Publications of Moreno Galleni
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See detailDetection and Characterization of VIM-52, a New Variant of VIM-1 from a Klebsiella pneumoniae Clinical Isolate
de Barsy, Marie; Mercuri, Paola ULiege; Oueslati, Saoussen et al

in Antimicrobial Agents and Chemotherapy (2021), 65(Issue 11), 02660-20

ABSTRACT Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-b-lactamases (MBLs), which inactivate virtually all b-lactams ... [more ▼]

ABSTRACT Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-b-lactamases (MBLs), which inactivate virtually all b-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime compared to VIM-1. These results were confirmed by steady-state kinetic measurements, where VIM-52 yielded a lower activity toward ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221 to 241), which borders the active site. As Arg 224 and Ser 228 both play an important and interrelated role in enzymatic activity, stability, and substrate specificity for the MBLs, targeted mutagenesis at both positions was performed and further confirmed their crucial role for substrate specificity. [less ▲]

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See detailConversion of electrospun chitosan into chitin: a robust strategy to tune the properties of 2D biomimetic nanofiber scaffolds
Toncheva-Moncheva, Natalia; Aqil, Abdelhafid ULiege; Galleni, Moreno ULiege et al

in Polysaccharides (2021), 2(2), 271-286

New biomimetic micro- and nano-CsU-based fibrous scaffolds electrospun from solution containing high purity-medical grade chitosan (CsU) of fungus origin (CsU1, Mv ~174,000 and CsU2, 205,000, degree of ... [more ▼]

New biomimetic micro- and nano-CsU-based fibrous scaffolds electrospun from solution containing high purity-medical grade chitosan (CsU) of fungus origin (CsU1, Mv ~174,000 and CsU2, 205,000, degree of deacetylation (DDA) ~65%) and polyethylene oxide (PEO, Mv ~ 900,000), in the presence of given amounts of Triton X-100 (from 0.01 to 0.5 wt%) as surfactant were fabricated. We demonstrate that by carefully selecting compositions and surfactant levels, porous mats with CsU content up to 90% (at this molecular weight and DDA) were achieved. Remarkable long-term stability in water or phosphate buffer solution storage were obtained by developing post-electrospinning treatment allowing the complete elimination of the PEO from the CsU-fibers as demonstrated by TGA, DSC and ESEM analysis. Subsequent reacetylation procedure was applied to convert 2D biomimetic chitosan mats to chitin (CsE)-based ones while preserving the nanofiber structure. This innovative procedure allows tuning and modifying the thermal, mechanical properties and more importantly the biodegradation abilities (fast enzymatic biodegradation in some cases and slower on the others) of the prepared nanofibrous mats. The established reproducible method offers the unique advantage to modulate the membrane properties leading to stable 2D biomimetic CsU and/or chitin (CsE) scaffolds tailor-made for specific purposes in the field of tissue engineering. [less ▲]

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See detailLaboratory Variants GESG170L, GESG170K, and GESG170H Increase Carbapenem Hydrolysis and Confer Resistance to Clavulanic Acid
Piccirilli, Alessandra; Mercuri, Paola ULiege; Segatore, Bernardetta et al

in Antimicrobial Agents and Chemotherapy (2021), 65(6), 01931-20

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See detailExploring the Role of L10 Loop in New Delhi Metallo- beta-lactamase (NDM-1): Kinetic and Dynamic Studies
Piccirilli, Alessandra; Criscuolo, Emanuele; Brisdelli, Fabrizia et al

in Molecules (2021), 26

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid ... [more ▼]

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some -lactams. Significant reduction of kcat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some -lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2- OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues. [less ▲]

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See detailMutational Effects on Carbapenem Hydrolysis of YEM-1, a New Subclass B2 Metallo-β-Lactamase from Yersinia mollaretii
Mercuri, Paola ULiege; Esposito, Roberto; Blétard, Sylvie et al

in Antimicrobial Agents and Chemotherapy (2020)

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See detaildi-Cysteine Residues of the Arabidopsis thaliana HMA4 C-Terminus Are Only Partially Required for Cadmium Transport
Ceasar, S. A.; Lekeux, Gilles ULiege; Motte, Patrick ULiege et al

in Frontiers in Plant Science (2020), 11

Cadmium (Cd) is highly toxic to the environment and humans. Plants are capable of absorbing Cd from the soil and of transporting part of this Cd to their shoot tissues. In Arabidopsis, the plasma membrane ... [more ▼]

