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Yuriy A. Knirel - One of the best experts on this subject based on the ideXlab platform.
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Serological characterization of the O-specific polysaccharide of Providencia alcalifaciens 023
Archivum Immunologiae Et Therapiae Experimentalis, 2020Co-Authors: Agnieszka Torzewska, Yuriy A. Knirel, Agnieszka Maszewska, Nina A. Kocharova, Antoni RozalskiAbstract:The genus Providencia belongs to the Enterobacteriaceae family and currently consists of five species: P. alcalifaciens, P. heimbachae, P. rettgerii, P. rustigianii and P. stuartii. The serological classification scheme of P. alcalifaciens, P. rustigianii and P. stuartii includes 63 O-serogroups and 30 H-serogroups. The O-antigenic specificity is defined by the structure of the O-antigen (O-specific polysaccharide - OPS), a part of the lipopolysaccharide (LPS, endotoxin), one of the major components of the outer membrane of Gram-negative bacteria and an important virulence factor of these bacteria. Among the bacteria of the Enterobacteriaceae family, the genus Providencia is one of the least studied in respect to its LPS structure and antigenic specificity. Studies of the chemical structures and the serological specificity of the O-antigens aim at the elucidation of the molecular basis of the serological classification of Providencia sp. LPS and alkali-treated LPS of P. alcalifaciens 023 and serologically related P. rustigianii 014, P. mirabilis 013 and P. myxofaciens as well as O-antiserum against P. alcalifaciens 023 were used. Serological characterization of P. alcalifaciens 023 O-specific polysaccharide was done by use enzyme immunosorbent assay (EIA), passive hemolysis test (PHT) as well as by inhibition and sodium deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) of LPS and Western blot. The OPS of P. alcalifaciens, 023, contains an N-(D-glucuronoyl)-N-[(R)-1-carboxyethyl]-L-lysine residue (GlcAAlaLys). The LPS of P. alcalifaciens, 023, and other LPSs containing AlaLys from Providencia and Proteus strains were tested with rabbit anti-P. alcalifaciens 023 serum. The serological data showed that a GlcAAlaLys-associated epitope plays a role as an antigenic determinant in the P. alcalifaciens 023 OPS and revealed the particular importance of glucuronic acid and the carboxyethyl group for the binding of 023-specific antibodies.
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structure of the o polysaccharide from the lipopolysaccharide of Providencia alcalifaciens o33
Carbohydrate Research, 2014Co-Authors: Olga G. Ovchinnikova, Antoni Rozalski, Alexander S Shashkov, Alexander O Chizhov, Magdalena Moryl, Yuriy A. KnirelAbstract:Abstract Mild acid degradation of the lipopolysaccharide from Providencia alcalifaciens O33 resulted in an O-polysaccharide along with core and O-unit-bearing core oligosaccharides. Composition of the oligosaccharides was inferred by ESI mass spectrometry. Based on sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy data, the following structure of the tetrasaccharide O-unit of the O-polysaccharide was established: Download : Download full-size image Another O-polysaccharide structure has been reported earlier for Providencia stuartii О33 but later found to belong to a P. stuartii О52 strain.
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o antigens of bacteria of the genus Providencia structure serology genetics and biosynthesis
Biochemistry, 2013Co-Authors: Olga G. Ovchinnikova, Antoni Rozalski, Yuriy A. KnirelAbstract:The genus Providencia consists of eight species of opportunistic pathogenic enterobacteria that can cause enteric diseases and urinary tract infections. The existing combined serological classification scheme of three species, P. alcalifaciens, P. stuartii, and P. rustigianii, is based on the specificity of O-antigens (O-polysaccharides) and comprises 63 O-serogroups. Differences between serogroups are related to polymorphism at a specific genome locus, the O-antigen gene cluster, responsible for O-antigen biosynthesis. This review presents data on structures of 36 O-antigens of Providencia, many of which contain unusual monosaccharides and non-carbohydrate components. The structural data correlate with the immunospecificity of the O-antigens and enable substantiation on a molecular level of serological relationships within the genus Providencia and between strains of Providencia and bacteria of the genera Proteus, Escherichia, and Salmonella. Peculiar features of the O-antigen gene cluster organization in 10 Providencia serogroups and biosynthetic pathways of nucleotide precursors of specific monosaccharide components of the O-antigens also are discussed.
