biology, Group of Molecular Microbiology , Rua do Campo Alegre, 823, 4150-180 Porto , Portugal
1 Instituto de Biologia Molecular e Celular, Universidade do Porto , Portugal
fisetin; Listeria monocytogenes; listeriolysin O; natural flavonoid; antivirulence strategies. EDITORIAL COMMENTARY
Since the discovery of penicillin,
antibiotics have been of critical importance in the
control of infectious diseases. However,
the extensive use and misuse of
antibiotics during recent decades led to the
widespread development and distribution of
resistance to multiple drugs among
bacteria. With the alarming levels of antibiotic
resistance and the difficulties associated
with discovering novel antibiotics,
researchers are now investigating alternatives to
treat infectious diseases. In contrast to
traditional strategies, which aim to kill
bacteria or prevent their growth, these
new approaches intend to block the
ability of bacteria to harm the host by directly
inhibiting bacterial virulence factors and
are thought to use less selective pressures
that limit the development of bacterial
resistance. These emerging strategies
benefit from the detailed knowledge of the
functional and molecular mechanisms
underlying key pathogenic determinants
acquired during the last decades of
research in host-pathogen interactions .
Bacterial toxins in particular are primary
targets for these novel antivirulence
strategies. In this issue of The Journal of
Infectious Diseases, Wang et al show that
fisetin, a natural flavonoid with negligible
antimicrobial activity, has effective
antivirulence activity against Listeria
monocytogenes by directly interfering with a
secreted bacterial toxin.
Listeria monocytogenes is a facultative
intracellular human foodborne pathogen
that causes gastroenteritis, meningitis,
encephalitis, and maternofetal infections.
Listeriosis is the most frequent cause of
death from consumption of
contaminated food in Europe and has the third
highest cost of illness and loss of quality of
life among foodborne infections [2, 3].
Listeria monocytogenes enters the host
via the ingestion of contaminated foods,
invades the intestine, translocates to
mesenteric lymph nodes, and spreads to the
liver, spleen, brain, and placenta. During
infection, L. monocytogenes has the
ability to cross the intestinal, blood-brain, and
placental barriers, entering, surviving,
and multiplying inside phagocytic and
nonphagocytic cells . To establish and
sustain infection, L. monocytogenes uses
an arsenal of virulence factors to hijack
host-signaling pathways [5, 6]. While
remaining a real public health concern,
L. monocytogenes has emerged as an
exceptional model to address the different
facets of host-pathogen interactions and
the design of new therapeutic strategies.
Listeriolysin O (LLO) is a crucial
virulence factor produced by L. monocytogenes
. It is a pore-forming toxin member of
the cholesterol-dependent cytolysin
family . LLO monomers are secreted by the
bacteria and oligomerize into a ring at the
surface of target cell . Membrane
insertion of LLO results in ion fluxes across
damaged membranes and ultimately
leads to cell lysis in conditions of
extensive damage and/or inefficient membrane
repair mechanisms . Inactivation of
LLO results in the inability of L.
monocytogenes to escape from the internalization
vacuole, thereby decreasing the virulence
potential of L. monocytogenes [11, 12].
Besides membrane lysis, it has become
apparent that LLO acting from the
intracellular or extracellular milieu exerts
additional effects on the host cell .
Intracellular LLO affects host cell
signaling [13, 14], induces autophagy ,
and suppresses reactive oxygen species
. LLO was also shown to deregulate
host SUMOylation , to induce
endoplasmic reticulum stress  and
mitochondrial fragmentation , and to
promote regulatory epigenetic changes
A previous study showed that
subinhibitory concentrations of plant essential
oils could inhibit LLO activity and
decrease L. monocytogenes virulence, but
the specific compound responsible for
this effect remained unknown .
