Structure and serological properties of the O-antigen of two clinical Proteus mirabilis strains classified into a new Proteus O77 serogroup

Two Proteus mirabilis strains, 3 B-m and 3 B-k, were isolated from urine and faeces of a hospitalized patient from Lodz, Poland. It was suggested that one strain originated from the other, and the presence of the bacilli in the patient's urinary tract was most probably a consequence of autoinfection. The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of P. mirabilis 3 B-m and studied by sugar analysis and nuclear magnetic resonance spectroscopy, including two-dimensional rotating frame Overhause effect spectroscopy (ROESY) and 1H,13C heteronuclear single quantum coherence (HSQC) experiments. The following structure of the linear trisaccharide-repeating unit of the O-polysaccharide was established:-->2)-beta-D-Glcp-(1-->3)-alpha-L-6dTalp2Ac-(1-->3)-beta-D-GlcpNAc-(1-->where 6dTal2Ac stands for 2-O-acetyl-6-deoxy-L-talose. It resembles the structure of the O-polysaccharide of Proteus penneri O66, which includes additional lateral residues of 2,3-diacetamido-2,3,6-trideoxy-L-mannose. The lipopolysaccharides from two P. mirabilis strains studied were serologically identical to each other but not to that from any of the existing 76 Proteus O-serogroups. Therefore, the strains were classified into a new O77 serogroup specially created in the genus Proteus. Serological studies using Western blot and enzyme-linked immunosorbent assay with intact and adsorbed O-antisera showed that the P. mirabilis O77 antigen is related to Proteus vulgaris O2 and P. penneri O68 antigens, and a putative disaccharide epitope responsible for the cross-reactivity was revealed.     

Structural and serological relatedness of the O-antigens of Proteus penneri 1 and 4 from a novel Proteus serogroup O72.

O-specific polysaccharides (O-antigens) of the lipopolysaccharides (LPS) of Proteus penneri strains 1 and 4 were studied using sugar analysis, (1)H and (13)C NMR spectroscopy, including 2D COSY, H-detected (1)H,(13)C HMQC, and rotating-frame NOE spectroscopy (ROESY). The following structures of the tetrasaccharide (strain 1) and pentasaccharide (strain 4) repeating units of the polysaccharides were established: [reaction: see text]. In the polysaccharide of P. penneri strain 4, glycosylation with the lateral Glc residue (75%) and O-acetylation of the lateral GalNAc residue (55%) are nonstoichiometric. This polysaccharide contains also other, minor O-acetyl groups, whose positions were not determined. The structural similarity of the O-specific polysaccharides was consistent with the close serological relatedness of the LPS, which was demonstrated by immunochemical studies with O-antisera against P. penneri 1 and 4. Based on these data, it was proposed to classify P. penneri strains 1 and 4 into a new Proteus serogroup, O72, as two subgroups, O72a and O72a,b, respectively. Serological cross-reactivity of P. penneri 1 O-antiserum with the LPS of P. penneri 40 and 41 was substantiated by the presence of an epitope(s) on the LPS core region shared by all P. penneri strains studied.     

Identification of a Homopolymer of 5-Acetamidino-7-acetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-nonulosonic Acid in the Lipopolysaccharides of Legionella pneumophila Non-1 Serogroups

O-Specific polysaccharides (OPS) were isolated by mild acid hydrolysis of the lipopolysaccharides (LPS) of strainsof Legionella pneumophila serogroups 2-14, as well as strains Lansing 3 and 16453-92 from newly proposed serogroups. TheOPS were studied by ¹H- and ¹³C-NMR spectroscopy, GLC/mass spectrometry, and chemical modifications (mild alkalineO-deacetylation and conversion of the N-acetimidoyl group into the N-acetyl group). All OPS were found to be ahomopolymer of a 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxynonulosonic acid, which in some strains is 8-O-acetylat-ed. In most strains studied, the monosaccharide has the D-glycero-D-talo configuration and is thus the C4 epimer of legion-aminic acid (4-epilegionaminic acid), which has been previously identified as the monomer in the OPS of L. pneumophilaserogroup 1. Poly(4-epilegionaminic acid) occurs as a minor polysaccharide in serogroups 5 (strain Dallas 1) and 13 and isabsent in serogroups 1 and 7. The chemical basis for serological differentiation of L. pneumophila strains is discussed.     

Structural and Immunochemical Studies of Two Cross-Reactive Proteus mirabilis O-Antigens,O6 and O23, Containing β1→3-Linked 2-Acetamido-2-Deoxy-d-Glucopyranose Residues

A marked serological cross-reactivity was observed by ELISA and a precipitation test between anti-Proteus mirabilis O23 serum and the lipopolysaccharide as well as the O-specific polysaccharide from the Proteus mirabilis strain belonging to serogroup O6. The structures of the O-specific polysaccharides were elucidated using chemical and NMR spectroscopic analyses, and the only common component, 2-acetamido-2-deoxy-β-d -glucopyranose (β-d -GlcNAc), was revealed, which was suggested to be responsible for the cross-reactivity observed. Both anti-O23 and anti-O6 sera were shown to react with 1, 3-linked β-d -GlcNAc-containing O-antigen from Salmonella enterica ssp. arizonae O59 also. The lack of reactivity of Smith-degraded P. mirabilis O6 O-specific polysaccharide with homologous antiserum indicated the crucial role of α-d -glucuronic acid in specific antibody binding.     

