Antigenic and Structural Properties of the Lipopolysaccharide of the Uropathogenic Proteus mirabilis Dm55 Strain Classified to a New O85 Proteus Serogroup.

The aim of the study was the serological and structural characterization of the lipopolysaccharide (LPS) O antigen from P. mirabilis Dm55 coming from the urine of a patient from Lodz. The Dm55 LPS was recognized in ELISA only by the O54 antiserum, suggesting a serological distinction of the Dm55 O antigen from all the 84 Proteus LPS serotypes described. The obtained polyclonal rabbit serum against P. mirabilis Dm55 reacted in ELISA and Western blotting with a few LPSs (including O54), but the reactions were weaker than those observed in the homologous system. The LPS of P. mirabilis Dm55 was subjected to mild acid hydrolysis, and the obtained high-molecular-mass O polysaccharide was chemically studied using sugar and methylation analyses, mass spectrometry, and 1H and 13C NMR spectroscopy, including 1H,1H NOESY, and 1H,13C HMBC experiments. The Dm55 O unit is a branched three-saccharide, and its linear fragment contains α-GalpNAc and ß-Galp, whereas α-GlcpNAc occupies a terminal position. The Dm55 OPS shares a disaccharide epitope with the Proteus O54 antigen. Due to the structural differences of the studied O antigen from the other described Proteus O polysaccharides, we propose to classify the P. mirabilis Dm55 strain to a new Proteus O85 serogroup.

Proteus mirabilis and Klebsiella pneumoniae as pathogens capable of causing co-infections and exhibiting similarities in their virulence factors.

The genera Klebsiella and Proteus were independently described in 1885. These Gram-negative rods colonize the human intestinal tract regarded as the main reservoir of these opportunistic pathogens. In favorable conditions they cause infections, often hospital-acquired ones. The activity of K. pneumoniae and P. mirabilis, the leading pathogens within each genus, results in infections of the urinary (UTIs) and respiratory tracts, wounds, bacteremia, affecting mainly immunocompromised patients. P. mirabilis and K. pneumoniae cause polymicrobial UTIs, which are often persistent due to the catheter biofilm formation or increasing resistance of the bacteria to antibiotics. In this situation a need arises to find the antigens with features common to both species. Among many virulence factors produced by both pathogens urease shows some structural similarities but the biggest similarities have been observed in lipids A and the core regions of lipopolysaccharides (LPSs). Both species produce capsular polysaccharides (CPSs) but only in K. pneumoniae these antigens play a crucial role in the serological classification scheme, which in Proteus spp. is based on the structural and serological diversity of LPS O-polysaccharides (OPSs). Structural and serological similarities observed for Klebsiella spp. and Proteus spp. polysaccharides are important in the search for the cross-reacting vaccine antigens.

The Contribution of Polysaccharide Antigens From Clinical Proteus spp. and Klebsiella spp. Isolates to the Serological Cross-Reactions.

Klebsiella spp. and Proteus spp. cause hospital-acquired urinary tract infections (UTIs), which are often related to the use of catheters. To create a vaccine preventing UTI, immunogenic bacterial antigens with common epitopes are still being looked for. In this work, the role of polysaccharide antigens of four Klebsiella spp. and eight Proteus spp. strains in serological cross-reactions with specific antisera was examined. Enzyme-linked immunosorbent assay (ELISA), Western blotting, and silver staining by Tsai method were performed. The Klebsiella and Proteus spp. LPSs and cells were used as antigens. Polyclonal rabbit sera specific to Klebsiella oxytoca 0.023 and 0.062 strains and four Klebsiella spp. LPSs were obtained. The ELISA and Western blotting results showed the strongest cross-reactions occurring between lipopolysaccharides (LPSs) from four Klebsiella strains and P. vulgaris O42 antiserum. The silver-staining procedure revealed the patterns typical of both slow- and fast-migrating mass species of the Klebsiella LPSs. The Klebsiella spp. antigens also cross-reacted with four P. penneri antisera, and most of the reactions were observed as low-migrating patterns. From two K. oxytoca antisera obtained in this work, only one, the K. oxytoca 0.062 antiserum, cross-reacted with satisfactory strength with P. penneri LPSs (19, 22, and 60). Obtaining cross-reactions between the antigens of Klebsiella strains and Proteus antisera and in the opposite systems is important for proving the immunogenic role of polysaccharide antigens in triggering the immunological response.

The prevailing O serogroups among the serologically differentiated clinical Proteus spp. strains in central Poland.

