Clinical Evolution of New Delhi Metallo-β-Lactamase (NDM) optimizes resistance under Zn(II) Deprivation

Carbapenem-resistant Enterobacteriaceae (CRE) are rapidly spreading and taking a staggering toll on all health care systems, largely due to the dissemination of genes coding for potent carbapenemases. An important family of carbapenemases are the Zn(II)-dependent β-lactamases, known as metallo-β-lactamases (MBLs). Among them, the New Delhi metallo-β-lactamase (NDM) has experienced the fastest and widest geographical spread. While other clinically important MBLs are soluble periplasmic enzymes, NDMs are lipoproteins anchored to the outer membrane in Gram-negative bacteria. This unique cellular localization endows NDMs with enhanced stability upon the Zn(II) starvation elicited by the immune system response at the sites of infection. Since the first report of NDM-1, new allelic variants (16 in total) have been identified in clinical isolates differing by a limited number of substitutions. Here, we show that these variants have evolved by accumulating mutations that enhance their stability or the Zn(II) binding affinity in vivo, overriding the most common evolutionary pressure acting on catalytic efficiency. We identified the ubiquitous substitution M154L as responsible for improving the Zn(II) binding capabilities of the NDM variants. These results also reveal that Zn(II) deprivation imposes a strict constraint on the evolution of this MBL, overriding the most common pressures acting on catalytic performance, and shed light on possible inhibitory strategies.Fil: Bahr, Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Vitor Horen, Luisina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bethel, Christopher R.. Louis Stokes Cleveland VA Medical Center; Estados UnidosFil: Bonomo, Robert A.. Louis Stokes Cleveland VA Medical Center; Estados UnidosFil: Gonzalez, Lisandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

A simple protocol to characterize bacterial cell-envelope lipoproteins in a native-like environment

Physiological conditions in living cells are strictly regulated to allow, optimize, and coordinate biological processes. The bacterial cell envelope is the compartment where the communication with the external environment takes place. This involves membrane proteins, key players in many biological processes that ensure bacterial survival. The biochemical characterization of membrane proteins, either integral, lipidated or peripheral is challenging due to their mixed protein-lipid nature, making it difficult to purify and obtain considerable amounts of samples. In contrast to integral membrane proteins, lipidated proteins are usually purified as truncated soluble versions, neglecting the impact of the membrane environment. Here we report a simple and robust protocol to characterize bacterial lipidated proteins in spheroplasts from Escherichia coli using a β-lactamase as a model. The Metallo-β-lactamase NDM-1 is an enzyme anchored to the inner leaflet of the outer membrane of Gram-negative bacteria. Kinetic parameters and stability of the lipidated NDM-1 and the soluble unbound version (NDM-1 C26A) were measured in spheroplasts and periplasm, respectively. These studies revealed that membrane anchoring increases the KM of the enzyme, consequently decreasing the catalytic efficiency, while not affecting its kinetic stability. This approach can be used to characterize lipidated proteins avoiding the purification step while mimicking its native environment. This approach also helps in filling the gap between in vitro and in vivo studies.Fil: Giannini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Gonzalez, Lisandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

Soil health -- a new challenge for microbiologists and chemists.

The original publication is available on LINK at http://revistes.iec.cat/index.php/IM/article/view/4c457c718d531.002 This work, including photographs and other illustrations, unless the contrary is indicated, is subject to an Attributions–Non-Commercial–ShareAlike 3.0 Creative Commons License, the full text of which can be consulted at http://creativecommons.org/licenses/by-nc-sa/3.0/. You are free to share, copy, distribute and transmit the work provided that the author is credited and reuse of the material is restricted to non-commercial purposes only and that if you alter, transform, or build upon this work, you may distribute the resulting work only under the same or similar license to this on

Sterilization by SCCO2 technology: Can we destroy bacterial spores while being gentle to absorbable biomaterials?

info:eu-repo/semantics/publishedVersio

Host-Specific Enzyme-Substrate Interactions in SPM-1 Metallo-beta-Lactamase are Modulated by Second Sphere Residues

Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-beta-lactamases (MBLs), enzymes able to hydrolyze most beta-lactam antibiotics. SPM-1 is an MBL produced only by P. aeruginosa, while other MBLs are found in different bacteria. Despite similar active sites, the resistance profile of MBLs towards beta-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MBLs in that in that it contains "atypical" second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards beta-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.Fil: Gonzalez, Lisandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Moreno, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; ArgentinaFil: Bonomo, Robert A.. Case Western Reserve University; Estados UnidosFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentin

