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Current
Drug Targets
ISSN: 1389-4501
Current Drug Targets
Volume 9, Number 9, September 2008
Contents
Control and Regulation of Permeability
of MDR Bacterial Pathogens to Antibiotics Presented by COST
Action BM0701
Guest Editors: L. Amaral and J.M. Pagès
Editorial Pp. 718
Structure and Mechanism of Drug Efflux Machinery in
Gram Negative Bacteria Pp.719-728
Z. Pietras, V.N. Bavro, N. Furnham, M. Pellegrini-Calace,
E.J. Milner-White and B.F. Luisi
[Abstract]
The AcrB Efflux Pump: Conformational Cycling
and Peristalsis Lead to Multidrug Resistance Pp.
729-749
M.A. Seeger, K. Diederichs, T. Eicher, L. Brandstätter,
A. Schiefner, F. Verrey and K.M. Pos
[Abstract]
Membrane Permeability and Regulation of Drug
“Influx and Efflux” in Enterobacterial Pathogens
Pp. 750-759
A. Davin-Regli, J-M. Bolla, C.E. James, J-P. Lavigne,
J. Chevalier, E. Garnotel, A. Molitor and J-M. Pagès
[Abstract]
New Methods for the Identification of Efflux
Mediated MDR Bacteria, Genetic Assessment of Regulators and
Efflux Pump Constituents, Characterization of Efflux Systems
and Screening for Inhibitors of Efflux Pumps Pp.
760-778
M. Viveiros, M. Martins, I. Couto, L. Rodrigues, G. Spengler,
A. Martins, J.E. Kristiansen, J. Molnar and L. Amaral
[Abstract]
Physical Insights into Permeation of and Resistance
to Antibiotics in Bacteria Pp. 779-788
M. Ceccarelli and P. Ruggerone
[Abstract]
Biophysical Characterization of In- and Efflux
in Gram-Negative Bacteria Pp. 789-796
H. Weingart, M. Petrescu and M. Winterhalter
[Abstract]
Clinical Impact of the Over-Expression of Efflux
Pump in Nonfermentative Gram-Negative Bacilli, Development
of Efflux Pump Inhibitors Pp. 797-807
J. Vila and J.L. Martínez
[Abstract]
Antimicrobial Resistance in Foodborne Pathogens
- A Cause for Concern? Pp. 808-815
C. Walsh and S. Fanning
[Abstract]
Promising Therapy of XDR-TB/MDR-TB with Thioridazine
an Inhibitor of Bacterial -Efflux Pumps Pp. 816-819
L. Amaral, M. Martins, M. Viveiros, J. Molnar and J. E.
Kristiansen
[Abstract]
Abstracts
[Back to top]
Editorial
Multi-drug resistance of Gram-negative and Gram-positive bacteria
is essentially due to functional changes of the cell envelope
which reduce the over-permeability of the bacterium to antibiotics
and in some cases, to biocides. The major components of the
Gram-negative cell envelope that control the permeability
of the bacterium to noxious agents (antibiotics, antimicrobials,
biocides and other toxins) are: i) the lipopolysaccharide
cover of the outer cell membrane whose effectiveness can be
increased by a two component regulatory system when the bacterium
is exposed to noxious compounds; ii) a reduction in the number
of channels (porins) that allow lipophobic to traverse the
outer cell envelope and reach internal compartments of the
cell when the bacterium is exposed to noxious compounds; and
iii) over-expressed efflux pumps that recognize noxious agents
that reach the periplasm or cytoplasm of the bacterium and
extrude these compounds prior to their reaching their intended
targets. The response of the Gram negative bacterium to noxious
compounds may involve all of these components. The manner
by which the bacterium organizes the individual response of
each of these regulators of permeability is highly relevant
to clinical medicine since these responses render the organism
increasingly resistant to two or more unrelated antibiotics,
and often times to entire families of antibiotics. Moreover,
it is now clear that regardless of how recent a new and effective
antibiotic is introduced for the therapy of Gram-negative
bacterial infections, the response of the global bacterial
population as a consequence of increased usage of the antibiotic,
soon renders the organisms multi-drug resistant.