Cadmium (Cd) is highly toxic to the environment and humans. Plants are capable of absorbing Cd from the soil and of transporting part of this Cd to their shoot tissues. In Arabidopsis, the plasma membrane Heavy Metal ATPase 4 (HMA4) transporter mediates Cd xylem loading for export to shoots, in addition to zinc (Zn). A recent study showed that di-Cys motifs present in the HMA4 C-terminal extension (AtHMA4c) are essential for high-affinity Zn binding and transport in planta. In this study, we have characterized the role of the AtHMA4c di-Cys motifs in Cd transport in planta and in Cd-binding in vitro. In contrast to the case for Zn, the di-Cys motifs seem to be partly dispensable for Cd transport as evidenced by limited variation in Cd accumulation in shoot tissues of hma2hma4 double mutant plants expressing native or di-Cys mutated variants of AtHMA4. Expression analysis of metal homeostasis marker genes, such as AtIRT1, excluded that maintained Cd accumulation in shoot tissues was the result of increased Cd uptake by roots. In vitro Cd-binding assays further revealed that mutating di-Cys motifs in AtHMA4c had a more limited impact on Cd-binding than it has on Zn-binding. The contributions of the AtHMA4 C-terminal domain to metal transport and binding therefore differ for Zn and Cd. Our data suggest that it is possible to identify HMA4 variants that discriminate Zn and Cd for transport. © Copyright © 2020 Ceasar, Lekeux, Motte, Xiao, Galleni and Hanikenne. [less ▲]

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See detailMutational Effects in Carbapenem Hydrolysis of YEM-1, a New Sub-Class B2 Metallo-beta-Lactamase from Yersinia mollaretii
Mercuri, Paola ULiege; Esposito, Roberto; Blétard, Sylvie et al

Poster (2019, June 22)

Background The genome of Yersinia mollaretii ATCC43969 was totally sequenced (NCBI NZ_AALD0200 0006.1). Its analysis indicated the presence of two β-lactamase genes, namely blaB and yem-1 coding for an ... [more ▼]

Background The genome of Yersinia mollaretii ATCC43969 was totally sequenced (NCBI NZ_AALD0200 0006.1). Its analysis indicated the presence of two β-lactamase genes, namely blaB and yem-1 coding for an AmpC-like and a metallo β-lactamase respectively. Up to date, many studies on BlaB were performed (1,2). Our work aimed to characterize the kinetic profile of YEM-1 and the residues essential for its activity. Methods YEM-1 was produced in E. coli Rosetta and purified to homogeneity. A survey of its kinetic properties was performed against different β-lactam antibiotics. A model of the three-dimensional structure of YEM-1 was build using the known structure of Aeromonas hydrophila CphA as template. Its analysis highlighted the major residues substitution between CphA and YEM-1. These residues were mutated by site-directed mutagenesis. The kinetic properties of the different mutants were analyzed. Results We noted that, at 37°C, YEM-1 is produced as inclusion bodies. We obtained a soluble form of YEM-1 when the culture was grown at 18°C in presence of IPTG 0.1 mM. The MBL was purified in three chromatographic steps that include a HiTRAP SP, an IMAC and a molecular sieve. The kinetic analysis of the YEM-1 showed that only imipenem was hydrolyzed efficiently. We confirmed that it belongs to the subclass B2 MBL family. As expected, the structure of YEM-1 has a similar fold than CphA. We found that the main differences between CphA and YEM-1 were the residues in position 67, 156 and 236 (BBL numbering) respectively. By site-directed mutagenesis, we made the YEM-1 single mutants (Y67V, T156F, S236F), the double mutant T156F-S236F and the triple mutant (Y67T156FS236F) in order to design the active site of CphA in YEM-1. The substitution Y67V yielded a global increase of the catalytic efficiency to carbapenems, with the exception of the imipenem. The mutations of T156 and/or S236 in phenylalanine globally decrease the enzymatic activity of the enzyme. Finally the double mutant and triple mutants did not increase the catalytic efficiencies of YEM-1 against carbapenems. Conclusions Despite a high sequence's similarity between CphA and YEM-1 (57%), we showed that the Yersinia MBL was less effective than CphA against carbapemens. We also highlighted the substitution Y67V in YEM-1 that increased its efficiency against all the carbapemen tested and in particular ertapenem and meropenem. [less ▲]

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See detailExploring the suitability of RanBP2-type Zinc Fingers for RNA-binding protein design
De Franco, Simona ULiege; Vandenameele, Julie ULiege; Brans, Alain ULiege et al

in Scientific Reports (2019), 9(1), 2484

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See detailHomology modeling and in vivo functional characterization of the zinc permeation pathway in a heavy metal P-type ATPase
Lekeux, Gilles ULiege; Crowet, Jean-Marc ULiege; Nouet, Cécile ULiege et al

in Journal of Experimental Botany (2019), 70

The P1B ATPase Heavy Metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in the plant Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements ... [more ▼]