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structural serological and genetic characterization of the o antigen of Providencia alcalifaciens o40
Fems Immunology and Medical Microbiology, 2012Co-Authors: Antoni Rozalski, Alexander S Shashkov, Nina A. Kocharova, Olga G. Ovchinnikova, Lu Feng, Lei Wang, Magdalena Bialczakkokot, Yuriy A. KnirelAbstract:The O-polysaccharide chain of the lipopolysaccharide (O-antigen) on the bacterial cell surface is one of the most structurally variable cell components and serves as a basis for serotyping of Gram-negative bacteria, including human opportunistic pathogens of the genus Providencia . In this work, the O-antigen of Providencia alcalifaciens O40 was obtained by mild acid degradation of the isolated lipopolysaccharide and studied by chemical methods and high-resolution NMR spectroscopy. The following structure of the O-polysaccharide was established: →4)-β-d-Qui p 3NFo-(1→3)-α-d-Gal p -(1→3)-β-d-Glc p A-(1→3)-β-d-Gal p NAc-(1→, where GlcA stands for glucuronic acid and Qui3NFo for 3,6-dideoxy-3-formamidoglucose. The O40-antigen was found to be structurally and serologically related to the O-antigens of P. alcalifaciens O5 and Providencia stuartii O18. The O40-antigen gene cluster between cpxA and yibK was sequenced, and the gene functions were predicted in silico . In agreement with the O-polysaccharide structure established, the genes for the synthesis of dTDP-d-Qui3NFo, UDP-d-Gal, UDP-d-GlcA, and UDP-d-GalNAc as well as those encoding three glycosyltransferases, flippase (Wzx), and O-antigen polymerase (Wzy) were recognized. In addition, homologues of wza, wzb , and wzc genes, which are required for the surface expression of capsular polysaccharides, were found within the gene cluster, suggesting that the O-polysaccharide studied is a part of the capsule-related form of the lipopolysaccharide called KLPS.
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genetic analysis of the o antigen of Providencia alcalifaciens o30 and biochemical characterization of a formyltransferase involved in the synthesis of a qui4n derivative
Glycobiology, 2012Co-Authors: Miao Chen, Yuriy A. Knirel, Antoni Rozalski, Olga G. Ovchinnikova, Lu Feng, Andrei V Perepelov, Dawei Zhou, Lei WangAbstract:O-Antigen is a component of the outer membrane of Gram-negative bacteria and one of the most variable cell surface constituents, giving rise to major antigenic variability. The diversity of O-antigen is almost entirely attributed to genetic variations in O-antigen gene clusters. Bacteria of the genus Providencia are facultative pathogens, which can cause urinary tract infections, wound infections and enteric diseases. Recently, the O-antigen gene cluster of Providencia was localized between the cpxA and yibK genes in the genome. However, few genes involved in the synthesis of Providencia O-antigens have been functionally identified. In this study, the putative O-antigen gene cluster of Providencia alcalifaciens O30 was sequenced and analyzed. Almost all putative genes for the O-antigen synthesis were found, including a novel formyltransferase gene vioF that was proposed to be responsible for the conversion of dTDP-4-amino-4,6- dideoxy-D-glucose (dTDP-D-Qui4N) to dTDP-4,6-dideoxy-4-formamido-D-glucose (dTDP-D-Qui4NFo). vioF was cloned, and the enzyme product was expressed as a His-tagged fusion protein, purified and assayed for its activity. High-performance liquid chromatography was used to monitor the enzyme-substrate reaction, and the structure of the product dTDP-D-Qui4NFo was established by electrospray ionization tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Kinetic parameters of VioF were determined, and effects of temperature and cations on its activity were also examined. Together, the functional analyses support the identification of the O-antigen gene cluster of P. alcalifaciens O30.
Antoni Rozalski - One of the best experts on this subject based on the ideXlab platform.
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Serological characterization of the O-specific polysaccharide of Providencia alcalifaciens 023
Archivum Immunologiae Et Therapiae Experimentalis, 2020Co-Authors: Agnieszka Torzewska, Yuriy A. Knirel, Agnieszka Maszewska, Nina A. Kocharova, Antoni RozalskiAbstract:The genus Providencia belongs to the Enterobacteriaceae family and currently consists of five species: P. alcalifaciens, P. heimbachae, P. rettgerii, P. rustigianii and P. stuartii. The serological classification scheme of P. alcalifaciens, P. rustigianii and P. stuartii includes 63 O-serogroups and 30 H-serogroups. The O-antigenic specificity is defined by the structure of the O-antigen (O-specific polysaccharide - OPS), a part of the lipopolysaccharide (LPS, endotoxin), one of the major components of the outer membrane of Gram-negative bacteria and an important virulence factor of these bacteria. Among the bacteria of the Enterobacteriaceae family, the genus Providencia is one of the least studied in respect to its LPS structure and antigenic specificity. Studies of the chemical structures and the serological specificity of the O-antigens aim at the elucidation of the molecular basis of the serological classification of Providencia sp. LPS and alkali-treated LPS of P. alcalifaciens 023 and serologically related P. rustigianii 014, P. mirabilis 013 and P. myxofaciens as well as O-antiserum against P. alcalifaciens 023 were used. Serological characterization of P. alcalifaciens 023 O-specific polysaccharide was done by use enzyme immunosorbent assay (EIA), passive hemolysis test (PHT) as well as by inhibition and sodium deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) of LPS and Western blot. The OPS of P. alcalifaciens, 023, contains an N-(D-glucuronoyl)-N-[(R)-1-carboxyethyl]-L-lysine residue (GlcAAlaLys). The LPS of P. alcalifaciens, 023, and other LPSs containing AlaLys from Providencia and Proteus strains were tested with rabbit anti-P. alcalifaciens 023 serum. The serological data showed that a GlcAAlaLys-associated epitope plays a role as an antigenic determinant in the P. alcalifaciens 023 OPS and revealed the particular importance of glucuronic acid and the carboxyethyl group for the binding of 023-specific antibodies.