In the years 2006-2011, 617 Proteus spp. strains isolated mostly from urine and wounds or other clinical sources were collected in Lódz, Poland, to determine the offensive O serotypes frequently occurring among patients. P. mirabilis exhibited the most intensive swarming growth and was dominating species (86.9%), followed by P. genomospecies, P. vulgaris, and P. penneri. Ninety four per cent strains were recognized as S (smooth) forms. Serological studies (involving ELISA-enzyme-linked immunosorbent assay and Western blotting using native and adsorbed rabbit antisera) enabled classification of 80% S isolates into respective Proteus O serogroups among the 83 ones, described so far. The remaining strains seemed to be serologically unique. Despite the observed big serological variety of Proteus spp. isolates, we found the O78 serogroup recently described in Poland as dominating and identified other widespread serotypes: O3, O6, O10, O11, O27, O28, and O30 reported earlier as predominating also in other countries; O77 and O79 detected lately in Poland; O16, O18, O20, and O50. No unique structural feature of the prevalent O serotypes has been indicated. However, the prevalence of some O serogroups indicates that particular serotypes may be in some ways beneficial to the strains producing these kinds of O antigen.

The New Structure of Core Oligosaccharide Presented by Proteus penneri 40A and 41 Lipopolysaccharides.

The new type of core oligosaccharide in Proteus penneri 40A and 41 lipopolysaccharides has been investigated by ¹H and 13C NMR spectroscopy, electrospray ionization mass spectrometry and chemical methods. Core oligosaccharides of both strains were chosen for structural analysis based on the reactivity of LPSs with serum against P. penneri 40A core oligosaccharide-diphtheria toxoid conjugate. Structural analyses revealed that P. penneri 40A and 41 LPSs possess an identical core oligosaccharide.

Systematic Identification of Lysine 2-hydroxyisobutyrylated Proteins in Proteus mirabilis.

Lysine 2-hydroxyisobutyrylation (Khib) is a novel post-translational modification (PTM), which was thought to play a role in active gene transcription and cellular proliferation. Here we report a comprehensive identification of Khib in Proteus mirabilis (P. mirabilis). By combining affinity enrichment with two-dimensional liquid chromatography and high-resolution mass spectrometry, 4735 2-hydroxyisobutyrylation sites were identified on 1051 proteins in P. mirabilis. These proteins bearing modifications were further characterized in abundance, distribution and functions. The interaction networks and domain architectures of these proteins with high confidence were revealed using bioinformatic tools. Our data demonstrate that many 2-hydroxyisobutyrylated proteins are involved in metabolic pathways, such as purine metabolism, pentose phosphate pathway and glycolysis/gluconeogenesis. The extensive distribution of Khib also indicates that the modification may play important influence to bacterial metabolism. The speculation is further supported by the observation that carbon sources can influence the occurrence of Khib Furthermore, we demonstrate that 2-hydroxyisobutyrylation on K343 was a negative regulatory modification on Enolase (ENO) activity, and molecular docking results indicate the regulatory mechanism that Khib may change the binding formation of ENO and its substrate 2-phospho-d-glycerate (2PG) and cause the substrate far from the active sites of enzyme. We hope this first comprehensive analysis of nonhistone Khib in prokaryotes is valuable for further functional investigation of this modification.

Classification of Proteus penneri lipopolysaccharides into core region serotypes.

The frequency of P. penneri isolation from hospital patients, mostly from urine and wounds, keeps on growing, and numerous isolates are multi-drug resistant. P. penneri rods produce lipopolysaccharide (LPS), which may lead to the septic shock. Until now, O-specific polysaccharide has been the best structurally and serologically characterized region of P. penneri LPS. It is worth having an insight into the serological specificity of both poly- and oligosaccharide parts of P. penneri LPS. The P. penneri core region is less structurally diverse than OPS, but still, among other enterobacterial LPS core regions, it is characterized by structural variability. In the present study, the serological reactivity of 25 P. penneri LPS core regions was analyzed by ELISA, passive immunohemolysis and Western blot technique using five polyclonal P. penneri antisera after or without their adsorption with the respective LPSs. The results allowed the assignment of the tested strains to five new core serotypes, which together with published serological studies led to the creation of the first serotyping scheme based on LPS core reactivities of 35 P. penneri and three P. mirabilis strains. Together with the O types scheme, it will facilitate assigning Proteus LPSs of clinical isolates into appropriate O and R serotypes.

A High-resolution Typing Assay for Uropathogenic Escherichia coli Based on Fimbrial Diversity.

Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, causing cystitis, pyelonephritis, and renal failure. Uropathogenic Escherichia coli (UPEC) is the leading cause of UTIs. Accurate and rapid discrimination of UPEC lineages is useful for epidemiological surveillance. Fimbriae are necessary for the adherence of UPEC strains to host uroepithelia, and seem to be abundant and diverse in UPEC strains. By analyzing all the possible fimbrial operons in UPEC strains, we found that closely related strains had similar types of chaperone-usher fimbriae, and the diversity of fimbrial genes was higher than that of multilocus sequence typing (MLST) genes. A typing assay based on the polymorphism of four gene sequences (three fimbrial genes and one housekeeping gene) and the diversity of fimbriae present was developed. By comparison with the MLST, whole-genome sequence (WGS) and fumC/fimH typing methods, this was shown to be accurate and have high resolution, and it was also relatively inexpensive and easy to perform. The assay can supply more discriminatory information for UPEC lineages, and have the potential to be applied in epidemiological surveillance of UPEC isolates.