The P1B ATPase Heavy Metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in the plant Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements enabling metal transport across the membrane. Thanks to high conservation level within the P-type ATPase family, the role of the HMA4 cytoplasmic catalytic domains can be inferred from well characterized pumps. In contrast, the function of its terminal cytosolic extensions as well as the metal permeation mechanism through the membrane remains elusive. Here, homology modeling of the HMA4 transmembrane region was conducted based on the crystal structure of a ZntA bacterial homolog. The analysis highlighted amino acids forming a metal permeation pathway, whose importance was subsequently investigated functionally through mutagenesis and complementation experiments in plants. Although the zinc pathway displayed overall conservation among the two proteins, significant differences were observed, especially in the entrance area with altered electronegativity and the presence of a salt bridge/H-bond network. The analysis also newly identified amino acids whose mutation results in total or partial loss of the protein function. In addition, comparison of zinc and cadmium accumulation in shoots of A. thaliana complemented lines revealed a number of HMA4 mutants exhibiting different abilities in zinc and cadmium translocation. These observations could be instrumental to design low cadmium accumulating crops, hence decreasing human cadmium exposure . [less ▲]

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See detailUnderstanding the significance and implications of antibody numbering and antigen-binding surface/residue definition
Dondelinger, Mathieu ULiege; Filée, Patrice; Sauvage, Eric ULiege et al

in Frontiers in Immunology (2018), 9(OCT),

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See detailCombinatorial Design of a Nanobody that Specifically Targets Structured RNAs
Cawez, Frédéric ULiege; Duray, Elodie ULiege; Hu, Y et al

in Journal of Molecular Biology (2018), 430(11), 1652-1670

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See detailThe use of a β-lactamase – based conductimetric biosensor assay to detect biomolecular interactions
Vandevenne, Marylène ULiege; Dondelinger, Mathieu ULiege; Yunus, Sami et al

in Journal of Visualized Experiments (2018), (132),

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See detailA Kinetic Study of the Replacement by Site Saturation Mutagenesis of Residue 119 in NDM-1 Metallo- beta-Lactamase
Marcoccia, Francesca; Mercuri, Paola ULiege; Galleni, Moreno ULiege et al

in Antimicrobial Agents and Chemotherapy (2018), 62(8),

New Delhi metallo- beta-lactamase 1 (NDM-1) is a subclass B1 metallo-beta - lactamase that exhibits a broad spectrum of activity against beta -lactam antibiotics. Here we report the kinetic study of 6 ... [more ▼]

New Delhi metallo- beta-lactamase 1 (NDM-1) is a subclass B1 metallo-beta - lactamase that exhibits a broad spectrum of activity against beta -lactam antibiotics. Here we report the kinetic study of 6 Q119X variants obtained by site-directed mutagenesis of NDM-1. All Q119X variants were able to hydrolyze carbapenems, penicillins and first-, second-, third-, and fourth-generation cephalosporins very efficiently. In particular, Q119E, Q119Y, Q119V, and Q119K mutants showed improvements in kcat/Km values for penicillins, compared with NDM-1. The catalytic efficiencies of the Q119K variant for benzylpenicillin and carbenicillin were about 65- and 70-fold higher, respectively, than those of NDM-1. The Q119K and Q119Y enzymes had kcat/Km values for ceftazidime about 25- and 89-fold higher, respectively, than that of NDM-1. [less ▲]

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See detailStudy of the outer membrane permeability of Pseudomonas aeruginosa to ß-lactam antibiotics
Amisano, Francesco ULiege; Silvestri, Mauro ULiege; Mercuri, Paola ULiege et al

Poster (2017, June 17)

Background The resistance of Gram negative bacteria toward β-lactam antibiotics is caused by the interplay between four independent factors: i) the alteration of the sensitivity of the target enzymes, the ... [more ▼]

