Jean-marie Pagès | Aix-Marseille University (original) (raw)

Papers by Jean-marie Pagès

Communications Biology

Gram-negative porins are the main entry for small hydrophilic molecules. We studied translocation... more Gram-negative porins are the main entry for small hydrophilic molecules. We studied translocation of structurally related cephalosporins, ceftazidime (CAZ), cefotaxime (CTX) and cefepime (FEP). CAZ is highly active on E. coli producing OmpF (Outer membrane protein F) but less efficient on cells expressing OmpC (Outer membrane protein C), whereas FEP and CTX kill bacteria regardless of the porin expressed. This matches with the different capacity of CAZ and FEP to accumulate into bacterial cells as quantified by LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry). Furthermore, porin reconstitution into planar lipid bilayer and zero current assays suggest permeation of ≈1,000 molecules of CAZ per sec and per channel through OmpF versus ≈500 through OmpC. Here, the instant killing is directly correlated to internal drug concentration. We propose that the net negative charge of CAZ represents a key advantage for permeation through OmpF porins that are less cation-selective than Om...

Subcellular Biochemistry, 2019

The transport of small molecules across membranes is essential for the import of nutrients and ot... more The transport of small molecules across membranes is essential for the import of nutrients and other energy sources into the cell and, for the export of waste and other potentially harmful byproducts out of the cell. While hydrophobic molecules are permeable to membranes, ions and other small polar molecules require transport via specialized membrane transport proteins . The two major classes of membrane transport proteins are transporters and channels. With our focus here on porins-major class of non-specific diffusion channel proteins , we will highlight some recent structural biology reports and functional assays that have substantially contributed to our understanding of the mechanism that mediates uptake of small molecules, including antibiotics, across the outer membrane of Enterobacteriaceae . We will also review advances in the regulation of porin expression and porin biogenesis and discuss these pathways as new therapeutic targets.

Antibiotics, 2021

Antibiotic efflux is a mechanism that is well-documented in the phenotype of multidrug resistance... more Antibiotic efflux is a mechanism that is well-documented in the phenotype of multidrug resistance in bacteria. Efflux is considered as an early facilitating mechanism in the bacterial adaptation face to the concentration of antibiotics at the infectious site, which is involved in the acquirement of complementary efficient mechanisms, such as enzymatic resistance or target mutation. Various efflux pumps have been described in the Gram-negative bacteria most often encountered in infectious diseases and, in healthcare-associated infections. Some are more often involved than others and expel virtually all families of antibiotics and antibacterials. Numerous studies report the contribution of these pumps in resistant strains previously identified from their phenotypes. The authors characterize the pumps involved, the facilitating antibiotics and those mainly concerned by the efflux. However, today no study describes a process for the real-time quantification of efflux in resistant clinic...

Communications Biology, 2020

With the spreading of antibiotic resistance, the translocation of antibiotics through bacterial e... more With the spreading of antibiotic resistance, the translocation of antibiotics through bacterial envelopes is crucial for their antibacterial activity. In Gram-negative bacteria, the interplay between membrane permeability and drug efflux pumps must be investigated as a whole. Here, we quantified the intracellular accumulation of a series of fluoroquinolones in population and in individual cells of Escherichia coli according to the expression of the AcrB efflux transporter. Computational results supported the accumulation levels measured experimentally and highlighted how fluoroquinolones side chains interact with specific residues of the distal pocket of the AcrB tight monomer during recognition and binding steps.

Antibiotics, 2020

This study reported the phytochemical composition of two hydroethanolic extracts of Acacia senega... more This study reported the phytochemical composition of two hydroethanolic extracts of Acacia senegal and Acacia seyal trees from Burkina Faso and their activities, alone or in combination with selected antibiotics, against multidrug resistant bacteria. High performance thin layer chromatography (HPTLC) method was used for phytochemical screening. Total phenolic and total flavonoid ant tannins in leaves extracts contents were assessed by spectrophotometric method. The minimal inhibitory concentrations (MICs) of plant extracts and antibiotics were determined using the microdilution method and p-iodonitrotetrazolium chloride. Combinations of extracts and antibiotics were studied using checkerboard assays. Screening revealed the presence of phenolic compounds, flavonoids, and tannins in the hydroethanolic extract (HE) of the leaves. The HE of A. seyal showed the highest total phenolic (571.30 ± 6.97 mg GAE/g), total flavonoids (140.41 ± 4.01 mg RTE/g), and tannins (24.72 ± 0.14%, condense...