Searching for the one(s): Using Probiotics as Anthelmintic Treatments

Helminths are a major health concern as over one billion people are infected worldwide and, despite the multiple efforts made, there is still no effective human vaccine against them. The most important drugs used nowadays to control helminth infections belong to the benzimidazoles, imidazothiazoles (levamisole) and macrocyclic lactones (avermectins and milbemycins) families. However, in the last 20 years, many publications have revealed increasing anthelmintic resistance in livestock which is both an economical and a potential health problem, even though very few have reported similar findings in human populations. To deal with this worrying limitation of anthelmintic drugs, alternative treatments based on plant extracts or probiotics have been developed. Probiotics are defined by the Food and Agriculture Organization as live microorganisms, which, when consumed in adequate amounts, confer a health benefit to the host. It has been proven that probiotic microbes have the ability to exert an immunomodulatory effect both at the mucosa and the systemic level. The immune response against gastrointestinal helminths is characterized as a type 2 response, with high IgE levels, increased numbers and/or activity of Th2 cells, type 2 innate lymphoid cells, eosinophils, basophils, mast cells, and alternatively activated macrophages. The oral administration of probiotics may contribute to controlling gastrointestinal helminth infections since it has been demonstrated that these microorganisms stimulate dendritic cells to elicit a type 2 or regulatory immune response, among other effects on the host immune system. Here we review the current knowledge about the use of probiotic bacteria as anthelmintic therapy or as a complement to traditional anthelmintic treatments. Considering all research papers reviewed, we may conclude that the effect generated by probiotics on helminth infection depends not only on the parasite species, their stage and localization but also on the administration scheme.Fil: Saracino, María Priscila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Inmunología; ArgentinaFil: Vila, Cecilia Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Inmunología; ArgentinaFil: Baldi, Pablo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Inmunología; ArgentinaFil: Gonzalez Maglio, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Inmunología; Argentin

Enrique Vila-Matas. Avec la participation de Dominique Gonzalez-Foerster

Enrique Vila-Matas et Dominique Gonzalez-Foerster Alain Prochiantz a évoqué, en guise d’introduction à la conférence, le dernier ouvrage d’Enrique Vila-Matas, Mac et son contretemps, dans lequel on trouve une phrase tirée du Degré zéro de l’écriture : « le roman est une mort qui fait de la vie un destin ». Ce livre est une réflexion sur l’écriture menée, pour ainsi dire, de l’intérieur de l’écriture. Une dislocation, une inspiration mallarméenne, non sans correspondances avec les arts contem..

Protein determinants of dissemination and host specificity of metallo-β-lactamases

The worldwide dissemination of metallo-β-lactamases (MBLs), mediating resistance to carbapenem antibiotics, is a major public health problem. The extent of dissemination of MBLs such as VIM-2, SPM-1 and NDM among Gram-negative pathogens cannot be explained solely based on the associated mobile genetic elements or the resistance phenotype. Here, we report that MBL host range is determined by the impact of MBL expression on bacterial fitness. The signal peptide sequence of MBLs dictates their adaptability to each host. In uncommon hosts, inefficient processing of MBLs leads to accumulation of toxic intermediates that compromises bacterial growth. This fitness cost explains the exclusion of VIM-2 and SPM-1 from Escherichia coli and Acinetobacter baumannii, and their confinement to Pseudomonas aeruginosa. By contrast, NDMs are expressed without any apparent fitness cost in different bacteria, and are secreted into outer membrane vesicles. We propose that the successful dissemination and adaptation of MBLs to different bacterial hosts depend on protein determinants that enable host adaptability and carbapenem resistance.Fil: López, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ayala, Juan A.. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; EspañaFil: Bonomo, Robert A.. Louis Stokes Cleveland Department of Veterans Affairs Medical Center; Estados Unidos. Case Western Reserve University; Estados Unidos. Center for Antimicrobial Resistance and Epidemiology; Estados UnidosFil: Gonzalez, Lisandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Vila, Alejandro Jose. Center for Antimicrobial Resistance and Epidemiology; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

The Metallo-β-lactamase GOB Is a Mono-Zn(II) Enzyme with a Novel Active Site

Metallo-β-lactamases (MβLs) are zinc-dependent enzymes able to hydrolyze and inactivate most β-lactam antibiotics. The large diversity of active site structures and metal content among MβLs from different sources has limited the design of a pan-MβL inhibitor. Here we report the biochemical and biophysical characterization of a novel MβL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MβLs. Contrasting all other related MβLs, GOB-18 is fully active against a broad range of β-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MβLs