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Development of a molecular serotyping scheme and a multiplexed luminex-based array for Providencia
Journal of Microbiological Methods, 2018Co-Authors: Yuhui Du, Antoni Rozalski, Agnieszka Torzewska, Huiying Li, Pan Yang, Chengqian Qian, Tingting Xu, Pan WuAbstract:Abstract Providencia is an opportunistic human pathogen that belongs to the Enterobacteriaceae family. The bacterial cell surface O-antigen is one of the most structurally variable cell constituents and serves as a basis for serotyping gram-negative bacteria. In this work, the genomes of 12 Providencia strains were sequenced, and genes driving O-antigen biosynthesis were analyzed. The O-antigen-synthesizing genes of Providencia are located in the O-antigen gene cluster (OGC) between the cpxA and yibK genes. The gene functions predicted in silico agreed with the known O-antigen structures. All clusters were found to contain both wzx and wzy and exhibit a high degree of heterogeneity. Based on the sero-specific genes, we developed a molecular serotyping system to detect 23 serotypes (from the present and previous studies) for the first time. Five Proteus strains, five Morganella strains, five uropathogenic Escherichia coli (UPEC) strains and 32 Providencia strains with other serotypes were used to assess the specificity of our multiplexed Luminex-based array. Five serogroups (O3, O8, O19, O38 and O52 strains) were used to determine the sensitivity of the suspension array. The detection sensitivity was 0.1 ng genomic DNA, 103 CFU/ml in pure culture, or 104 CFU/ml in mock urine specimens. Furthermore, 29 publicly available Providencia genomes (which have not been serotyped) were analyzed, and 23 novel putative OGC types were identified. In total, we identified 35 new OGCs and developed a molecular serotyping system based on the sero-specific genes. The established classification system can support promising applications in basic research, clinical diagnosis, and epidemiological surveillance.
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structure of the o polysaccharide from the lipopolysaccharide of Providencia alcalifaciens o33
Carbohydrate Research, 2014Co-Authors: Olga G. Ovchinnikova, Antoni Rozalski, Alexander S Shashkov, Alexander O Chizhov, Magdalena Moryl, Yuriy A. KnirelAbstract:Abstract Mild acid degradation of the lipopolysaccharide from Providencia alcalifaciens O33 resulted in an O-polysaccharide along with core and O-unit-bearing core oligosaccharides. Composition of the oligosaccharides was inferred by ESI mass spectrometry. Based on sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy data, the following structure of the tetrasaccharide O-unit of the O-polysaccharide was established: Download : Download full-size image Another O-polysaccharide structure has been reported earlier for Providencia stuartii О33 but later found to belong to a P. stuartii О52 strain.
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o antigens of bacteria of the genus Providencia structure serology genetics and biosynthesis
Biochemistry, 2013Co-Authors: Olga G. Ovchinnikova, Antoni Rozalski, Yuriy A. KnirelAbstract:The genus Providencia consists of eight species of opportunistic pathogenic enterobacteria that can cause enteric diseases and urinary tract infections. The existing combined serological classification scheme of three species, P. alcalifaciens, P. stuartii, and P. rustigianii, is based on the specificity of O-antigens (O-polysaccharides) and comprises 63 O-serogroups. Differences between serogroups are related to polymorphism at a specific genome locus, the O-antigen gene cluster, responsible for O-antigen biosynthesis. This review presents data on structures of 36 O-antigens of Providencia, many of which contain unusual monosaccharides and non-carbohydrate components. The structural data correlate with the immunospecificity of the O-antigens and enable substantiation on a molecular level of serological relationships within the genus Providencia and between strains of Providencia and bacteria of the genera Proteus, Escherichia, and Salmonella. Peculiar features of the O-antigen gene cluster organization in 10 Providencia serogroups and biosynthetic pathways of nucleotide precursors of specific monosaccharide components of the O-antigens also are discussed.
