Serological Studies of an Acid-Labile O-Polysaccharide of Proteus vulgaris OX19 Lipopolysaccharide Using Human and Rabbit Antibodies

In a Weil-Felix test, sera from patients infected with Rickettsia sp. agglutinate Proteus OX types of bacteria and Proteus lipopolysaccharide (LPS) are responsible for the cross-reaction. Data on the character of LPS of one of the OX group strains, Proteus vulgaris OX19, are contradictory, and it remained unclear whether it has an O-polysaccharide (OPS) and is thus LPS of the smooth type (S) or not (rough-type LPS). Our studies showed that P. vulgaris OX19 (strain PZH-24) produces a smooth-type LPS that contains a long-chain OPS, but it undergoes depolymerization during mild acid hydrolysis conventionally used for LPS delipidation and loses the serological activity. An elucidation of the complete structure of OPS demonstrated the presence of a glycosyl phosphate linkage responsible for the acid-lability of the polysaccharide chain. In ELISA, both IgM type antibodies in a Weil-Felix test with human anti-Rickettsia typhi sera and rabbit anti-P. vulgaris OX19 antibodies reacted with OPS. Rabbit antibodies did not inhibit the cross-reaction with human antibodies and thus bind to different epitopes.     

Serological Studies of the Antigenic Similarity between Typhus Group Rickettsiae and Weil-Felix Test Antigens

The sera from two patients with murine typhus reacted with whole cells of Rickettsia prowazekii, R. typhi, and Proteus vulgaris OX19, and with lipopolysaccharides (LPS) from the spotted fever group rickettsia strain TT-118 and P. vulgaris OX19 in the enzyme-linked immunosorbent assay. Sera from these patients reacted with ladder-like bands of LPS from R. prowazekii and R. typhi in the immunoblot, whereas the reactivity of these sera with LPS from P. vulgaris OX19 differed from each other. These results indicate that LPS from the typhus group rickettsiae and P. vulgaris OX19 contain similar epitopes.     

Monoclonal antibodies as probes to examine serotype-specific and cross-reactive epitopes of lipopolysaccharides from serotypes O2, O5, and O16 of Pseudomonas aeruginosa.

Serotypes O2, O5, and O16 of Pseudomonas aeruginosa are chemically related, and the O antigens of their lipopolysaccharides share a similar trisaccharide repeat backbone structure. Serotype-specific monoclonal antibodies (MAbs) MF71-3, MF15-4, and MF47-4 against the O2, O5, and O16 serotypes, respectively, were isolated. MAb 18-19, which is cross-reactive with all strains of this chemically related serogroup, was also produced. When column chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated lipopolysaccharide (LPS) samples from each of the serotypes were probed with the MAbs in Western immunoblots, each of the serotype-specific MAbs interacted only with high-molecular-weight bands of the homologous LPS, with a minimum O-antigen chain length of at least 6 to 10 repeats. In contrast, cross-reactive MAb 18-19 was shown to interact in Western immunoblots with the entire LPS banding pattern except the fastest-running band, which lacks O antigen. Chemical modification of P. aeruginosa LPS by alkali treatment and carboxyl reduction abolished reactions between LPS and MAb 18-19, while reactions of modified LPS with serotype-specific MAbs were not affected. Therefore, cross-reactive MAb 18-19 likely recognizes the chemical backbone structure of the O repeat that is common to all three serotypes of the O2-O5-O16 group, while the O-specific MAbs appeared to recognize LPS epitopes that could be presented when 6 to 10 or more O-antigen repeat units are present on the LPS molecule. Thus, the O-specific LPS epitopes likely involve unique chemical structures, glycosidic linkages, and some order of folding of the O side chains.     

Immunological characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test and reactivity with patient sera of tsutsugamushi diseases.

