RESUMO
The outer membrane permeability of Serratia marcescens was studied by comparing porin-deficient mutants with their parental strains. Omp1-deficient strains were selected by moxalactam resistance, whereas mutants lacking the Omp2 porin were obtained by experimental infection with the SMP2 phage, whose primary receptor is the Omp2 porin. The role of porins was demonstrated in quinolone accumulation assays, where semiquantitative differences in accumulation were observed. Permeability coefficients to cephaloridine of Omp1 mutants were determined and compared with those of the parental strain. The clinical isolates S. marcescens HCPR1 and 866 showed 30- to 200-fold reduced permeability coefficients when Omp1 porin was absent.
Assuntos
Antibacterianos/farmacologia , Proteínas de Peixes , Porinas/fisiologia , Serratia marcescens/efeitos dos fármacos , Antibacterianos/metabolismo , Anti-Infecciosos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/fisiologia , Bacteriófagos/efeitos dos fármacos , Bacteriófagos/genética , Bacteriófagos/ultraestrutura , Ciprofloxacina/metabolismo , Meios de Cultura , Farmacorresistência Bacteriana , Eletroforese em Gel de Poliacrilamida , Proteínas da Matriz Extracelular/fisiologia , Cinética , Testes de Sensibilidade Microbiana , Mutação , Permeabilidade , Porinas/genética , Serratia marcescens/genética , Serratia marcescens/metabolismo , beta-Lactamases/metabolismoRESUMO
Serratia marcescens outer membrane contains three different general diffusion porins: Omp1, Omp2 and Omp3. Omp1 was cloned and sequenced and it shows a great homology to the family of outer membrane porins that comprises the general porins of enteric bacteria. The gene for Omp1 was transferred into an expression plasmid and was expressed in Escherichia coli UH302 (E. coli UH302 pOM100), a porin deficient strain. Its expression confers a higher susceptibility towards different antibiotics to this strain. Omp1 was purified to homogeneity from outer membrane of E. coli UH302 pOM100. Reconstitution of the purified protein into black lipid bilayers demonstrated that it is a channel-forming component with a single-channel conductance of approximately 2 nS in 1 M KCl similar to that of other porins from enteric bacteria. Omp1 is slightly cation-selective. Its homology to already crystallised members of the family of enteric porins whose three-dimensional-structures are known and allowed the design of a topology model for Omp1. The charge distribution within a porin monomer is similar as in other general diffusion pores. The positively charged amino acids localised at the beta-strands opposite the external loop L3, which restrict the pore diameter in the porin monomer.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Porinas/química , Serratia marcescens/genética , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/fisiologia , Clonagem Molecular , Condutividade Elétrica , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Potenciais da Membrana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Porinas/genética , Porinas/fisiologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
In this work the porin Omp1 of Serratia marcescens was expressed in a porin deficient mutant (Escherichia coli UH302) and its functionality studied following the accumulation of ciprofloxacin in bacteria. The protein was extracted, purified and reconstituted in proteoliposomes of different composition (lipopolysaccharide (LPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)). Maximum extraction of the detergent was achieved applying different steps of dialysis and centrifugation. Proteolipid sheets with different composition were spread onto mica and observed by atomic force microscopy. Two-dimensional crystal of Omp1 was not observed in any case due to low resolution achieved. Judging from the images features POPC is the most suitable phospholipid to enhance 2D lattice formation for Omp1.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Dimiristoilfosfatidilcolina/metabolismo , Microscopia de Força Atômica , Fosfatidilcolinas/metabolismo , Proteolipídeos/metabolismo , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Ciprofloxacina/isolamento & purificação , Ciprofloxacina/metabolismo , Cristalização , Escherichia coli/genética , Escherichia coli/metabolismo , Indicadores e Reagentes/metabolismo , Bicamadas Lipídicas , Membranas , Porinas/deficiência , Serratia marcescens/químicaRESUMO
BACKGROUND: Populations of Pseudomonas aeruginosa have been extensively studied, although there is no general agreement concerning their genetic structure. It has been proposed that P. aeruginosa is a very homogeneous species with 90% of individuals within the same clonal group; nonetheless, other results suggested that Pseudomonas populations are panmictic. Here we compared P. aeruginosa populations from clinical and environmental samples, both isolated from the Bellvitge Hospital of the University of Barcelona in Spain. METHODS: Antibiotic susceptibility determination as well as whole cell and outer membrane protein denaturing gel electrophoresis, pulsed-field electrophoresis, and random amplified polymorphic DNA analysis were performed. RESULTS: Environmental isolates were much more susceptible to antibiotics than those isolated from clinical specimens. The remainder of the analyses revealed high degree of diversity. CONCLUSIONS: Whole-cell proteins, outer-membrane proteins, and pulsed field electrophoresis did not support a close relationship between clinical and environmental isolates. Random amplified polymorphic DNA (RAPD) confirmed the distance between isolates from both sources. This suggests that the origin of hospital infections by P. aeruginosa is due mainly to growth of bacterial strains acquired by patients prior to hospital admission or from patient-to-patient through healthcare workers (HCWs).
Assuntos
Técnicas de Tipagem Bacteriana , Infecção Hospitalar , Resistência Microbiana a Medicamentos , Infecções por Pseudomonas/diagnóstico , Pseudomonas aeruginosa/genética , Antibacterianos/farmacologia , Eletroforese em Gel de Campo Pulsado , Eletroforese em Gel de Poliacrilamida , Genótipo , Hospitais , Humanos , Polimorfismo Genético , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
Unclassifiable lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma is a new category of B-cell lymphoma appearing in the new World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. This lymphoma usually shows MYC rearrangements with non-IGH genes in the setting of a complex karyotype possibly involving BCL2 and, less frequently, BCL6 rearrangements. According to the presence of two or three rearrangements, these lymphomas are called double-hit lymphomas or triple-hit lymphomas (THL), respectively. Here we report two cases of THL with MYC, BCL2, and BCL6 rearrangements and t(3;8)(q27;q24) diagnosed in one center in the last two years.