Background The resistance of Gram negative bacteria toward β-lactam antibiotics is caused by the interplay between four independent factors: i) the alteration of the sensitivity of the target enzymes, the penicillin-binding proteins, ii) the properties and concentration of the periplasmic β-lactamases, iii) the permeability of the outer membrane, iv) the efficiency of the active efflux system. On this basis, Zimmermann and Rosselet [1] proposed a model yelding a quantitative prediction of the MICs for gram-negative bacteria which was successfully applied to Escherichia coli and Enterobacter cloacae. This model seems to be less suitable in Pseudomonas aeruginosa due to its low outer membrane permeability which is mostly influenced by both a remarkable reduction of functional porins expression and an over-expression of efflux systems [2]. This decreased permeability causes difficulties in obtaining permeability coefficient direct measures. Moreover, the few published coefficients for P. aeruginosa are highly variable. For this purpose, BlaR-CTD, the C-terminal domain of a highly sensitive penicillin binding protein from Bacillus licheniformis, expressed in the periplasmic space, has been used in order to directly determinate of the concentrations of different β-lactams in this cell compartment and, consequently to obtain reliable measures of the permeability coefficient [3]. Methods P. aeruginosa PAO1 cells were incubated with different β-lactams, whose penetration into the periplasm is rapidly followed by the formation of a stable complex with BlaR-CTD. This latter was quantified in cells lysate by densitometric analysis, countermarking the free BlaR-CTD with a fluorescent β-lactam. The excess of the antibiotics will be hydrolysed by the addition of a class B β-lactamase. We used the same protocol for P. aeruginosa TNP004 [4], a PAO1 strain with a selective deletion of OprD porin, in order to study the influence of this single mutation for the antibiotic permeability. Results By the approach described above we determined the permeability coefficients of the external membrane of P. aeruginosa for different antibiotics belonging to the penicillin, cephalosporin and carbapenem sub-families. The comparison with the porin mutant strain showed similar coefficients for all the antibiotic tested except, as expected, for Imipenem Conclusion This work allowed a preliminary characterization of antibiotic permeability in P. aeruginosa which was poorly studied until now. Furthermore, we could apply this method to correlate the permeability with the role of porin deletions and/or efflux pumps overexpression in antibiotic resistant strains of clinical relevance. References 1 Zimmermann, W. and A. Rosselet. 1977. Function of the outer membrane of Escherichia coli as a permeability barrier to beta-lactam antibiotics. Antimicrob. Agents Chemother. 12:368–372. 2 Livermore D. M., and K. W. M. Davy. 1991. Invalidity for Pseudomonas aeruginosa of an accepted model of bacterial permeability to β-lactam antibiotics. Antimicrob. Agents Chemother. 35:916-921. 3 Lakaye B., Dubus A., Joris B., and J.M. Frère. 2002. Method for estimation of low outer membrane permeability to β-lactam antibiotics. Antimicrob. Agents Chemother. 46:2901-2907. 4 Yoneyama H., Yamano Y and T. Nakae. 1995 Role of porins in the antibiotic susceptibility of Pseudomonas aeruginosa: construction of mutants with deletions in the multiple porin genes. Biochem Biophys Res Commun. 213:88-95. [less ▲]

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See detailCharacterisation of the sub-class B2 metallo-β-lactamase of Yersinia mollaretii Wauters
Mercuri, Paola ULiege; Blétard, Sylvie; Kerff, Frédéric ULiege et al

Poster (2017, June)

Background. The sub class B2 metallo-β- lactamases are Zn2+-dependent enzymes that efficiently hydrolyzes only carbapenem antibiotics. We study the metallo-β-lactamase (MBL) produced by a soil bacteria ... [more ▼]

Background. The sub class B2 metallo-β- lactamases are Zn2+-dependent enzymes that efficiently hydrolyzes only carbapenem antibiotics. We study the metallo-β-lactamase (MBL) produced by a soil bacteria named Yersinia mollaretii Wauters DMS 18520 (1). The enzyme exhibits two potential zinc-binding sites, the conserved residues Zn1 site (N116-H118-H196), and Zn2 site (D120-C221-H263), tipycals of the sub-class B2 metallo-β- lactamases. Compared to CphA, we found the similar α3 helix located near the active-site and three prolines of the "rich prolines loop" were conserved (2). Materials&methods. The gene coding for the metallo-β-lactamase was isolated by PCR from the genomic DNA as template. The MBL was produced in E. coli Rosetta (DE3) pLysS with the help of overexpression vector as pET26b. The enzyme was produced in TB medium at 18°C in presence of 100 µM IPTG. The metallo-β- lactamases was purified by three steps, an ion exchange chromatography (Sepharose SP-HP column), a Pentadentate Chelator (PDC) Zn2+ column, followed by a molecular sieve column (Superdex 75). pH dependence of activity was study. The enzymatic profile was studied on a representative numbers of carbapenems and measured in the presence of increasing concentrations of zinc. The influence of chelating agents on the β-lactamase activity was also assessed. Results. The MBL from Yersinia mollaretii was overproduced in E. coli Rosetta (DE3) pLysS and purified in three chromatography steps as described in Materials & Methods. The active fractions were pooled and concentrated. The enzyme showed the best activity in MES buffer at pH 6.0. The steady state kinetic parameters were determined for a representative set of carbapenems antibiotics. We showed that Imipenem was the best substrate among the tested substrates. Conclusion. Compared to CphA the new sub class B2 MBL shows a narrow antibiotics profile, a reduced susceptibility toward high zinc concentration and to zinc chelators. [less ▲]

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See detailNDM-1 could still evolve: analysis of a random mutagenesis in position 123
Marcoccia, Francesca; Melchiorre, C; Robert, Charly ULiege et al

Poster (2017, June)

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See detailHuman chitotriosidase: Catalytic domain or carbohydrate binding module, who's leading HCHT's biological function
Crasson, Oscar ULiege; Courtade, Gaston; Léonard, Raphaël ULiege et al

in Scientific Reports (2017), 7(1),

Detailed reference viewed: 51 (12 ULiège)