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structural serological and genetic characterization of the o antigen of Providencia alcalifaciens o40
Fems Immunology and Medical Microbiology, 2012Co-Authors: Antoni Rozalski, Alexander S Shashkov, Nina A. Kocharova, Olga G. Ovchinnikova, Lu Feng, Lei Wang, Magdalena Bialczakkokot, Yuriy A. KnirelAbstract:The O-polysaccharide chain of the lipopolysaccharide (O-antigen) on the bacterial cell surface is one of the most structurally variable cell components and serves as a basis for serotyping of Gram-negative bacteria, including human opportunistic pathogens of the genus Providencia . In this work, the O-antigen of Providencia alcalifaciens O40 was obtained by mild acid degradation of the isolated lipopolysaccharide and studied by chemical methods and high-resolution NMR spectroscopy. The following structure of the O-polysaccharide was established: →4)-β-d-Qui p 3NFo-(1→3)-α-d-Gal p -(1→3)-β-d-Glc p A-(1→3)-β-d-Gal p NAc-(1→, where GlcA stands for glucuronic acid and Qui3NFo for 3,6-dideoxy-3-formamidoglucose. The O40-antigen was found to be structurally and serologically related to the O-antigens of P. alcalifaciens O5 and Providencia stuartii O18. The O40-antigen gene cluster between cpxA and yibK was sequenced, and the gene functions were predicted in silico . In agreement with the O-polysaccharide structure established, the genes for the synthesis of dTDP-d-Qui3NFo, UDP-d-Gal, UDP-d-GlcA, and UDP-d-GalNAc as well as those encoding three glycosyltransferases, flippase (Wzx), and O-antigen polymerase (Wzy) were recognized. In addition, homologues of wza, wzb , and wzc genes, which are required for the surface expression of capsular polysaccharides, were found within the gene cluster, suggesting that the O-polysaccharide studied is a part of the capsule-related form of the lipopolysaccharide called KLPS.
Brian P Lazzaro - One of the best experts on this subject based on the ideXlab platform.
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Providencia sneebia sp. nov. and Providencia burhodogranariea sp. nov., isolated from wild Drosophila melanogaster.
International journal of systematic and evolutionary microbiology, 2020Co-Authors: Punita Juneja, Brian P LazzaroAbstract:Multiple isolates of the genus Providencia were obtained from the haemolymph of wild-caught Drosophila melanogaster fruit flies. Sixteen isolates were distinguished from the six previously described species based on 16S rRNA gene sequences. These isolates belonged to two distinct groups, which we propose each comprise previously undescribed species. Two isolates, designated A(T) and B(T), were characterized by DNA sequences of the fusA, lepA, leuS, gyrB and ileS housekeeping genes, whole-genome DNA-DNA hybridizations with their nearest relatives and utilization of substrates for metabolism. The closest phylogenetic relatives of strain A(T) are strain B(T) (86.9 % identity for the housekeeping genes) and Providencia stuartii DSM 4539(T) (86.0 % identity). The closest phylogenetic relatives of strain B(T) are strain A(T) (86.9 % identity) and P. stuartii DSM 4539(T) (86.6 % identity). The type strains of described species in this genus shared between 84.1 and 90.1 % identity for these sequences. DNA-DNA hybridization between the strain pairs A(T)-B(T), A(T)-P. stuartii DSM 4539(T) and B(T)-P. stuartii DSM 4539(T) all resulted in less than 25 % relatedness. In addition, patterns of utilization of amygdalin, arbutin, aesculin, salicin, d-sorbitol, trehalose, inositol, d-adonitol and d-galactose distinguish strains A(T) and B(T) from other members of this genus. Strains A(T) and B(T) therefore represent novel species, for which the names Providencia sneebia sp. nov. (type strain A(T) =DSM 19967(T) =ATCC BAA-1589(T)) and Providencia burhodogranariea sp. nov. (type strain B(T) =DSM 19968(T) =ATCC BAA-1590(T)) are proposed.
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comparative genomics of bacteria in the genus Providencia isolated from wild drosophila melanogaster
BMC Genomics, 2012Co-Authors: Madeline R Galac, Brian P LazzaroAbstract:Comparative genomics can be an initial step in finding the genetic basis for phenotypic differences among bacterial strains and species. Bacteria belonging to the genus Providencia have been isolated from numerous and varied environments. We sequenced, annotated and compared draft genomes of P. rettgeri, P. sneebia, P. alcalifaciens, and P. burhodogranariea. These bacterial species that were all originally isolated as infections of wild Drosophila melanogaster and have been previously shown to vary in virulence to experimentally infected flies. We found that these Providencia species share a large core genome, but also possess distinct sets of genes that are unique to each isolate. We compared the genomes of these isolates to draft genomes of four Providencia isolated from the human gut and found that the core genome size does not substantially change upon inclusion of the human isolates. We found many adhesion related genes among those genes that were unique to each genome. We also found that each isolate has at least one type 3 secretion system (T3SS), a known virulence factor, though not all identified T3SS belong to the same family nor are they in syntenic genomic locations. The Providencia species examined here are characterized by high degree of genomic similarity which will likely extend to other species and isolates within this genus. The presence of T3SS islands in all of the genomes reveal that their presence is not sufficient to indicate virulence towards D. melanogaster, since some of the T3SS-bearing isolates are known to cause little mortality. The variation in adhesion genes and the presence of T3SSs indicates that host cell adhesion is likely an important aspect of Providencia virulence.