Antimicrobial Agents and Chemotherapy, 2020

The Cpx stress response is widespread among Enterobacteriaceae . We previously reported a mutatio... more The Cpx stress response is widespread among Enterobacteriaceae . We previously reported a mutation in cpxA in a multidrug-resistant strain of Klebsiella aerogenes isolated from a patient treated with imipenem. This mutation yields a single-amino-acid substitution (Y144N) located in the periplasmic sensor domain of CpxA. In this work, we sought to characterize this mutation in Escherichia coli by using genetic and biochemical approaches.

Research in Microbiology, 2018

To understand the antibiotic resistance in Gram-negative bacteria, a key point is to investigate ... more To understand the antibiotic resistance in Gram-negative bacteria, a key point is to investigate antibiotic accumulation which is defined by influx and efflux. Several methods exist to evaluate the membrane permeability and efflux pump activity but they present some disadvantages and limitations. An optimized spectrofluorimetric method using the intrinsic tryptophan fluorescence as internal standard as well as a complementary microfluorimetric assay following the time-course accumulation in intact individual cells have been developed. Comparing the latter population and single cell approaches can lead to the understanding of the phenotypic heterogeneity within a population. The two methodologies lead to the determination of parameters, concentration, accumulation rates, localization that contribute to emerging concepts (RTC2T, SICAR) with the aim to identify and detail the antibiotic chemotypes that are involved in influx/efflux.

Nature Protocols, 2018

the efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrati... more the efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. this is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMsF). these different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (oM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. the three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (Wcls) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d.

Antibiotics, 2017

Antibiotic resistant Gram-negative bacteria are a serious threat for public health. The permeatio... more Antibiotic resistant Gram-negative bacteria are a serious threat for public health. The permeation of antibiotics through their outer membrane is largely dependent on porin, changes in which cause reduced drug uptake and efficacy. Escherichia coli produces two major porins, OmpF and OmpC. MicF and MicC are small non-coding RNAs (sRNAs) that modulate the expression of OmpF and OmpC, respectively. In this work, we investigated factors that lead to increased production of MicC. micC promoter region was fused to lacZ, and the reporter plasmid was transformed into E. coli MC4100 and derivative mutants. The response of micC-lacZ to antimicrobials was measured during growth over a 6 h time period. The data showed that the expression of micC was increased in the presence of β-lactam antibiotics and in an rpoE depleted mutant. Interestingly, the same conditions enhanced the activity of an ompN-lacZ fusion, suggesting a dual transcriptional regulation of micC and the quiescent adjacent ompN. Increased levels of OmpN in the presence of sub-inhibitory concentrations of chemicals could not be confirmed by Western blot analysis, except when analyzed in the absence of the sigma factor σ E. We suggest that the MicC sRNA acts together with the σ E envelope stress response pathway to control the OmpC/N levels in response to β-lactam antibiotics.

Microbiology, 2018

Bacteria have evolved several strategies to survive a myriad of harmful conditions in the environ... more Bacteria have evolved several strategies to survive a myriad of harmful conditions in the environment and in hosts. In Gramnegative bacteria, responses to nutrient limitation, oxidative or nitrosative stress, envelope stress, exposure to antimicrobials and other growth-limiting stresses have been linked to the development of antimicrobial resistance. This results from the activation of protective changes to cell physiology (decreased outer membrane permeability), resistance transporters (drug efflux pumps), resistant lifestyles (biofilms, persistence) and/or resistance mutations (target mutations, production of antibiotic modification/degradation enzymes). In targeting and interfering with essential physiological mechanisms, antimicrobials themselves are considered as stresses to which protective responses have also evolved. In this review, we focus on envelope stress responses that affect the expression of outer membrane porins and their impact on antimicrobial resistance. We also discuss evidences that indicate the role of antimicrobials as signaling molecules in activating envelope stress responses.

Nature Microbiology, 2017

PloS one, 2015

Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in... more Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations ...