Immunological analyses of lipopolysaccharides (LPS) isolated from Proteus strains OX2, OX19, and OXK used as antigens of Weil-Felix (WF) test, were performed by quantitative agglutination, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Antisera against LPS and whole cells (WC) of the three Proteus strains reacted with homologous LPS but not with heterologous LPS, and the reaction was inhibited by the O-polysaccharide fraction isolated from the homologous LPS except OX19-LPS, which lacked O-polysaccharide moiety. The immunological data support the findings that the O-polysaccharide moieties of LPS from OX2 and OXK strains possess different chemical composition (Mizushiri, Amano, Fujii, Fukushi, and Watanabe, Microbiol. Immunol. 34: 121-133, 1990). Antisera against Proteus strains reacted weakly with WC of Rickettsia prowazekii, Rickettsia typhi, and Rickettsia tsutsugamushi. Antisera from patients with tsutsugamushi disease reacted with OXK-WC by WF test when the sera were obtained 13 days after onset of fever. The immunoperoxidase (IP) test titers of these antisera began to rise 6 days after the onset of fever. By ELISA tests these antisera reacted with OXK-WC and OXK-LPS independently of the titers of WF or IP tests.     

Serological Reactivity of Sera from Scrub Typhus Patients against Weil-Felix Test Antigens

Sera from 17 patients of scrub typhus in the acute and convalescent phases were tested by indirect immunoperoxidase test, Weil-Felix (WF) test, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. In the comparison of antibody titers between acute- and convalescent-phase sera, we recognized a parallelism of increment between the titers in WF test and titers of immunoglobulin M (IgM) in ELISA against Proteus mirabilis strain OXK-whole cells and OXK-lipopolysaccharides (Proteus OXK-LPS). Furthermore, IgM antibodies from almost all of WF test-positive sera recognized LPS from Proteus OXK in immunoblotting. Based on these results, it was concluded that IgM antibody rather than IgG may participate in WF test, and that Proteus OXK-LPS may have one of antigenic epitopes common to the components of R. tsutsugamushi.     

Monoclonal antibodies that distinguish inner core, outer core, and lipid A regions of Pseudomonas aeruginosa lipopolysaccharide.

In order to examine the immunochemistry of the core-lipid A region of Pseudomonas aeruginosa lipopolysaccharide (LPS), monoclonal antibodies (MAbs) specific for this region were produced in mice. Immunogen was prepared by coating a rough mutant of P. aeruginosa with column-purified core oligosaccharide fractions in order to enhance the immune response to the LPS core-lipid A region. Fourteen hybridoma clones were isolated, characterized, and further divided into three groups on the basis of their reactivities to rough LPS antigens in both enzyme-linked immunosorbent assays and Western immunoblots. In addition, another MAb, 18-19, designated group 1, was included in this study for defining core-lipid A epitopes. MAb 18-19 recognizes the LPS core-plus-one O-repeat unit of the serologically cross-reactive P. aeruginosa O2, O5, and O16. Group 2 MAbs are specific for the LPS outer core region and reacted with P. aeruginosa O2, O5, O7, O8, O10, O16, O18, O19, and O20, suggesting that these serotypes share a common outer core type. Group 3 MAbs recognize the inner core region and reacted with all 20 P. aeruginosa serotypes as well as with other Pseudomonas species, revealing the conserved nature of this region. Group 4 MAbs are specific for lipid A and reacted with all gram-negative organisms tested. Immunoassays using these MAbs and well-defined rough mutants, in addition to the recently determined P. aeruginosa core structures, have allowed us to precisely define immunodominant epitopes within the LPS core region.     

Chemical characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test

The lipopolysaccharides (LPS) extracted from Proteus strains OX2, OX19, and OXK used as antigens in the Weil-Felix test, were characterized by chemical analysis and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To separate the O-polysaccharide, core-oligosaccharide, and lipid A moieties, each LPS was treated with 2% acetic acid, centrifuged, and applied to Sephadex G-50 column. The core-oligosaccharides contained L-glycero-D-mannoheptose, D-glycero-D-mannoheptose, glucose (Glc), galactose, 3-deoxy-D-mannooctulosonic acid, uronic acid, phosphate, glucosamine (GlcN), and galactosamine (GalN). The lipid A preparations contained GlcN, GlcN-phosphate, and three fatty acids (myristic, plamitic, and beta-hydroxymyristic acids). However, the O-polysaccharides of OX2- and OXK-LPS had different chemical compositions which consisted of Glc, GlcN, and quinovosamine, and Glc, uronic acid, and GalN, respectively, while OX19-LPS seemed to lack O-polysaccharide.     

