Antoni Rozalski

Structural Study of O-Specific Polysaccharides of Proteus

Abstract Proteus bacteria are important human opportunistic pathogens which frequently cause urinary tract infections. According to Bergeys Manual of Systematic Bacteriology,1 this genus includes three species: P. mirabilis, P. vulgaris, and P. myxofaciens. A novel species of P. penneri has been recently proposed2,3 for strains formerly called P. vulgaris biogroup I. Proteus is an antigenically heterogeneous group of bacteria, and this is mainly associated with diverse composition and structures of O-specific polysaccharide chains of outer-membrane lipopolysaccharides (O-antigens). The Kauffman-Perch serological classification4 of P. mirabilis and P. vulgaris includes 49 O-serogroups. However, a number of S-strains remain unclassified,5 including strains of P. penneri.

Structure and serology of O-antigens as the basis for classification of Proteus strains:

This review is devoted to structural and serological characteristics of the O-antigens (O-polysaccharides) of the lipopolysaccharides of various Proteus species, which provide the basis for classifying Proteus strains to Oserogroups. The antigenic relationships of Proteus strains within and beyond the genus as well as their O-antigenrelated bioactivities are also discussed.

Determination of the epitope specificity of monoclonal antibodies against the inner core region of bacterial lipopolysaccharides by use of 3-deoxy-d-manno-octulosonate-containing synthetic antigens☆

Partial structures of enterobacterial lipopolysaccharides (LPS) of the Rechemotype, consisting of lipid A and 3-deoxy-D-manno-2-octulosonic acid (Kdo), as well as oligosaccharides and derivative of Kdo were synthesized and used to characterize the epitope specificity of monoclonal antibodies against Re-mutant LPS. High-molecular-weight antigens, obtained after copolymerization of the respective allyl glycosides with acrylamide, and the haptenic oligosaccharides were used in immunoprecipitation, immune hemolysis, and in inhibition assays. A monoclonal antibody (clone 20, igM) recognizing a terminal Kdop group was shown to require for its binding the alpha-anomeric configuration and OH-4 and OH-5 groups, whereas the C-7 - C-8 chain was of minor importance. Another monoclonal antibody (clone 25, IgG3), which recognizes a (2--4)-linked Kdo disaccharide, was shown to require for its binding the alpha-anomeric configuration of both residues. The isomer having a reducing beta-Kdo residue was significantly less active, and that with a terminal beta-Kdo group was completely inactive. The OH-5 group of the reducing residue was shown to be not important for the specificity of this antibody, since it could be replaced by a hydrogen atom without loss of serological reactivity. The alpha-(2--8)-linked Kdo disaccharide was strongly cross-reactive with its (2--4)-linked isomer. The antibody recognized also parts of the 2-amino-2-deoxy-D-glucose backbone of lipid A.

Structure of the O-specific polysaccharide of Proteus vulgaris O4 containing a new component of bacterial polysaccharides, 4,6-dideoxy-4-{N-[(R)-3-hydroxybutyryl]-l-alanyl}amino-d-glucose

Abstract A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O4 lipopolysaccharide followed by GPC. The polysaccharide was studied by chemical methods along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, and 1H,13C HMBC experiments. Solvolysis of the polysaccharide with trifluoromethanesulfonic (triflic) acid resulted in a GlcpA-(1→3)-GlcNAc disaccharide and a novel amino sugar derivative, 4,6-dideoxy-4-{N-[(R)-3-hydroxybutyryl]- l -alanyl}amino- d -glucose [Qui4N(HbAla)]. On the basis of the data obtained, the following structure of the tetrasaccharide repeating unit of the O-specific polysaccharide was established: →4 )- β - d - Glc p A -(1 →3 )- β - d - Glc p NAc -(1 →2 )- β - d - Qui p4 N ( HbAla )-(1 →3 )- α - d - Gal p-(1 → This structure is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied in a separate Proteus serogroup.

New structures of the O-specific polysaccharides of bacteria of the genus Proteus. 1. Phosphate-containing polysaccharides.