免疫法筛选酵母HSA-IL-11融合蛋白转化子

报道了一种筛选高表达融合蛋白HSA-IL-11的毕赤酵母转化子的免疫双膜筛选法.将生长在醋酸纤维素滤膜上的转化子进行原位诱导,再用硝酸纤维素滤膜对表达的蛋白进行原位捕捉,并经封闭过夜后使用抗HSA抗体进行免疫杂交,再用标记二抗进行显色.根据显色强弱将转化子分为强阳性、中等和阴性三类,再用抗IL-11抗体进行复筛验证.结...     

Structural and genetic characterization of the Escherichia coli O180 O antigen and identification of a UDP-GlcNAc 6-dehydrogenase

The O antigen is an essential component of the lipopolysaccharides on the surface of Gram-negative bacteria and its variation provides a major basis for serotyping schemes. The Escherichia coli O-antigen form O180 was first designated in 2004, and O180 strains were found to contain virulence factors and cause diarrhea. Different O-antigen forms are almost entirely due to genetic variations in the O-antigen gene clusters. In this study, the chemical structure and gene cluster of E. coli O180 O antigen were investigated. A tetrasaccharide repeating unit with the following structure: →4)-β-d-ManpNAc3NAcA-(1?→?2)-α-l-Rhap(I)-(1?→?3)-β-l-Rhap(II)-(1?→?4)-α-d-GlcpNAc-(1→was identified in the E. coli O180 O antigen, including the residue d-ManpNAc3NAcA (2,3-diacetamido-2,3-dideoxy-d-mannopyranuronic acid) that had not been hitherto identified in E. coli. Genes in the O-antigen gene cluster were assigned functions based on their similarities with those from available databases, and five genes involved in the synthesis of UDP-d-ManpNAc3NAcA (the nucleotide-activated form of d-ManpNAc3NAcA) were identified. The gnaA gene, encoding the enzyme involved in the initial step of the UDP-d-ManpNAc3NAcA biosynthetic pathway, was cloned and the enzyme product was expressed, purified and assayed for its activity. GnaA was characterized using capillary electrophoresis and electrospray ionization mass spectrometry and identified as a UDP-GlcNAc 6-dehydrogenase. The kinetic and physicochemical parameters of GnaA also were determined.     

Structural and genetic characterization of enterohemorrhagic Escherichia coli O145 O antigen and development of an O145 serogroup-specific PCR assay

Enterohemorrhagic Escherichia coli O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the E. coli O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamidoylamino-2,6-dideoxy-L-galactose, and N-acetylneuraminic acid. It is very closely related to Salmonella enterica serovar Touera and S. enterica subsp. arizonae O21 antigen. The E. coli O145 gene cluster is located between the JUMPStart sequence and the gnd gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the E. coli O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An E. coli O145 serogroup-specific PCR assay based on the genes wzx and wzy was also developed by screening E. coli and Shigella isolates of different serotypes.     

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->     

Structural and genetic relationships between the O-antigens of Escherichia coli O118 and O151

O-antigen (O-polysaccharide) is a highly variable part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, which is used as the basis for bacterial serotyping and is essential for the full function and virulence of bacteria. In this work, the structure and genetics of the O-antigens of Escherichia coli O118 and O151 were investigated. Both O-polysaccharides were found to contain ribitol phosphate and have similar structures, the only difference between their backbones being one linkage mode (β1→3 in E. coli O118 vs. β1→2 in E. coli O151), which, most probably, is the linkage between the oligosaccharide repeats (O-units). The O-antigen gene clusters of the two bacteria are organized in the same manner and share high-level identity (>99%). Analysis of the wzy genes from E. coli O118 and O151 strains, which are responsible for the linkage between O-units, revealed only one nucleotide substitution, resulting in one amino acid residue substitution. The possible genetic events that may lead to the structural difference between two O-antigen structures are discussed. Salmonella O47 has the same O-unit backbone and a similar O-antigen gene cluster (OGC) (the DNA identity ranges from 74% to 83%) as E. coli O118 and O151. It was suggested that the OGCs of the three bacteria studied originated from a common ancestor.     

Structural and genetic characterization of the O-antigen of Salmonella enterica O56 containing a novel derivative of 4-amino-4,6-dideoxy-d-glucose

Based on the O-antigens (O-polysaccharides), one of the most variable cell constituents, 46 O-serogroups have been recognized in the Kauffmann-White serotyping scheme for Salmonella enterica. In this work, the structure of the O-polysaccharide and the genetic organization of the O-antigen gene cluster of S. enterica O56 were investigated. As judged by sugar and methylation analyses, along with NMR spectroscopic data, the O-polysaccharide has a linear tetrasaccharide O-unit, which consists of one residue each of d-ribofuranose, N-acetyl-d-glucosamine, N-acetyl-d-galactosamine, and a novel sugar derivative, 4-(N-acetyl-l-seryl)amino-4,6-dideoxy-d-glucose (d-Qui4NSerAc). The following structure of the O-polysaccharide was established:→3)-β-d-Quip4NSerAc-(1→3)-β-d-Ribf-(1→4)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen gene cluster of S. enterica O56 having 12 open reading frames was found between the housekeeping genes galF and gnd. A comparison with databases and using the O-antigen structure data enabled us to ascribe functions to genes for (i) synthesis of d-GalNAc and d-Qui4NSerAc, (ii) sugar transfer, and (iii) O-antigen processing, including genes for O-unit flippase (Wzx) and O-antigen polymerase (Wzy).