Journal of medical microbiology, Jan 3, 2015

The continuing emergence of multidrug resistance phenotype in Gram-negative bacteria make the dev... more The continuing emergence of multidrug resistance phenotype in Gram-negative bacteria make the development of rapid susceptibility tests mandatory. To achieve this goal, proprietary specific media for bacterial growth can be used but may have some adverse effects. In this study, we dissect the role of media on porin, efflux pump and ß-lactamase expression. Depending on the medium used, we observed a change in piperacillin-tazobactam (TZP) susceptibility for some isolates, such as increases in MIC values. No significant alteration in efflux activity or in ß-lactamase production was detected after changing the incubation medium. The ratio piperacillinase/nitrocefinase showed no specific alteration, indicating that the media do not affect significantly the relative enzymatic affinity for the substrates. In contrast, osmotic variation is able to modulate both porin expression and OmpC/OmpF balance thus modulating the antibiotic uptake.This study suggests that porin expression may be impa...

Targets, Mechanisms and Resistance, 2013

ABSTRACT The worldwide dissemination of resistant bacteria has severely reduced the efficacy of o... more ABSTRACT The worldwide dissemination of resistant bacteria has severely reduced the efficacy of our antibiotic arsenal and increased the frequency of therapeutic failure. Modifications of membrane permeability by changing the expression of transporters alter the mechanical barrier to control the intracellular concentration of antibiotics. This first line of bacterial defense actively participates in the dissemination of multidrug-resistance phenotype.The regulation of membrane permeability and the expression of appropriate channel-forming proteins such as porins or efflux pumps is a key way controlling the intracellular concentration of β-lactams and quinolones, two prominent classes of our antibiotic arsenal. It is necessary to decipher functional, structural, and genetic aspects of the membrane transporters to understand their involvement in membrane physiology and permeability. Regarding the clinical aspect, resistant bacterial strains exhibit significant variations in transporters' expression under antibiotic pressure, demonstrating their role in the adaptation of membrane permeability.Faced with bacterial membrane adaptation, a number of scientific challenges address the drug influx and efflux in resistant isolates. By what means can we circumvent the bacterial membrane controls and bypass the barrier: by using permeabilizers and increasing the influx rate? How can we submerge the efflux activity: by synthesizing efflux blockers? Several studies carried out in these large areas have provided information for the development of a new generation of antibacterial agents exhibiting a variety of chemical–biological properties. Consequently, different chemical or natural groups of modulators of the bacterial membrane permeability have been characterized to increase the intracellular concentration of antibiotics as “escort or adjuvant” molecules. Used in combination, they can restore the activity of old antibiotics in multidrug-resistant gram-negative bacteria.

Revue Francophone des Laboratoires, 2011

ABSTRACT Pseudomonas aeruginosa est un pathogène important en médecine humaine et il est surtout ... more ABSTRACT Pseudomonas aeruginosa est un pathogène important en médecine humaine et il est surtout associé aux infections rencontrées chez les patients hospitalisés. La faible perméabilité de sa membrane externe, l’expression de pompes d’efflux et son adaptation rapide à la présence d’antibiotiques sont autant d’obstacles majeurs dans le traitement des infections. Le mécanisme d’efflux MexAB-OprM est avec AcrAB-TolC l’archétype des pompes d’efflux à trois composants couramment rencontrées chez les isolats résistants des bactéries à Gram négatif. Les travaux concernant la résolution de la structure de la pompe et de ses composants, son assemblage, et les bases moléculaires du transport des molécules reconnues ont permis de proposer des modèles pour tenter de comprendre le fonctionnement de ce complexe membranaire expulsant les agents antibactériens. Durant ces dernières années, ces données ont été la base de la synthèse et de la sélection de nouvelles molécules destinées à bloquer ces mécanismes de résistance par efflux. Summary Pseudomonas aeruginosa is a nosocomial and community-acquired pathogen associated with considerable patient morbidity and mortality. Multidrug resistance in P. aeruginosa is a concern owing to the limited therapeutic options available to treat infections due to this organism. The continuous dissemination of «multi-drug resistant» (MDR) bacteria drastically reduces the efficacy of our antibiotic “arsenal” and consequently increases the frequency of therapeutic failure. In Pseudomonas aeruginosa MDR bacteria the over-expression of efflux pumps, as MexAB-OprM, expel structurally-unrelated antibiotics decreasing their intracellular concentration. Several clinical data indicate an increasing contribution of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter and to combat this mechanism.