Proteus mirabilis RMS 203 as a new representative of the O13 Proteus serogroup.

The unique feature of some Proteus O-polysaccharides is occurrence of an amide of galacturonic acid with N(ε)-[(S/R)-1-Carboxyethyl]-L-lysine, GalA6(2S,8S/R-AlaLys). The results of the serological studies presented here, with reference to known O-antigens structures suggest that GalA6(2S,8S/R-AlaLys) or 2S,8R-AlaLys contribute to cross-reactions of O13 Proteus antisera, and Proteeae LPSs. It was also revealed that the Proteus mirabilis RMS 203 strain can be classified into the O13 serogroup, represented so far by two strains: Proteus mirabilis 26/57 and Proteus vulgaris 8344. The O13 LPS is a serologically important antigen with a fragment common to LPSs of different species in the Proteeae tribe.

The antigens contributing to the serological cross-reactions of Proteus antisera with Klebsiella representatives.

Proteus sp. and Klebsiella sp. mainly cause infections of the urinary and respiratory tracts or wounds in humans. The representatives of both genera produce virulence factors like lipopolysaccharide (LPS) or outer membrane proteins (OMPs) having much in common in the structures and/or functions. To check how far this similarity is revealed in the serological cross-reactivity, the bacterial masses of 24 tested Klebsiella sp. strains were tested in ELISA with polyclonal rabbit antisera specific to the representatives of 79 Proteus O serogroups. The strongest reacting systems were selected to Western blot, where the majority of Klebsiella masses reacted in a way characteristic for electrophoretic patterns of proteins. The strongest reactions were obtained for proteins of near 67 and 40 kDa and 12.5 kDa. Mass spectrometry analysis of the proteins samples of one Proteus sp. and one Klebsiella sp. strain showed the GroEL like protein of a sequence GI number 2980926 to be similar for both strains. In Western blot some Klebsiella sp. masses reacted similarly to the homologous Proteus LPSs. The LPS contribution in the observed reactions of the high molecular-mass LPS species was confirmed for Klebsiella oxytoca 0.062.

The amide of galacturonic acid with lysine as an immunodominant component of the lipopolysaccharide core region from Proteus penneri 42 strain.

Most Proteus lipopolysaccharides (LPSs) contain uronic acids or their amides with different amino acids, which together with other negatively charged components account for the acidic character of such LPS molecules. Previous studies have shown the significance of an amide of galacturonic acid with lysine [D-GalA(L-Lys)] for serological specificity of O-antigens from few P. mirabilis strains. In this work, the immunodominant role of GalALys was indicated for the P. penneri 42 LPS core region. The studies also showed the serological identity of core oligosaccharides from P. penneri 42 (O71), P. mirabilis 51/57 (O28) and R14/S1959 strains.

Serological studies of Proteus penneri strains determining qualification to appropriate O-serogroup.

Our Department of General Microbiology created a wide collection of P. penneri isolates and classified most of them into 19 different O-serogroups. This work describes the classification of 12 remaining P. penneri strains. The lipopolysaccharides extracted from P. penneri strains were tested in an enzyme-linked immunosorbent assay (ELISA) with selected O-antisera against Proteus sp. strains. Homologous and cross-reacting systems were checked in: passive immunohemolysis (PIH), inhibition of ELISA and PIH and Western blot procedure. These studies led to the qualification of tested P. penneri strains to five Proteus sp. O-serogroups, thus completing the serological classification of the whole collection.

Structure of a Kdo-containing O polysaccharide representing Proteus O79, a newly described serogroup for some clinical Proteus genomospecies isolates from Poland.

From 41 Proteus genomospecies strains isolated in Poland, seven displayed similar serospecificity in ELISA with intact and adsorbed O antisera as well as in Western blot. The cross-reacting strains were found to belong to Proteus genomospecies 5/6 and classified into a new Proteus serogroup, O79, which seems to be widespread among Proteus genomospecies clinical isolates in Lodz, Poland. The O polysaccharide of the lipopolysaccharide of a representative O79 strain, 11 B-r, was studied by chemical analyses and (1)H and (13)C NMR spectroscopy, and the following structure of the repeating unit was established: →4)-α-D-GlcpNAlaAc-(1→5)-α-Kdop-(2→2)-α-D-Glcp-(1→3)-ß-D-GlcpNAc-(1→ where AlaAc indicates N-acetyl-L-alanyl and Kdo indicates 3-deoxy-D-manno-oct-2-ulosonic acid. The O polysaccharide was unstable under mild acidic conditions and cleaved by acid-labile linkages of Kdo residues to yield a tetrasaccharide with Kdo at the reducing end. The structure established is unique among Proteus O polysaccharides, which is in agreement with the lack of any significant cross-reactivity for the lipopolysaccharide of strain 11 B-r and O antisera against strains of all known Proteus O serogroups and vice versa.