Understanding the mechanisms by which bacteria trigger the
development of multi-drug resistance requires the concerted
efforts of many widely diverse areas of science and technology.
From clinical bacteriology, molecular and structural biology,
biochemistry and bacterial physiology, molecular modeling
and chemical synthesis, much knowledge must be achieved regarding
the regulation of membrane permeability, the structure, the
membrane topology and the activity of the involved drug transporters.
The main objective of the COST1
Action ATENS (BM0701) is to organize a framework of collaboration
between well-known experts in these scientific areas necessary
for understanding membrane-mediated resistance at the molecular
and genetic levels and to translate this knowledge into the
development of diagnostic tests and antimicrobials that will,
in the future, help control MDR infectious diseases. This
program links the research disciplines of biology, physical-chemistry
and medicine through pools of teams belonging to several European
countries and different chapters of this issue have been written
by these partners. This multi- and inter- disciplinary aspect
will be a prominent advantage of ATENS that will yield major
weapons to be used against our favourite enemy, the microbe.
This issue of the Current Drug Targets contains articles written
by European scientists that are leaders in the field of efflux
mediated multi-drug resistance of clinically important bacteria.
The articles discuss the structural biology of the efflux
pumps of Gram negative bacteria, the control of permeability
by the joint action of efflux pumps and the outer membrane
channels of Gram negative bacteria, the regulation of efflux
pumps by regulator genes, the relevance of efflux pump mediated
mdr of bacteria, the development of molecular models that
measure on a real-time basis the movement of antibiotic molecules
through a porin channel, the development of methods that identify
efflux mediated mdr bacteria, and the potential therapy of
efflux mediated mdr infections by agents that have been used
for decades for non-infectious pathology. Each of the articles
provides a substantial listing of references that by itself,
yields the information that will readily result in the acquired
expertise in what was once a most difficult to understand
area of infectious disease-namely, the control and regulation
of permeability of mdr bacteria to antibiotics. We hope the
readers will enjoy this issue and direct their students to
the contents of this special issue of “Current Drug
Targets”.
Leonard Amaral
President, International Society of Non-Antibiotics (ISN)
Professor and Director
Unit of Mycobacteriology/UPMM
Institute of Hygiene and Tropical Medicine
Universidade Nova de Lisboa
Jean Marie Pagès
Chairperson, COST Action BM0701 (ATENS)
Director of Research
UMR-MD1, Transporteurs Membranaires
Chimiorésistance et Drug-Design
Faculté de Médecine, Université de la
Méditerranée
[Back to top]
Structure and Mechanism of Drug Efflux Machinery in
Gram Negative Bacteria
Z. Pietras, V.N. Bavro, N. Furnham, M. Pellegrini-Calace,
E.J. Milner-White and B.F. Luisi
In Gram-negative bacteria, multi-component machines that
span the inner and outer membranes actively extrude drugs
and other toxic small compounds. Many of these machines are
assembled principally from three different types of components:
i) an outer membrane protein that acts as a channel and opens
from a sealed resting state during the transport process,
ii) an inner membrane protein that transduces proton electrochemical
energy into vectorial displacement of the transported compounds,
and iii) a bridging, periplasmic component that links the
inner and outer membrane proteins. The pumps may assemble
transiently, and the association of components is favoured
by engaged substrate and the trans-membrane electrochemical
potential. We describe recent structural and functional studies
on the individual pump components and discuss models that
explain how they associate in the dynamic, active assembly.
Based on the available data, we suggest that the assembly
of these multi-drug efflux pumps is accompanied by induced
fit of the outer membrane component driven mainly by accommodation
of the periplasmic component.