Monoclonal Antibodies againstAntigens of Hydra vulgaris and Hydra oligactis

The goal of the study was to obtain a panel of monoclonal antibodies (MAb) against antigens of freshwater polyps of the genus Hydra. Hybrid mice F₁(Balb/c × SJL/J) were immunized with cell membrane fraction of H. vulgaris and three months later their splenocytes were fused with cultured mouse myeloma cells 653A. Testing of culture fluids in ELISA with immobilized H. vulgaris cells, 82 hybridomas producing MAb were revealed. Study of MAb specificity in ELISA with H. vulgaris and H. oligactis cells indicated that 22% of them recognized only H. vulgaris antigens. About 50% of MAb recognized equally antigens of the both species. The rest of MAb reacted with H. vulgaris and H. oligactis antigens to different degree. Eight hybridomas producing MAb of all three above groups were adapted for growth as ascitic tumors. The distribution of antigens binding these MAb was studied in indirect immunofluorescence on fixed polyps, living or fixed cells, and on paraffin- embedded sections. Among the best studied MAb, of the greatest interest were the following reagents. One of them (1A10) revealed an antigen on surface membranes of ectodermal epithelial cells of H. vulgaris. The second one (1G10) was specific of the antigen located in mesoglea and basal cytoplasmic areas of ectodermal and entodermal epithelial cells of the both hydra species. The MAb 4G3 interacted with cytoplasmic antigen of ectodermal epithelia-muscular cells of the both hydra species. MAb 4H1 revealed nematocytes in H. vulgaris and H. oligactis. The data obtained indicate that in two species of hydra the epitopes binding the same MAb might be located in cells of different types.     

Epitope Identification for a Panel of Anti-Sinorhizobium meliloti Monoclonal Antibodies and Application to the Analysis of K Antigens and Lipopolysaccharides from Bacteroids

In two published reports using monoclonal antibodies (MAbs) generated against whole cells, Olsen et al. showed that strain-specific antigens on the surface of cultured cells of Sinorhizobium meliloti were diminished or absent in the endophytic cells (bacteroids) recovered from alfalfa nodules, whereas two common antigens were not affected by bacterial differentiation (P. Olsen, M. Collins, and W. Rice, Can. J. Microbiol. 38:506–509, 1992; P. Olsen, S. Wright, M. Collins, and W. Rice, Appl. Environ. Microbiol. 60:654–661, 1994). The nature of the antigens (i.e., the MAb epitopes), however, were not determined in those studies. For this report, the epitopes for five of the anti-S. meliloti MAbs were identified by polyacrylamide gel electrophoresis-immunoblot analyses of the polysaccharides extracted from S. meliloti and Sinorhizobium fredii. This showed that the strain-specific MAbs recognized K antigens, whereas the strain-cross-reactive MAbs recognized the lipopolysaccharide (LPS) core. The MAbs were then used in the analysis of the LPS and K antigens extracted from S. meliloti bacteroids, which had been recovered from the root nodules of alfalfa, and the results supported the findings of Olsen et al. The size range of the K antigens from bacteroids of S. meliloti NRG247 on polyacrylamide gels was altered, and the epitope was greatly diminished in abundance compared to those from the cultured cells, and no K antigens were detected in the S. meliloti NRG185 bacteroid extract. In contrast to the K antigens, the LPS core appeared to be similar in both cultured cells and bacteroids, although a higher proportion of the LPS fractionated into the organic phase during the phenol-water extraction of the bacteroid polysaccharides. Importantly, immunoblot analysis with an anti-LPS MAb showed that smooth LPS production was modified in the bacteroids.     