Immunochemical properties of Proteus penneri lipopolysaccharides--one of the major Proteus sp. virulence factors.

Proteus penneri, like the other seven species from the genus, are Gram-negative, peritrichously flagellated rods capable of swarming growth on humid solid media. These bacteria are human opportunistic pathogens involved in many infections but they mainly affect the urinary tract of hospitalized, long-term catheterized patients. P. penneri rods produce a lot of virulence factors, among which the lipopolysaccharide seems to be the most interesting due to its structural and serological diversity. From the three LPS regions of P. penneri strains only the core region and O-specific polysaccharide (OPS) were structurally and serologically examined. P. penneri LPS core region is characterized by a common inner part representing the III glycoform and a diverse distal part (12 different structures). The P. penneri O-antigens contain sugar and non-sugar compounds and some of them rarely occur in nature. In both P. penneri LPS regions putative epitopes have been pointed out. Serospecificity of OPS allowed classifying many P. penneri isolates to different Proteus sp. O-serogroups, among which 12 contain P. penneri strains only.

Characterization of epitope specificity of Proteus penneri 7 lipopolysaccharide core region.

To extend the knowledge on the fragments of Proteus penneri lipopolysaccharide core regions, which determine the cross-reactions with specific antibodies, serological studies were performed by use of P. penneri 7 core-specific antiserum and Proteus sp. lipopolysaccharides. Different reactivity of the tested antiserum with three groups of antigens suggested differences in their core regions' epitope specificity. Comparing the results of the serological investigations with the previously determined structures of the core regions of the tested P. penneri lipopolysaccharides allowed distinguishing two potential tri- and tetrasaccharide epitopes and a third fragment which could not be determined precisely.

Serological characterization of the core region of lipopolysaccharides of rough Proteus sp. strains.

INTRODUCTION: Both smooth and rough Proteus sp. strains can be found. The latter are characterized by their lack of an O-polysaccharide chain in the lipopolysaccharide (LPS) molecule, which makes them suitable for obtaining anti-core sera. Using this kind of material enables identifying fragments of the Proteus LPS core region that might be involved in cross-reactions. To date only a few similar epitopes have been established for the genus Proteus. MATERIALS AND METHODS: Polyclonal rabbit antisera directed against three rough strains of Proteus sp. were tested by enzyme-linked immunosorbent assay (ELISA) with a set of LPSs. The reactivity of the selected cross-reactive and homologous systems was checked by the Western blot technique and by a passive immunohemolysis assay preceded by the absorption of each antiserum with appropriate cross-reactive and homologous alkalized LPSs. RESULTS: On the basis of the ELISA results, 19 cross-reactive antigens were selected among which both smooth and rough LPS forms were found. All the observed reactions involved the core region of the LPS. Using the antisera absorbed with the appropriate LPSs allowed identification of four groups of antigens with serologically identical core regions. CONCLUSIONS: Comparing the results of the serological studies with the known chemical structures of the core regions of the LPSs used enabled the identification of a few core oligosaccharide fragments probably involved in the observed cross-reactions. All were located in the most distal part of LPS core region, which made them more easily recognized by specific antibodies.

Application of two different kinds of sera against the Proteus penneri lipopolysaccharide core region in search of epitopes determining cross-reactions with antibodies.

INTRODUCTION: Proteus penneri lipopolysaccharide (LPS) core regions are characterized by a greater structural variability than that observed in other Enterobacteriaceae. This fact and the small amount of published data concerning the serological activity of this part of P. penneri LPS prompted an examination of which fragment might determine cross-reactions with antibodies. To date, such epitopes have been found in the LPS core regions of P. mirabilis and P. vulgaris strains. MATERIALS AND METHODS: Proteus sp. LPSs were tested with unabsorbed rabbit antisera by enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot, and once again by ELISA or passive immunohemolysis after the absorption of these antisera with selected LPSs. RESULTS: The serological studies of P. penneri 8 LPS demonstrated antibodies in the tested antisera recognizing a common epitope located in the core regions of six of the LPSs, i.e. P. penneri 8, 34, 133, 7, 14, and 15. Additionally, another type of antibody directed against some fragment of P. penneri 13 and the core regions of other LPSs investigated was observed in one antiserum. CONCLUSIONS: A distal, trisaccharide fragment of the P. penneri 8 LPS core region is suggested to determine the cross-reactions of the tested antisera with the six P. penneri LPSs.