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comparative pathology of bacteria in the genus Providencia to a natural host drosophila melanogaster
Microbes and Infection, 2011Co-Authors: Madeline R Galac, Brian P LazzaroAbstract:Abstract Bacteria in the genus Providencia are pathogens of many organisms, including humans and insects. We and colleagues have isolated five different strains belonging to four distinct Providencia species as natural infections of Drosophila melanogaster captured in the wild. We found that these isolates vary considerably in pathology to infected D. melanogaster, differing in the level of mortality they cause, their ability to replicate within the host and the level that the fly’s immune response is elicited. One interesting bacterium was Providencia sneebia, which causes nearly complete mortality and reaches large numbers in the fly but does not elicit a comparably strong immune response. Through coinfection experiments, we determined that P. sneebia avoids recognition by the immune system. We tested for biofilm formation and replication within D. melanogaster cells as possible mechanisms for P. sneebia escape from host immunity, but did not find evidence for either. D. melanogaster and Providencia provide a powerful system for studying general host–pathogen interactions, and for understanding how the well-studied immune model host D. melanogaster interacts with its natural bacterial pathogens.
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Providencia sneebia sp nov and Providencia burhodogranariea sp nov isolated from wild drosophila melanogaster
International Journal of Systematic and Evolutionary Microbiology, 2009Co-Authors: Punita Juneja, Brian P LazzaroAbstract:Multiple isolates of the genus Providencia were obtained from the haemolymph of wild-caught Drosophila melanogaster fruit flies. Sixteen isolates were distinguished from the six previously described species based on 16S rRNA gene sequences. These isolates belonged to two distinct groups, which we propose each comprise previously undescribed species. Two isolates, designated A T and B T , were characterized by DNA sequences of the fusA, lepA, leuS, gyrB and ileS housekeeping genes, whole-genome DNA–DNA hybridizations with their nearest relatives and utilization of substrates for metabolism. The closest phylogenetic relatives of strain A T are strain B T (86.9% identity for the housekeeping genes) and Providencia stuartii DSM 4539 T (86.0% identity). The closest phylogenetic relatives of strain B T are strain A T (86.9% identity) and P. stuartii DSM 4539 T (86.6% identity). The type strains of described species in this genus shared between 84.1 and 90.1% identity for these sequences. DNA–DNA hybridization between the strain pairs A T –B T ,A T –P. stuartii DSM 4539 T and B T –P. stuartii DSM 4539 T all resulted in less than 25% relatedness. In addition, patterns of utilization of amygdalin, arbutin, aesculin, salicin, D-sorbitol, trehalose, inositol, D-adonitol and D-galactose distinguish strains A T and B T from other members of this genus. Strains A T and B T therefore represent novel species, for which the names Providencia sneebia sp. nov. (type strain A T 5DSM 19967 T 5ATCC BAA-1589 T ) and Providencia burhodogranariea sp. nov. (type strain B T 5DSM 19968 T 5ATCC BAA-1590 T ) are
Olga G. Ovchinnikova - One of the best experts on this subject based on the ideXlab platform.
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structure of the o polysaccharide from the lipopolysaccharide of Providencia alcalifaciens o33
Carbohydrate Research, 2014Co-Authors: Olga G. Ovchinnikova, Antoni Rozalski, Alexander S Shashkov, Alexander O Chizhov, Magdalena Moryl, Yuriy A. KnirelAbstract:Abstract Mild acid degradation of the lipopolysaccharide from Providencia alcalifaciens O33 resulted in an O-polysaccharide along with core and O-unit-bearing core oligosaccharides. Composition of the oligosaccharides was inferred by ESI mass spectrometry. Based on sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy data, the following structure of the tetrasaccharide O-unit of the O-polysaccharide was established: Download : Download full-size image Another O-polysaccharide structure has been reported earlier for Providencia stuartii О33 but later found to belong to a P. stuartii О52 strain.
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o antigens of bacteria of the genus Providencia structure serology genetics and biosynthesis
Biochemistry, 2013Co-Authors: Olga G. Ovchinnikova, Antoni Rozalski, Yuriy A. KnirelAbstract:The genus Providencia consists of eight species of opportunistic pathogenic enterobacteria that can cause enteric diseases and urinary tract infections. The existing combined serological classification scheme of three species, P. alcalifaciens, P. stuartii, and P. rustigianii, is based on the specificity of O-antigens (O-polysaccharides) and comprises 63 O-serogroups. Differences between serogroups are related to polymorphism at a specific genome locus, the O-antigen gene cluster, responsible for O-antigen biosynthesis. This review presents data on structures of 36 O-antigens of Providencia, many of which contain unusual monosaccharides and non-carbohydrate components. The structural data correlate with the immunospecificity of the O-antigens and enable substantiation on a molecular level of serological relationships within the genus Providencia and between strains of Providencia and bacteria of the genera Proteus, Escherichia, and Salmonella. Peculiar features of the O-antigen gene cluster organization in 10 Providencia serogroups and biosynthetic pathways of nucleotide precursors of specific monosaccharide components of the O-antigens also are discussed.