Wang et al now report the discovery of
the natural flavonoid fisetin as an
effective antivirulence agent against LLO
activity. They showed that fisetin inhibits
the hemolysis capacity of LLO and is able
to protect mice from lethal infection by
L. monocytogenes. Molecular modeling
studies demonstrated that fisetin directly
engages LLO, causing a conformational
shift of the LLO domains critical for its
binding to cholesterol and
oligomerization. Coupling mutational and
biochemical approaches, they identified LLO
amino acid residues involved in
sensitivity to fisetin. This work establishes fisetin
as a novel antivirulence compound that
targets LLO by a unique mechanism,
counteracting toxin binding to host cells
and oligomerization. Interestingly, fisetin
is able to decrease L. monocytogenes
virulence in tissue-cultured cells and animal
infection models, without affecting the
L. monocytogenes growth or the
phagocytic capacity of macrophages. Notably,
while new roles have been frequently
assigned to LLO, this study supports the
concept that, during infection, pore
formation remains the toxin’s principal
Antivirulence strategies targeting
bacterial toxins to prevent their deleterious
effects were previously developed. Such
approaches were based on the use of
soluble analogs or specific antibodies against
toxins, thereby preventing their
interaction with their receptors at the host cell
membrane. Alternatively, they aim at
blocking toxin pores, using synthetic
compounds . In the case of LLO,
neutralizing monoclonal antibodies were
previously described to control L.
monocytogenes intracellular growth and virulence
. One important caveat, however, is
that these strategies are often associated
with high costs of production and
maintenance. In contrast, fisetin is present in
many fruits and vegetables associated
with low costs of production and was
shown to have broad biological properties,
ranging from antioxidative to cancer
therapeutic effects. The bioavailability and
toxicity of fisetin are also well established
. In addition, an important advantage
associated with dietary plant flavonoids
is that they are perceived as nontoxic and
have wide human acceptance . This
study thus paves the way for the
development of broad antivirulence approaches
based on natural products. Importantly, as
cholesterol-dependent cytolysins generally
have structural similarities, it would be
interesting to test the effect of fisetin on
produced by other bacterial pathogens, such
as Streptococcus pyogenes, Streptococcus
pneumoniae, Arcanobacterium pyogenes,
and Clostridium perfringens . Indeed,
an optimal therapeutic agent would target
virulence factors present in several
pathogens. Results presented by Wang et al
regarding the recent determination of the
LLO crystal structure  could also
allow the generation of fisetin derivatives
with improved activity against
Because of their rapid evolution rate,
bacteria are experts at finding alternative
routes to achieve growth and infection.
Because most virulence traits are
nonessential for bacterial survival, therapeutic
strategies based on the inhibition of
virulence should apply mild evolutionary
pressure and limit the development of
resistance. These promising approaches are
sought to dampen pathogen progression,
allowing the control of infection through
an effective host immune response or
increasing the efficacy of classic therapies
targeting bacterial growth. Indeed, in
the presence of antivirulence compounds,
bacteria can still grow and produce the
targeted virulence determinants and are
thus able to reinjure the host in the
absence of suitable inhibitors. Coupled with
traditional antibiotics, antivirulence
therapies may provide an important advantage
in the fight against infectious diseases.
Although resistance development and
side effects, including disruption of
microbiota, are always possible, the report
by Wang et al opens new possibilities
for natural compounds as effective
antivirulence strategies against human
Financial support. This work was supported
by the Fundo Europeu de Desenvolvimento
Regional – Programa Operacional Factores de
Competitividade (FEDER-COMPETE) and
Fundação para a Ciência e a Tecnologia (FCT)
InfectERA PROANTILIS/0001/2013 to D. C.), the
European Molecular Biology Organization (EMBO) (to
F. S. M.), and European Society of Clinical
Microbiology and Infectious Diseases (ESCMID) (to
Potential conflicts of interest. All authors:
No reported conflicts.
All authors have submitted the ICMJE Form
for Disclosure of Potential Conflicts of Interest.
Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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