Bactericidal Monoclonal Antibody Against <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis> Lipooligosaccharide Cross-Reacts with <Emphasis Type="Italic">Haemophilus Spp.</Emphasis>

Monoclonal antibodies (MAbs) against lipooligosaccharide (LOS) determinants after immunization of BALB/c mice with heat inactivated Moraxella catarrhalis serotype A were generated. MAb 219A9 was specific for a common epitope of A, B, and C M. catarrhalis serotypes in ELISA and immunofluorescent test (IFT). In both tests it also cross-reacted with whole bacteria and LPS antigens isolated from non-typeable H. influenzae and H. parainfluenzae strains. IgM antibody clone 219A9 possessed a strong bactericidal effect against the three serotypes in the presence of complement. Our results demonstrate that antibodies directed to a single LOS epitope common for A, B, and C serotype could be highly protective. This suggests that the common determinants are very promising in the development of LOS-based vaccine against M. catarrhalis. The cross-reactions of MAb 219A9 with Haemophilus spp. also show that immunization could result in immune response to epitopes conserved in other important respiratory pathogens.     

The structure of the carbohydrate backbone of the core–lipid A region of the lipopolysaccharide from Proteus penneri strain 40: new Proteus strains containing open-chain acetal-linked N-acetylgalactosamine in the core part of the LPS

Analysis of the core part of the LPS from several strains of Proteus revealed that P. penneri strains 2, 11, 19, 107, and P. vulgaris serotypes O4 and O8 have the same structure with a new type of linkage between monosaccharides–an open-chain acetal — that was previously determined for P. vulgaris OX2 and P. penneri 17. The LPS from P. penneri strain 40 contains the same structure substituted with one additional monosaccharide:

Full-size image (5K)

where (1S)-GalaNAc¹ is a residue of N-acetyl- -galactosamine in the open-chain form. It is connected as a cyclic acetal to positions 4 and 6 of the galactosamine residue having a free amino group. All other sugars are in the pyranose form.     

Effect of the Proteolytic Enzymes of Bacillus licheniformis and the Lysoamidase of Lysobacter sp. XL1 on Proteus vulgaris and Proteus mirabilis Cells

Preparations of culture liquid of three Bacullus licheniformis strains (S, 103, and 60.4) and the enzymatic preparation lysoamidase from culture liquid of Lysobacter sp. strain XL1 actively lysed pre-autoclaved cells of the gram-negative bacteria Proteus vulgaris and P. mirabilis. Living Proteus cells treated with these enzymatic preparations were lysed during their subsequent autoclaving. Inoculation of enzyme-treated Proteus cells, taken either separately or in combination with one another and polymyxin B, into a rich medium led to cell repair and restoration of the culture viability. __________ Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 5, 2005, pp. 558–563. Original Russian Text Copyright ? 2005 by Ryazanova, Ledova, Tsurikova, Stepnaya, Sinitsyn, Kulaev.     

A new bacteriophage typing scheme forProteus mirabilis andProteus vulgaris strains

The lytic properties of 21 bacteriophages constituting a new typing set forProteus were examined in 507Proteus mirabilis and 29P. vulgaris strains isolated from patients and healthy subjects. Comparison of their morphological, serological, genetic and lytic properties showed that, in theMyoviridae andPodoviridae families, some phages were so closely related that the presence of all of them in the set was redundant. Analysis of the lytic properties revealed that some of the bacteriophages were not active enough to facilitate the differentiation ofProteus strains The size of the final typing set was reduced from 21 to 12 phages but it was suggested that, in order to improve the differentiation capacity of the set, new phages should be included. Second part:Folia Microbiol. 41, 137–140 (1996).     