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structural serological and genetic characterization of the o antigen of Providencia alcalifaciens o40
Fems Immunology and Medical Microbiology, 2012Co-Authors: Antoni Rozalski, Alexander S Shashkov, Nina A. Kocharova, Olga G. Ovchinnikova, Lu Feng, Lei Wang, Magdalena Bialczakkokot, Yuriy A. KnirelAbstract:The O-polysaccharide chain of the lipopolysaccharide (O-antigen) on the bacterial cell surface is one of the most structurally variable cell components and serves as a basis for serotyping of Gram-negative bacteria, including human opportunistic pathogens of the genus Providencia . In this work, the O-antigen of Providencia alcalifaciens O40 was obtained by mild acid degradation of the isolated lipopolysaccharide and studied by chemical methods and high-resolution NMR spectroscopy. The following structure of the O-polysaccharide was established: →4)-β-d-Qui p 3NFo-(1→3)-α-d-Gal p -(1→3)-β-d-Glc p A-(1→3)-β-d-Gal p NAc-(1→, where GlcA stands for glucuronic acid and Qui3NFo for 3,6-dideoxy-3-formamidoglucose. The O40-antigen was found to be structurally and serologically related to the O-antigens of P. alcalifaciens O5 and Providencia stuartii O18. The O40-antigen gene cluster between cpxA and yibK was sequenced, and the gene functions were predicted in silico . In agreement with the O-polysaccharide structure established, the genes for the synthesis of dTDP-d-Qui3NFo, UDP-d-Gal, UDP-d-GlcA, and UDP-d-GalNAc as well as those encoding three glycosyltransferases, flippase (Wzx), and O-antigen polymerase (Wzy) were recognized. In addition, homologues of wza, wzb , and wzc genes, which are required for the surface expression of capsular polysaccharides, were found within the gene cluster, suggesting that the O-polysaccharide studied is a part of the capsule-related form of the lipopolysaccharide called KLPS.
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genetic analysis of the o antigen of Providencia alcalifaciens o30 and biochemical characterization of a formyltransferase involved in the synthesis of a qui4n derivative
Glycobiology, 2012Co-Authors: Miao Chen, Yuriy A. Knirel, Antoni Rozalski, Olga G. Ovchinnikova, Lu Feng, Andrei V Perepelov, Dawei Zhou, Lei WangAbstract:O-Antigen is a component of the outer membrane of Gram-negative bacteria and one of the most variable cell surface constituents, giving rise to major antigenic variability. The diversity of O-antigen is almost entirely attributed to genetic variations in O-antigen gene clusters. Bacteria of the genus Providencia are facultative pathogens, which can cause urinary tract infections, wound infections and enteric diseases. Recently, the O-antigen gene cluster of Providencia was localized between the cpxA and yibK genes in the genome. However, few genes involved in the synthesis of Providencia O-antigens have been functionally identified. In this study, the putative O-antigen gene cluster of Providencia alcalifaciens O30 was sequenced and analyzed. Almost all putative genes for the O-antigen synthesis were found, including a novel formyltransferase gene vioF that was proposed to be responsible for the conversion of dTDP-4-amino-4,6- dideoxy-D-glucose (dTDP-D-Qui4N) to dTDP-4,6-dideoxy-4-formamido-D-glucose (dTDP-D-Qui4NFo). vioF was cloned, and the enzyme product was expressed as a His-tagged fusion protein, purified and assayed for its activity. High-performance liquid chromatography was used to monitor the enzyme-substrate reaction, and the structure of the product dTDP-D-Qui4NFo was established by electrospray ionization tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Kinetic parameters of VioF were determined, and effects of temperature and cations on its activity were also examined. Together, the functional analyses support the identification of the O-antigen gene cluster of P. alcalifaciens O30.