[Back to top]
The AcrB Efflux Pump: Conformational Cycling and Peristalsis
Lead to Multidrug Resistance
M.A. Seeger, K. Diederichs, T. Eicher, L. Brandstätter,
A. Schiefner, F. Verrey and K.M. Pos
Antimicrobial resistance of human pathogenic bacteria
is an emerging problem for global public health. This resistance
is often associated with the overproduction of membrane transport
proteins that are capable to pump chemotherapeutics, antibiotics,
detergents, dyes and organic solvents out of the cell. In
Gram-negative bacteria such as Escherichia coli and
Pseudomonas aeruginosa, tripartite multidrug efflux
systems extrude a large variety of cytotoxic substances from
the cell membrane directly into the medium bypassing the periplasm
and the outer membrane. In E. coli, the tripartite
efflux system AcrA/AcrB/TolC is the pump in charge of the
efflux of multiple antibiotics, dyes, bile salts and detergents.
The trimeric outer membrane factor (OMF) TolC forms a β-barrel
pore in the outer membrane and exhibits a long periplasmic
α-helical
conduit. The periplasmic membrane fusion protein (MFP) AcrA
serves as a linker between TolC and the trimeric resistance
nodulation cell division (RND) pump AcrB, located in the inner
membrane acting as a proton/drug antiporter.
The newly elucidated asymmetric structure of trimeric AcrB
reveals three different monomer conformations representing
consecutive states in a transport cycle. The monomers show
tunnels with occlusions at different sites leading from the
lat-eral side through the periplasmic porter (pore) domains
towards the funnel of the trimer and TolC. The structural
changes create a hydrophobic pocket in one monomer, which
is not present in the other two monomers. Minocyclin and doxorubicin,
both AcrB substrates, specifically bind to this pocket substantiating
its role as drug binding pocket. The energy transduction from
the proton motive force into drug efflux includes proton binding
in (and release from) the transmembrane part. The conformational
changes observed within a triad of essential, titratable residues
(Asp407/Asp408/Lys940) residing in the hydrophobic transmembrane
domain appear to be transduced by transmembrane helix 8 and
associated with the conformational changes seen in the periplasmic
domain.
From the asymmetric structure a possible peristaltic pump
transport mechanism based on a functional rotation of the
AcrB trimer has been postulated. The novel transport model
merges Jardetzky’s alternate access pump mechanism with
the rotating site catalysis of F1Fo
ATPase and suggests a working hypothesis for the transport
mechanism of RND transporters in general.
[Back to top]
Membrane Permeability and Regulation of Drug “Influx
and Efflux” in Enterobacterial Pathogens
A. Davin-Regli, J-M. Bolla, C.E. James, J-P. Lavigne,
J. Chevalier, E. Garnotel, A. Molitor and J-M. Pagès
In Enterobacteriaceae, membrane permeability
is a « key » in the level of susceptibility to
antibiotics. Modification of the bacterial envelope by decreasing
the porin production or increasing the expression of efflux
pump systems has been reported. These phenomena are frequently
associated with other resistance mechanisms such as alteration
of antibiotics or modification of the drug targets, in various
clinical isolates showing a MultiDrugResistant phenotype (MDR).
In Escherichia coli, Enterobacter aerogenes, Klebsiella
pneumoniae and Salmonella enterica several genes and
external factors are involved in the emergence of MDR isolates.
These bacterial isolates exhibit a noticeable reduction of
func-tional porins per cell due to a decrease, a complete
shutdown of synthesis, or the expression of an altered porin
and a high expression of efflux systems (e.g. overexpression
of the pump). The combined action of these mechanisms during
an infection confers a significant decrease in bacterial sensitivity
to antibiotherapy ensuring dissemination and colonization
of the patient and favours the acquisition of additional mechanisms
of resistance. MarA and ramA are involved in a complex
regulation cascade controlling membrane permeability and actively
participate in the triggering of the MDR phenotype. Mutations
in regulator genes have been shown to induce the overproduction
of efflux and the down-regulation of porin synthesis. In addition,
various compounds such as salicylate, imipenem or chloramphenicol
are able to activate the MDR response. This phenomenon has
been observed both in vitro during culture of bacteria
in the presence of drugs and in vivo during antibiotic
treatment of infected patients. These effectors activate the
expression of specific global regulators, marA, ramA,
or target other genes located downstream in the regulation
cascade.