Structural studies on the lipopolysaccharide core of Proteus OX strains used in Weil-Felix test: a mass spectrometric approach

The core region of the lipopolysaccharides of Proteus group OX bacteria, which are used as antigens in Weil-Felix test for serodiagnosis of rickettsiosis, were studied by chemical degradations in combination with ESI FTMS, including infrared multi-photon dissociation (IRMPD) MS/MS and capillary skimmer dissociation. Structural variants of the inner core region were found to be the same as in Proteus non-OX strains that have been studied earlier. The outer core region has essentially the same structure in Proteus vulgaris OX19 (serogroup O1) and OX2 (serogroup O2) and a different structure in Proteus mirabilis OXK (serogroup O3). A fragmentation due to the rupture of the linkage between GlcN or GalN and GalA was observed in IRMPD-MS/MS of core oligosaccharides and found to be useful for screening of Proteus strains to assign structures of the relatively conserved inner core region and to select for further studies strains with distinct structures of a more variable outer core region.     

Epitope identification for a panel of anti-Sinorhizobium meliloti monoclonal antibodies and application to the analysis of K antigens and lipopolysaccharides from bacteroids.

In two published reports using monoclonal antibodies (MAbs) generated against whole cells, Olsen et al. showed that strain-specific antigens on the surface of cultured cells of Sinorhizobium meliloti were diminished or absent in the endophytic cells (bacteroids) recovered from alfalfa nodules, whereas two common antigens were not affected by bacterial differentiation (P. Olsen, M. Collins, and W. Rice, Can. J. Microbiol. 38:506-509, 1992; P. Olsen, S. Wright, M. Collins, and W. Rice, Appl. Environ. Microbiol. 60:654-661, 1994). The nature of the antigens (i.e., the MAb epitopes), however, were not determined in those studies. For this report, the epitopes for five of the anti-S. meliloti MAbs were identified by polyacrylamide gel electrophoresis-immunoblot analyses of the polysaccharides extracted from S. meliloti and Sinorhizobium fredii. This showed that the strain-specific MAbs recognized K antigens, whereas the strain-cross-reactive MAbs recognized the lipopolysaccharide (LPS) core. The MAbs were then used in the analysis of the LPS and K antigens extracted from S. meliloti bacteroids, which had been recovered from the root nodules of alfalfa, and the results supported the findings of Olsen et al. The size range of the K antigens from bacteroids of S. meliloti NRG247 on polyacrylamide gels was altered, and the epitope was greatly diminished in abundance compared to those from the cultured cells, and no K antigens were detected in the S. meliloti NRG185 bacteroid extract. In contrast to the K antigens, the LPS core appeared to be similar in both cultured cells and bacteroids, although a higher proportion of the LPS fractionated into the organic phase during the phenol-water extraction of the bacteroid polysaccharides. Importantly, immunoblot analysis with an anti-LPS MAb showed that smooth LPS production was modified in the bacteroids.     

Chicken Mhc alloantiserum cross-reactivity analysis by hemagglutination and flow cytometry

 The major histocompatibility complex (Mhc) haplotype in the chicken is generally determined by the use of alloantisera in a hemagglutination assay. This method restricts haplotype determination to antigens expressed on the surface of erythrocytes which includes class I (B – F) and class IV (B – G) antigens as well as any other polymorphic molecules on these cells. Alloantisera can result in complex cross-reactivity patterns. We describe here the analysis of 53 alloantisera made within Mhc-congenic lines. Each antiserum was tested by hemagglutination with erythrocytes and by flow cytometry with erythrocytes and peripheral white blood cells of seven Mhc haplotypes; B ² , B ⁵ , B ¹² , B ¹³ , B ¹⁵ , B ¹⁹ , and B ²¹ . Five types of antiserum were identified based on their reactivity to different cell subpopulations of the peripheral blood of the donor haplotype as well as in cross-reactivity for different haplotypes. RBC specific cross-reactive antigens attributed to B – G molecules were demonstrated for the B5 : B19, B12 : B19, and B19 : B21 cross-reactions. Cross-reactive antigens detected on RBC and thrombocytes attributable to B – G molecules on both types of cells were demonstrated for the B2 : B12, B2 : B15, B2 : B19, and B2 : B21 cross-reactions. In addition, cross-reactive antigens occurring on RBC and WBC were attributed to B – F (or RBC and lymphocyte-expressed B – G loci) and included the B12 : B13, B13 : B19, and B15 : B19 cross-reactions. Several antisera with specificity for B cells purportedly identifying B – L epitopes were found but their numbers were limited and cross-reactivities were not defined. The identities described here may be useful in understanding B haplotype similarities and differences in disease resistance and immune response. Received: 18 September 1995 / 15 November 1995     