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localization and molecular characterization of putative o antigen gene clusters of Providencia species
Microbiology, 2012Co-Authors: Yuriy A. Knirel, Antoni Rozalski, Alexander S Shashkov, Nina A. Kocharova, Olga G. Ovchinnikova, Miao Chen, Lu Feng, Lei WangAbstract:Enterobacteria of the genus Providencia are opportunistic human pathogens associated with urinary tract and wound infections, as well as enteric diseases. The lipopolysaccharide (LPS) O antigen confers major antigenic variability upon the cell surface and is used for serotyping of Gram-negative bacteria. Recently, Providencia O antigen structures have been extensively studied, but no data on the location and organization of the O antigen gene cluster have been reported. In this study, the four Providencia genome sequences available were analysed, and the putative O antigen gene cluster was identified in the polymorphic locus between the cpxA and yibK genes. This finding provided the necessary information for designing primers, and cloning and sequencing the O antigen gene clusters from five more Providencia alcalifaciens strains. The gene functions predicted in silico were in agreement with the known O antigen structures; furthermore, annotation of the genes involved in the three-step synthesis of GDP-colitose (gmd, colD and colC) was supported by cloning and biochemical characterization of the corresponding enzymes. In one strain (P. alcalifaciens O39), no polysaccharide product of the gene cluster in the cpxA–yibK locus was found, and hence genes for synthesis of the existing O antigen are located elsewhere in the genome. In addition to the putative O antigen synthesis genes, homologues of wza, wzb, wzc and (in three strains) wzi, required for the surface expression of capsular polysaccharides, were found upstream of yibK in all species except Providencia rustigianii, suggesting that the LPS of these species may be attributed to the so-called K LPS (KLPS). The data obtained open a way for development of a PCR-based typing method for identification of Providencia isolates.
Nina A. Kocharova - One of the best experts on this subject based on the ideXlab platform.
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Serological characterization of the O-specific polysaccharide of Providencia alcalifaciens 023
Archivum Immunologiae Et Therapiae Experimentalis, 2020Co-Authors: Agnieszka Torzewska, Yuriy A. Knirel, Agnieszka Maszewska, Nina A. Kocharova, Antoni RozalskiAbstract:The genus Providencia belongs to the Enterobacteriaceae family and currently consists of five species: P. alcalifaciens, P. heimbachae, P. rettgerii, P. rustigianii and P. stuartii. The serological classification scheme of P. alcalifaciens, P. rustigianii and P. stuartii includes 63 O-serogroups and 30 H-serogroups. The O-antigenic specificity is defined by the structure of the O-antigen (O-specific polysaccharide - OPS), a part of the lipopolysaccharide (LPS, endotoxin), one of the major components of the outer membrane of Gram-negative bacteria and an important virulence factor of these bacteria. Among the bacteria of the Enterobacteriaceae family, the genus Providencia is one of the least studied in respect to its LPS structure and antigenic specificity. Studies of the chemical structures and the serological specificity of the O-antigens aim at the elucidation of the molecular basis of the serological classification of Providencia sp. LPS and alkali-treated LPS of P. alcalifaciens 023 and serologically related P. rustigianii 014, P. mirabilis 013 and P. myxofaciens as well as O-antiserum against P. alcalifaciens 023 were used. Serological characterization of P. alcalifaciens 023 O-specific polysaccharide was done by use enzyme immunosorbent assay (EIA), passive hemolysis test (PHT) as well as by inhibition and sodium deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) of LPS and Western blot. The OPS of P. alcalifaciens, 023, contains an N-(D-glucuronoyl)-N-[(R)-1-carboxyethyl]-L-lysine residue (GlcAAlaLys). The LPS of P. alcalifaciens, 023, and other LPSs containing AlaLys from Providencia and Proteus strains were tested with rabbit anti-P. alcalifaciens 023 serum. The serological data showed that a GlcAAlaLys-associated epitope plays a role as an antigenic determinant in the P. alcalifaciens 023 OPS and revealed the particular importance of glucuronic acid and the carboxyethyl group for the binding of 023-specific antibodies.
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structural serological and genetic characterization of the o antigen of Providencia alcalifaciens o40
Fems Immunology and Medical Microbiology, 2012Co-Authors: Antoni Rozalski, Alexander S Shashkov, Nina A. Kocharova, Olga G. Ovchinnikova, Lu Feng, Lei Wang, Magdalena Bialczakkokot, Yuriy A. KnirelAbstract:The O-polysaccharide chain of the lipopolysaccharide (O-antigen) on the bacterial cell surface is one of the most structurally variable cell components and serves as a basis for serotyping of Gram-negative bacteria, including human opportunistic pathogens of the genus Providencia . In this work, the O-antigen of Providencia alcalifaciens O40 was obtained by mild acid degradation of the isolated lipopolysaccharide and studied by chemical methods and high-resolution NMR spectroscopy. The following structure of the O-polysaccharide was established: →4)-β-d-Qui p 3NFo-(1→3)-α-d-Gal p -(1→3)-β-d-Glc p A-(1→3)-β-d-Gal p NAc-(1→, where GlcA stands for glucuronic acid and Qui3NFo for 3,6-dideoxy-3-formamidoglucose. The O40-antigen was found to be structurally and serologically related to the O-antigens of P. alcalifaciens O5 and Providencia stuartii O18. The O40-antigen gene cluster between cpxA and yibK was sequenced, and the gene functions were predicted in silico . In agreement with the O-polysaccharide structure established, the genes for the synthesis of dTDP-d-Qui3NFo, UDP-d-Gal, UDP-d-GlcA, and UDP-d-GalNAc as well as those encoding three glycosyltransferases, flippase (Wzx), and O-antigen polymerase (Wzy) were recognized. In addition, homologues of wza, wzb , and wzc genes, which are required for the surface expression of capsular polysaccharides, were found within the gene cluster, suggesting that the O-polysaccharide studied is a part of the capsule-related form of the lipopolysaccharide called KLPS.