[Back to top]
New Methods for the Identification of Efflux Mediated MDR
Bacteria, Genetic Assessment of Regulators and Efflux Pump
Constituents, Characterization of Efflux Systems and Screening
for Inhibitors of Efflux Pumps
M. Viveiros, M. Martins, I. Couto, L. Rodrigues, G. Spengler,
A. Martins, J.E. Kristiansen, J. Molnar and L. Amaral
Immunology, Institute of Medical Microbiology, University
of Szeged, Hungary Abstract: We have developed a number of
methods that identify efflux pump mediated multi-drug resistant
bacteria, characterize efflux systems and screen for inhibitors
of efflux pumps. These approaches were complemented by the
quantification of the expression of genes that regulate and
code for constituents of efflux pumps. The methods described
are easy to use, reproducible and for the most part, require
instrumentation normally present in a clinical bacteriology
laboratory. Because each method provides good reproducibility,
they lend themselves for interlaboratory use.
[Back to top]
Physical Insights into Permeation of and Resistance
to Antibiotics in Bacteria
M. Ceccarelli and P. Ruggerone
Bacteria can resist antibiotics simply by hindering physical
access to the interior, where in general antibiotic targets
are located. Gram-negative bacteria, protected by the outer
membrane, possess in the latter several porins that act as
a gate for the exchange of small hydrophilic molecules. These
porins are water-filled membrane-protein channels that are
considered to be the main pathway for different class of antibiotics,
such as beta-lactams and fluoroquinolones. Bacterial strains
resistant to antibiotics can either decrease the density of
porins expressed in the outer membrane or decrease the porin
internal size by mutating a few amino acids. In both cases,
understanding how antibiotics diffuse through bacterial porins
can help the design of new antibiotics that have better penetrating
power. A considerable contribution can be offered by molecular
dynamics simulations since reliability of force fields, computer
power, and algorithms have consid-erably increased the predictive
power thereof. Large systems, as pores inserted in a membrane,
and long simulation runs are now feasible, and the time scale
can be even extended via the use of accelerated techniques,
such as metadynamics, and combined strategies. The details
of interactions and processes, extracted from the simulations,
complement experimental findings and also deepen aspects not
accessible to experiments. In this paper we will review the
results obtained by our group on this topic with a particular
focus on possible general criteria that can guide the rational
design of new anti-bacterial compounds.
[Back to top]
Biophysical Characterization of In- and Efflux in Gram-Negative
Bacteria
H. Weingart, M. Petrescu and M. Winterhalter
Gram-negative bacteria developed a number of tools to
avoid accumulation of cell-toxic compounds. The outer membrane
as a first defense system is tightly packed reducing permeation
through the lipid membrane. Water-soluble compounds may penetrate
through membrane channels called porins. Once inside the periplasmic
space special enzymes may welcome the foreign molecule for
inactivation. The molecules entering the inner membrane will
be harvested by efflux pumps and ejected back to the extra-cellular
space. Bacteria modulate all these barriers through the level
of protein expression or mutations. In order to understand
the function of the involved proteins a quantification of
the individual transport elements is necessary. Here we describe
recent biophysical methods to characterize molecular transport
across membranes.
[Back to top]
Clinical Impact of the Over-Expression of Efflux Pump in Nonfermentative
Gram Negative Bacilli, Development of Efflux Pump Inhibitors
J. Vila and J.L. Martínez
In this manuscript, we want to review the biochemical
and genetic characteristics of the different efflux pumps
involved in both intrinsic and acquired multiresistance in
non-fermentative Gram-negative bacteria such as Pseudomonas
aeruginosa, Acinetobacter baumannii, and Stenotrophomonas
maltophilia, as well as the regulation of their expression.