Establishment and Application of a Dual TaqMan Real-Time PCR Method for Proteus Mirabilis and Proteus Vulgaris

Proteus species are common opportunistic bacteria and foodborne pathogens. The proper detection of Proteus can effectively reduce the occurrence of food-borne public health events. Proteus mirabilis and Proteus vulgaris are the two most important pathogens in the Proteus genus. In this study, a dual TaqMan Real-Time PCR method was established to simultaneously detect and distinguish P. mirabilis and P. vulgaris in samples. The method exhibited good specificity, stability, and sensitivity. Specifically, the minimum detection concentrations of P. mirabilis and P. vulgaris in pure bacterial cultures were 6.08 × 10² colony forming units (CFU)/ml and 4.46 × 10² CFU/ml, respectively. Additionally, the minimum detectable number of P. mirabilis and P. vulgaris in meat and milk was 10³ CFU/g. In addition, the method can be used to distinguish between strains of P. mirabilis and P. vulgaris within two hours. Overall, it is a sensitive, easy-to-use, and practical test for the identification and classification of Proteus in food.Key words: Proteus mirabilis, Proteus vulgaris, TaqMan Real-Time PCR, food-borne pathogens, food poisoning     

Production of superoxide dismutases from Proteus mirabilis and Proteus vulgaris

Proteus mirabilis and Proteus vulgaris expressed a combination of superoxide dismutase (Sod) activities, which was assigned to FeSod1, FeSod2 and MnSod for P. mirabilis, and FeSod, MnSod and CuZnSod for P. vulgaris. Production of the Sod proteins was dependent on the availability of iron, whether cells were grown under anaerobiosis or aerobiosis and growth phase. Nalidixic acid and chloramphenicol inhibited cell growth and the iron- and dioxygen-dependent production of Sod. These results support the involvement of metal ions and redox status in the production of Proteus Sods.     

Neutralization of Chlamydia psittaci with Monoclonal Antibodies

Neutralization of Chlamydia (C.) psittaci avian strain P-1041 was examined in vitro using monoclonal antibodies (MAbs). Of the 10 MAbs used, 6 were found to exhibit neutralizing capability. These include 3 against major outer membrane protein (MOMP), 1 against lipopolysaccharide (LPS) and 2 against other protein molecules [90 kilodalton (kDa) and 90/50 kDa]. Most neutralizing MAbs were dependent on complement for efficient neutralization, while a strain-specific MAb (2B5) against the 90 kDa protein displayed a different requirement for complement and neutralized the infectivity of the P-1041 at high concentrations without complement. By competitive inhibition enzyme-linked immunosorbent assay (competitive inhibition ELISA), all 3 neutralizing anti-MOMP MAbs were demonstrated to recognize different epitopes found in very close proximity to each other on the outer membrane.     

New Structures of the O-Specific Polysaccharides of Proteus. 2*. Polysaccharides Containing O-Acetyl Groups

Structures of five new O-specific polysaccharides of Proteus bacteria were established. Four of them, Proteus penneri 4 (O72), Proteus vulgaris 63/57 (O37), Proteus mirabilis TG 277 (O69), and Proteus penneri 20 (O17), contain O-acetyl groups in non-stoichiometric quantities, and the polysaccharide of P. penneri 1 is structurally related to that of P. penneri 4. The structures were elucidated using NMR spectroscopy, including one dimensional ¹H- and ¹³C-NMR spectroscopy, two-dimensional ¹H, ¹H correlation (COSY, TOCSY), H-detected ¹H, ¹³C heteronuclear multiple-quantum coherence (HMQC), heteronuclear multiple-bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY or ROESY), along with chemical methods. The structural data obtained are useful as the chemical basis for the creation of the classification scheme for Proteus strains.