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localization and molecular characterization of putative o antigen gene clusters of Providencia species
Microbiology, 2012Co-Authors: Yuriy A. Knirel, Antoni Rozalski, Alexander S Shashkov, Nina A. Kocharova, Olga G. Ovchinnikova, Miao Chen, Lu Feng, Lei WangAbstract:Enterobacteria of the genus Providencia are opportunistic human pathogens associated with urinary tract and wound infections, as well as enteric diseases. The lipopolysaccharide (LPS) O antigen confers major antigenic variability upon the cell surface and is used for serotyping of Gram-negative bacteria. Recently, Providencia O antigen structures have been extensively studied, but no data on the location and organization of the O antigen gene cluster have been reported. In this study, the four Providencia genome sequences available were analysed, and the putative O antigen gene cluster was identified in the polymorphic locus between the cpxA and yibK genes. This finding provided the necessary information for designing primers, and cloning and sequencing the O antigen gene clusters from five more Providencia alcalifaciens strains. The gene functions predicted in silico were in agreement with the known O antigen structures; furthermore, annotation of the genes involved in the three-step synthesis of GDP-colitose (gmd, colD and colC) was supported by cloning and biochemical characterization of the corresponding enzymes. In one strain (P. alcalifaciens O39), no polysaccharide product of the gene cluster in the cpxA–yibK locus was found, and hence genes for synthesis of the existing O antigen are located elsewhere in the genome. In addition to the putative O antigen synthesis genes, homologues of wza, wzb, wzc and (in three strains) wzi, required for the surface expression of capsular polysaccharides, were found upstream of yibK in all species except Providencia rustigianii, suggesting that the LPS of these species may be attributed to the so-called K LPS (KLPS). The data obtained open a way for development of a PCR-based typing method for identification of Providencia isolates.
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elucidation of the full o polysaccharide structure and identification of the core type of the lipopolysaccharide of Providencia alcalifaciens o9
Carbohydrate Research, 2011Co-Authors: Olga G. Ovchinnikova, Antoni Rozalski, Alexander S Shashkov, Nina A. Kocharova, Nikolay P Arbatsky, Yuriy A. KnirelAbstract:Abstract Opportunistic human pathogens of the genus Providencia from the family Enterobacteriaceae are serotyped by their O-antigens, which represent the O-polysaccharide chains of the lipopolysaccharides (LPSs) on the cell surface. In this work, the O-polysaccharide of Providencia alcalifaciens O9 was obtained by mild acid degradation of a long-chain S-form LPS. The structure of the hexasaccharide repeat (O-unit) of the O-polysaccharide containing one d -Gal, two d -Glc, and three d -GalNAc residues was established by sugar and methylation analyses along with one- and two-dimensional 1 H and 13 C NMR spectroscopy. Another degradation product was derived from a short-chain SR-form LPS and found to consist of a core oligosaccharide bearing one O-unit. Its studies by NMR spectroscopy and electrospray ionization mass spectrometry enabled identification of one of the GalNAc residues as the first monosaccharide of the O-unit, whose glycosidic linkage links the O-units to each other and the first O-unit to the core. The core is distinguished by the occurrence of two glycoforms differing in the nature of a lateral monosaccharide, which is either d -Glc or d -GlcNAc. Although composed of common monosaccharides, the O-polysaccharide of P. alcalifaciens O9 has a unique structure among bacterial polysaccharides, whereas the oligosaccharide region belongs to one of several core types recognized in the LPSs of Providencia .
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The Full Structure of the Carbohydrate Chain of the Lipopolysaccharide of Providencia alcalifaciens O19
Journal of Carbohydrate Chemistry, 2008Co-Authors: Nina A. Kocharova, Antoni Rozalski, Alexander S Shashkov, Anna N Kondakova, Evgeny Vinogradov, Yuriy A. KnirelAbstract:An oligosaccharide isolated from the lipopolysaccharide of Providencia alcalifaciens O19 was found to consist of a single O‐antigen repeating unit linked to the core. The full oligosaccharide structure was elucidated by 2D 1H, 13C, and 31P NMR spectroscopy. It was shown that the inner core region has the same structure as in other Providencia strains studied, whereas the outer core is structurally diverse between Providencia strains. A pyruvic acid acetal was found in the isolated oligosaccharide and in the long‐chain O‐antigen, whose structure has been established earlier. The biological O‐unit structure was elucidated in the short‐chain lipopolysaccharide.