Moreover, the clinical impact of the over-expression of these
efflux pumps and the investigation developed to define efflux
pump inhibitors will be discussed. In this review it will
be stated that antimicrobial resistance associated with the
over-expression of MDR efflux pumps is widely recognised as
a frequent multidrug resistant determinant in nonfermentative
Gram-negative bacilli. Moreover, MDR pumps contribute to the
intrinsic resistance of these bacterial pathogens. Cir-cumventing
the activity of efflux pumps will thus have clear benefits
for therapy, since this will increase the susceptibility of
nonfermentative Gram-negative bacilli, thereby increasing
the therapeutic efficacy of antibiotics used for treating
such infections by those pathogens. In addition, it has been
shown that the lack of activity of MDR pumps impedes selection
of mutants showing high-level antibiotic resistance to antiotics
like quinolones or beta-lactams. Thus, besides reducing intrinsic
resistance, inhibitors of efflux pumps will reduce the emergence
of mutants that acquire antibiotic resistance as the consequence
of mutations in MDR-regulatory elements or in other targets.
Recent advances on the search for inhibitors of MDR pumps
will also be finally discussed.
[Back to top]
Antimicrobial Resistance in Foodborne Pathogens - A Cause
for Concern?
C. Walsh and S. Fanning
The widespread use of antibiotics in food animal production
systems has resulted in the emergence of antibiotic resistant
zoonotic bacteria that can be transmitted to humans through
the food chain. Infection with antibiotic resistant bacteria
negatively impacts on public health, due to an increased incidence
of treatment failure and severity of disease. Development
of resistant bacteria in food animals can result from chromosomal
mutations but is more commonly associated with the horizontal
transfer of resistance determinants borne on mobile genetic
elements. Food may represent a dynamic environment for the
continuing transfer of antibiotic resistance determinants
between bacteria. Current food preservation systems that use
a combination of environmental stresses to reduce growth of
bacteria, may serve to escalate development and dissemination
of antibiotic resistance among food related pathogens. The
increasing reliance on biocides for pathogen control in food
production and processing, heightens the risk of selection
of biocide-resistant strains. Of particular concern is the
potential for sublethal exposure to biocides to select for
bacteria with enhanced multi-drug efflux pump activity capable
of providing both resistance to biocides and cross-resistance
to multiple antibiotics. Although present evidence suggests
that biocide resistance is associated with a physiological
cost, the possibility of the development of adaptive mutations
conferring increased fitness cannot be ruled-out. Strategies
aimed at inhibiting efflux pumps and eliminating plasmids
could help to restore therapeutic efficacy to antibiotics
and reduce the spread of antibiotic resistant food-borne pathogens
through the food chain.
[Back to top]
Promising Therapy of XDR-TB/MDR-TB with Thioridazine an Inhibitor
of Bacterial Efflux Pumps
L. Amaral, M. Martins, M. Viveiros, J. Molnar and J. E.
Kristiansen
Global rates of pulmonary tuberculosis (TB) continue
to increase. Moreover, resistance of the causative organism
Mycobacterium tuberculosis to the two most effective
anti-TB medications continue to rise. Now, multi-drug resistant
TB (MDR-TB) has progressed to extensively drug resistant TB
(XDR-TB) - a M. tuberculosis organism that is resistant
to the most effective second line drugs available for the
treatment of TB. This review provides detailed, significant
evidence that supports the use of an old neuroleptic compound,
thioridazine (TZ), for the management of MDR-TB and XDR-TB
infections and which has been shown to inhibit efflux pumps
of bacteria. The argument has been previously presented but
no one seems to be listening - and the disease continues unabated
when there is a very good probability that the suggested drug
will prove to be effective. When the prognosis is poor, available
therapy predictably ineffective and death is inevitable, compassionate
therapy with TZ should be contemplated. The risks are small
and the rewards great.
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