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1.
Nat Chem Biol ; 16(4): 450-457, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152541

RESUMO

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-ß-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.


Assuntos
Glicosiltransferases/metabolismo , Antígenos O/metabolismo , Antígenos O/ultraestrutura , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Glicosiltransferases/fisiologia , Hexosiltransferases , Klebsiella pneumoniae/metabolismo , Lipopolissacarídeos/química , Polissacarídeos Bacterianos/química
2.
Clin Vaccine Immunol ; 21(7): 930-9, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24789798

RESUMO

The Escherichia coli lineage sequence type 131 (ST131)-O25b:H4 is a globally spread multidrug-resistant clone responsible for a great proportion of extraintestinal infections. Driven by the significant medical needs associated with this successful pathogenic lineage, we generated murine monoclonal antibodies (MAbs) against its lipopolysaccharide (LPS) O25b antigen in order to develop quick diagnostic tests. Murine monoclonal antibodies were generated by immunizing mice with whole killed nonencapsulated ST131-O25b E. coli cells and screening hybridoma supernatants for binding to purified LPS molecules obtained from an E. coli ST131-O25b clinical isolate. The MAbs selected for further study bound to the surface of live E. coli O25b strains irrespective of the capsular type expressed, while they did not bind to bacteria or purified LPS from other serotypes, including the related classical O25 antigen (O25a). Using these specific MAbs, we developed a latex bead-based agglutination assay that has greater specificity and is quicker and simpler than the currently available typing methods. The high specificities of these MAbs can be explained by the novel structure of the O25b repeating unit elucidated in this article. Based on comparative analysis by nuclear magnetic resonance (NMR) and mass spectrometry, the N-acetyl-fucose in the O25a O-antigen had been replaced by O-acetyl-rhamnose in the O25b repeating unit. The genetic determinants responsible for this structural variation were identified by aligning the corresponding genetic loci and were confirmed by trans-complementation of a rough mutant by the subserotype-specific fragments of the rfb operons.


Assuntos
Anticorpos Monoclonais/imunologia , Infecções por Escherichia coli/diagnóstico , Antígenos O/genética , Antígenos O/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Farmacorresistência Bacteriana Múltipla , Escherichia coli/imunologia , Infecções por Escherichia coli/imunologia , Imunização , Testes de Fixação do Látex , Camundongos , Dados de Sequência Molecular , Antígenos O/ultraestrutura
3.
Water Res ; 46(6): 1838-46, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22285041

RESUMO

In wastewater treatment plants, optimizing bacterial flocculation and bacterial sludge dewatering requires a detailed understanding of the concomitant biological and physico-chemical processes governing the action of flocculating agent on living cells. Here we investigate the interactions between polyethyleneimine (PEI, 60,000g/mol) and Shewanella oneidensis MR-1 lacking or not the lipopolysaccharide (LPS) O-antigen surface structure. Flocculation tests were performed on bacteria with/without LPS O-antigen after being exposed to 0-100mg/L PEI concentrations. Measurements of electrophoretic mobility and bacterial aggregates size were complemented by transmission electron micrographs and atomic force microscopy images. While low PEI concentrations (<20mg/L) lead to flocculation of both bare and LPS O-antigen-decorated bacterial strains, the lysis of bacterial membranes occurred at larger polymer concentrations for the latter, which highlights the protective role of LPS O-antigen against harmful PEI-mediated membrane alterations. Depending on polymer concentration, two types of bacterial aggregates are identified: one that solely integrates bacterial cells, and another that includes both cells and cell residues resulting from lysis (membrane and/or LPS fragments, and inner cell content materials). The latter is expected to significantly contribute to water entrapping in sludge and thus lower dewatering process efficiency.


Assuntos
Extensões da Superfície Celular/efeitos dos fármacos , Polietilenoimina/farmacologia , Shewanella/citologia , Shewanella/efeitos dos fármacos , Extensões da Superfície Celular/metabolismo , Condutividade Elétrica , Eletroforese , Floculação/efeitos dos fármacos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Modelos Moleculares , Antígenos O/metabolismo , Antígenos O/ultraestrutura , Shewanella/ultraestrutura
4.
J Bacteriol ; 187(22): 7619-30, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16267286

RESUMO

High-pressure freeze-substitution and transmission electron microscopy have been used for high-resolution imaging of the natural structure of a gram-negative biofilm. Unlike more conventional embedding techniques, this method confirms many of the observations seen by confocal microscopy but with finer structural detail. It further reveals that there is a structural complexity to biofilms at both the cellular and extracellular matrix levels that has not been seen before. Different domains of healthy and lysed cells exist randomly dispersed within a single biofilm as well as different structural organizations of exopolymers. Particulate matter is suspended within this network of fibers and appears to be an integral part of the exopolymeric substance (EPS). O-side chains extending from the outer membrane are integrated into EPS polymers so as to form a continuum. Together, the results support the concept of physical microenvironments within biofilms and show a complexity that was hitherto unknown.


Assuntos
Biofilmes , Pseudomonas aeruginosa/ultraestrutura , Biopolímeros/metabolismo , Matriz Extracelular/ultraestrutura , Substituição ao Congelamento , Microscopia Eletrônica de Transmissão , Antígenos O/metabolismo , Antígenos O/ultraestrutura , Polissacarídeos Bacterianos/metabolismo , Polissacarídeos Bacterianos/ultraestrutura , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/crescimento & desenvolvimento
5.
Proc Natl Acad Sci U S A ; 93(20): 10584-8, 1996 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-8855221

RESUMO

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and D1 serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two O-antigenic repeating units from Salmonella typhimurium, Salmonella enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.


Assuntos
Bacteriófago P22/ultraestrutura , Glicosídeo Hidrolases/ultraestrutura , Antígenos O/ultraestrutura , Polissacarídeos Bacterianos/ultraestrutura , Receptores Virais/ultraestrutura , Salmonella/ultraestrutura , Proteínas da Cauda Viral/ultraestrutura , Sítios de Ligação , Sequência de Carboidratos , Cristalografia por Raios X , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Antígenos O/metabolismo , Salmonella/imunologia , Proteínas da Cauda Viral/metabolismo
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