Current Drug Target -Infectious Disorders, 2005

The emergence and spread of antiparasitic drug resistance pose a severe and increasing public hea... more The emergence and spread of antiparasitic drug resistance pose a severe and increasing public health threat. Failures in prophylaxis or those in treatment with quinolines, hydroxynaphtoquinones, sesquiterpenic lactones, antifolate drugs, arsenic and antimony containing drugs sulfamides induce reemergence of parasitic-related morbidity and mortality. Resistance is often associated with alteration of drug accumulation into parasites, which results from a reduced uptake of the drug, an increased efflux or, a combination of the two processes. Resistance to quinolines, artemisinin derivatives and arsenicals and expression of an active efflux mechanism are more or less correlated in protozoa like Plasmodium spp., Leishmania spp., and Trypanosoma spp. Various parasite candidate genes have been proposed to be involved in drug resistance, each concerned in membrane transport. Genes encoding membrane glycoproteins, orthologue to the P-glycoproteins identified in MDR human cancer cells, have been described in these resistant pathogens in addition to various membrane proteins involved in drug transport. Several compounds have demonstrated, in the past decade, promising capability to reverse the drug resistance in parasite isolates in vitro, in animal models and for human malaria. These drugs belong to different pharmacological classes such as calcium channel blockers, tricyclic antidepressants, antipsychotic calmodulin antagonists, histamine H1-receptor antagonists, analgesic antipyretic drugs, non-steroidal anti-inflammatory drugs, and to different chemical classes such as synthetic surfactants, alkaloids from plants used in traditional medicine, pyrrolidinoaminoalkanes and derivatives, and anthracene derivatives. Here, are summarized the molecular bases of antiparasitic resistance emphasizing recent developments with compounds acting on trans-membrane proteins involved in drug efflux or uptake.

Trends in Molecular Medicine, 2005

In Gram-negative bacteria, efflux complexes, consisting of an inner-membrane pump, a periplasmic ... more In Gram-negative bacteria, efflux complexes, consisting of an inner-membrane pump, a periplasmic adaptor protein and outer-membrane channel, provide an efficient means for the export of structurally unrelated drugs, causing the multidrug-resistance phenotype. Resistance due to this antibiotic efflux is an increasing problem worldwide. A new molecular challenge is to combat this transport by searching for new molecules to block efflux and thus restore drug susceptibility to resistant clinical strains. Recent data shed new light on the structure and activity of the archetypal efflux pumps AcrAB-TolC and MexAB-OprM. Here, we describe recent insights into the molecular mechanisms of bacterial efflux pumps and their inhibitors. Current progress for the clinical use of efflux-pump inhibitors and new strategies to combat the drug-efflux mechanisms will be discussed.

Journal of Chromatography B, 2003

The tripartite AcrA-AcrB-TolC system is the major efflux pump of the nosocomial pathogen Enteroba... more The tripartite AcrA-AcrB-TolC system is the major efflux pump of the nosocomial pathogen Enterobacter aerogenes. AcrA is a trimeric periplasmic lipoprotein anchored in the inner membrane, AcrB is an inner membrane transporter and TolC is a trimeric outer membrane channel. In order to reconstitute the AcrA-AcrB-TolC system of E. aerogenes in artificial membranes, we overexpressed and purified the three proteins. The E. aerogenes acrA, acrB and tolC open reading frames were individually inserted in the expression vector pET24a(+), in frame with a sequence coding a C-terminal hexahistidine tag to allow purification by INAC (Immobilized Nickel Affinity Chromatography). The mature AcrA-6His was overproduced in a soluble form in the cytoplasm of Escherichia coli BL21(DE3). AcrA-6His was purified under native conditions in two steps using INAC and gel permeation chromatography. We obtained about 25 mg of 97% pure AcrA-6His per liter of culture. AcrB-6His was solubilized from the membrane fraction of E. coli C43(DE3) in 300 mM NaCl, 5% Triton X-100 and purified in one step by INAC. The AcrB-6His enriched fraction was eluted with 100 mM imidazole. The final yield was 1-2 mg of 95% pure AcrB-6His per liter of culture. The membrane fraction of E. coli BL21(DE3)pLysS containing TolC-6His was first treated with 2% Triton X-100, 30 mM MgCl(2) to solubilize the inner membrane proteins. After ultracentrifugation, the pellet was treated with 5% Triton X-100, 5 mM EDTA to solubilize the outer membrane proteins. Approximately 5 mg of 95% pure TolC-6His trimers per liter of culture was purified by INAC.