The O-specific polysaccharide chains (O-antigens) of the lipopolysaccharides of five Proteus strains, P. vulgaris O17, P. mirabilis O16 and O33, and P. penneri 31 and 103, were found to contain phosphate groups that link the non sugar components, e.g., ethanolamine and ribitol. The polysaccharides of P. mirabilis O16 and P. penneri 103 include ribitol phosphate in the main chain and thus resemble ribitol teichoic acids of Gram-positive bacteria. The structures of the polysaccharides were elucidated using NMR spectroscopy, including two-dimensional 1H, 1H correlation spectroscopy (COSY and TOCSY), nuclear Overhauser effect spectroscopy (NOESY or ROESY), and H-detected 1H, 13C and 1H, 31P heteronuclear multiple-quantum coherence spectroscopy (HMQC), along with chemical methods. The structures determined are unique among the bacterial polysaccharides and, together with the data obtained earlier, represent the chemical basic for classification of Proteus strains. Based on structural similarities of the O-specific polysaccharides and serological relationships between the O-antigens, we propose to extend Proteus serogroups O17 and O19 by including P. penneri strains 16 and 31, respectively.

O-antigens of bacteria of the genus Providencia: Structure, serology, genetics, and biosynthesis

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.

Localization and molecular characterization of putative O antigen gene clusters of Providencia species.

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 (K(LPS)). The data obtained open a way for development of a PCR-based typing method for identification of Providencia isolates.

Proteus sp. – an opportunistic bacterial pathogen – classification, swarming growth, clinical significance and virulence factors

ABSTRACT The genus Proteus belongs to the Enterobacteriaceae family, where it is placed in the tribe Proteeae, together with the genera Morganella and Providencia. Currently, the genus Proteus consists of five species: P. mirabilis, P. vulgaris, P. penneri, P. hauseri and P. myxofaciens, as well as three unnamed Proteus genomospecies. The most defining characteristic of Proteus bacteria is a swarming phenomenon, a multicellular differentiation process of short rods to elongated swarmer cells. It allows population of bacteria to migrate on solid surface. Proteus bacteria inhabit the environment and are also present in the intestines of humans and animals. These microorganisms under favorable conditions cause a number of infections including urinary tract infections (UTIs), wound infections, meningitis in neonates or infants and rheumatoid arthritis. Therefore, Proteus is known as a bacterial opportunistic pathogen. It causes complicated UTIs with a higher frequency, compared to other uropathogens. Proteus infections are accompanied by a formation of urinary stones, containing struvite and carbonate apatite. The virulence of Proteus rods has been related to several factors including fimbriae, flagella, enzymes (urease - hydrolyzing urea to CO2 and NH3, proteases degrading antibodies, tissue matrix proteins and proteins of the complement system), iron acqusition systems and toxins: hemolysins, Proteus toxin agglutinin (Pta), as well as an endotoxin - lipopolysaccharide (LPS). Proteus rods form biofilm, particularly on the surface of urinary catheters, which can lead to serious consequences for patients. In this review we present factors involved in the regulation of swarming phenomenon, discuss the role of particular pathogenic features of Proteus spp., and characterize biofilm formation by these bacteria.

Full Structure of the Carbohydrate Chain of the Lipopolysaccharide of Providencia rustigianii O34

A lipopolysaccharide isolated from an opportunistic pathogen of the Enterobacteriaceae family Providencia rustigianii O34 was found to be a mixture of R-, SR-, and S-forms consisting of a lipid moiety (lipid A) that bears a core oligosaccharide, a core with one O-polysaccharide repeating unit attached, and a long-chain O-polysaccharide, respectively. The corresponding carbohydrate moieties were released from the lipopolysaccharide by mild acid hydrolysis and studied by sugar and methylation analyses along with one- and two-dimensional NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. As a result, the structures of the core and the O-polysaccharide were established, including the structure of the biological repeating unit (an oligosaccharide that is preassembled and polymerized in biosynthesis of the O-polysaccharide), as well as the mode of the linkage between the O-polysaccharide and the core. Combining the structure of the carbohydrate moiety thus determined and the known structure of lipid A enabled determination of the full lipopolysaccharide structure of P. rustigianii O34.

Mass‐Spectrometric Studies of Providencia SR‐Form Lipopolysaccharides and Elucidation of the Biological Repeating Unit Structure of Providencia rustigianii O14‐Polysaccharide

Enterobacteria Providencia are opportunistic human pathogens causing multiple types of infections. Earlier we have studied the S‐ and R‐form lipopolysaccharides (LPSs) of Providencia strains of various O‐serogroups and established the structures of the O‐polysaccharides (O‐antigens) and core‐region oligosaccharides, respectively. Now we report on mass spectrometric studies of oligosaccharides consisting of the core moiety with one O‐polysaccharide repeating unit attached, which were derived from the SR‐form LPSs of Providencia strains. The site of attachment of the O‐polysaccharide to the core and the structure of the O‐polysaccharide biological repeating unit were elucidated in Providencia rustigianii O14 using NMR spectroscopy.