Journal of Biological Chemistry, 2010

An integrative approach combining biophysical and microbiological methods was used to characteriz... more An integrative approach combining biophysical and microbiological methods was used to characterize the antibiotic translocation through the outer membrane of Providencia stuartii. Two novel members of the General Bacterial Porin family of Enterobacteriaceae, named OmpPst1 and OmpPst2, were identified in P. stuartii. In the presence of ertapenem (ERT), cefepime (FEP), and cefoxitin (FOX) in growth media, several resistant derivatives of P. stuartii ATCC 29914 showed OmpPst1-deficiency. These porin-deficient strains showed significant decrease of susceptibility to ␤-lactam antibiotics. OmpPst1 and OmpPst2 were purified to homogeneity and reconstituted into planar lipid bilayers to study their biophysical characteristics and their interactions with ␤-lactam molecules. Determination of ␤-lactam translocation through OmpPst1 and OmpPst2 indicated that the strength of interaction decreased in the order of ertapenem Ͼ Ͼ cefepime > cefoxitin. Moreover, the translocation of these antibiotics through OmpPst1 was more efficient than through OmpPst2. Heterologous expression of OmpPst1 in the porin-deficient E. coli strain BL21(DE3)omp8 was associated with a higher antibiotic susceptibility of the E. coli cells to ␤-lactams compared with expression of OmpPst2. All our data enlighten the involvement of porins in the resistance of P. stuartii to ␤-lactam antibiotics. Providencia stuartii is an opportunistic pathogen involved in community-acquired as well as hospital-acquired infectious diseases. Clinical strains of P. stuartii are mostly isolated from urinary tract infections of patients with long-term indwelling urinary catheters and, in fewer cases, from respiratory and skin infections (1, 2). P. stuartii is reported as one of the most resistant species in the family of Enterobacteriaceae (3). P. stuartii strains show high levels of resistance to the majority of antibiotic classes but were found to remain susceptible to carbapenems (3, 4). P. stuartii produces a chromosomally encoded cephalosporinase, AmpC, which causes the natural resistance

Journal of Antimicrobial Chemotherapy, 2007

After several decades of continuously successful antibiotic therapy against bacterial infections,... more After several decades of continuously successful antibiotic therapy against bacterial infections, we are now facing a worrying prospect: the accelerated evolution of antibiotic resistance to important human pathogens and the scarcity of new anti-infective drug families under development. Efflux is a general mechanism responsible for bacterial resistance to antibiotics. This active drug transport is involved in low intrinsic susceptibility, cross-resistance to chemically unrelated classes of molecules, and selection/acquisition of additional mechanisms of resistance. Thus, inhibition of bacterial efflux mechanisms appears to be a promising target in order to (i) increase the intracellular concentration of antibiotics that are expelled by efflux pumps, (ii) restore the drug susceptibility of resistant clinical strains, and (iii) reduce the capability for acquired additional resistance. Structurally unrelated classes of efflux pump inhibitors (EPIs) have been described and tested in the last decade, including some analogues of antibiotic substrates and new chemical molecules. Among the current collection of EPIs, only a few compounds have been studied taking into account the structure-activity relationships and the spectrum of activity in terms of antibiotics, pumps and bacteria. While large efforts have characterized an increasing number of bacterial efflux pumps and generated several potentially active EPIs, they have not elucidated the molecular basis of efflux transport and inhibition. Recent studies of pumpsubstrate complexes, the 3D resolution of the efflux pumps, the synthesis of novel compounds and molecular dynamic studies may generate new clues to decipher and select novel targets inside the efflux mechanisms and, finally, may result in a clinically useful molecule.